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This is to certify that the
dissertation entitled

BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF .

NATURALLY OCCURRING RECOMBINANT AVIAN
LEUKOSIS VIRUS ISOLATED FROM COMMERCIAL
LAYERS AFFECTED WITH MYELOID LEUKOSIS

presented by

Ghida R. Banat

has been accepted towards fulfillment
of the requirements for the

Ph.D degree in Pathology

 

 

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BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF NATURALLY
OCCURRING RECOMBINANT AVIAN LEUKOSIS VIRUS ISOLATED FROM
COMMERCIAL LAYERS AFFECTED WITH MY ELOID LEUKOSIS

By

Ghida R. Banat

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Pathology

2009

Copyright by
GHIDA R. BANAT
2009

ABSTRACT
BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF NATURALLY
OCCURRING RECOMBINANT AVIAN LEUKOSIS VIRUS ISOLATED FROM
COMMERCIAL LAYERS AFFECTED WITH MYELOID LEUKOSIS
By

Ghida R. Banat

Avian leukosis virus (ALV) is an economically important poultry pathogen
resulting in low productivity and tumor mortality in chickens. Limited information is
currently available about natural recombination among different ALV subgroups and
pathogenesis in chicken. Several biological and molecular studies were conducted to
characterize seven isolates (AF 115-1, AF 115-5, AF 115-7, AF 115-10, AF 115-13, AF
115-14 and AF 115-16) of a naturally occurring recombinant ALV from commercial
layers affected with myeloid leukosis.

The first aim was to characterize seven isolates (AF 115-1, AF 115-5, AF 115-7,
AF 115-10, AF 115-13, AF 115-14 and AF 115-16) ofa naturally occurring recombinant
ALV by biological assays and polymerase chain reaction. Closely and distinctly related
strains of naturally occurring recombinant ALVs were identified using various diagnostic
techniques. The host range and antigenic relationships of the seven AF 115 isolates were
comparable with those of the first characterized naturally occurring recombinant ALV-
B/J (AF 115-4). However, the PCR amplification results, using different primer sets,
indicated the presence of recombinant ALV-B/J sequences in proviral DNA of all AF 1 15
isolates. Moreover, three isolates (AF 115-5, AF 115-7 and AF 115-10) demonstrated

different patterns of PCR amplification from the recombinant ALV-B/J (AF 115-4) and

the rest of the isolates, suggesting the presence of mutations and rearrangements in
different genomic regions.

The second aim was to molecularly characterize these seven AF 115 isolates by
DNA sequence analyses of various genomic regions. All AF 115 isolates were confirmed
to be recombinant ALVs with different genomic regions representing different ALV
subgroups. Five isolates (AF 115-1, AF115-7, AF 115-l3, AF 115-l4 & AF 115-16)
were closely related to the naturally occurring recombinant ALV-B/J, containing a
subgroup B gp85, subgroup E gp37 and subgroup J 3’ UTR (including DR] and E
element) and LTR. On the other hand, AF 115-5 had a subgroup A gpSS and is believed
to be contain a mixture of two recombinant ALVs (ALV-A/J & ALV-B/J). The 3’ UTR
and LTR of AF 115-10 contained base deletions and insertions and were highly
associated with a recombinant ALV isolate (ALV TymS_90). In addition, all AF 115
isolates contained important potential sites for protein structure, stability and function in
the envelope glycoproteins as well as transcription regulatory elements in the 3’ UTR and
LTR. It is believed that recombination hotspots exist between SU and TM domains of the
envelope, between the gp37 and the 3’ UTR upstream of the DR] region as well as 3’
UTR and LTR.

Therefore, these research studies have added new information on ALV diversity
(mutations, rearrangement and recombination) and should aid in improving the diagnosis

and control of ALV infection in field flocks.

I would like to dedicate this work
to

my family especially my mother Mrs. Kamleh Ibrahim Al-Baitam
and my father Mr. Rakad Saleh Banat

ACKNOWLEDGMENTS

Thanks to God, The Greatest and The Merciful, who inspired and guided me
through my entire life, especially through my Ph.D. journey.

I would like to express my gratitude and appreciation to my current academic
advisor Dr. Scott Fitzgerald, my research advisor Dr. Aly F adly and my previous
academic advisor Dr. Willie Reed for providing me the opportunity to pursue my
graduate Ph.D. studies in the collaborative program between USDA-ARS-ADOL and
DCPAH. In addition, I would like to thank Dr. Robert Silva, Dr. John Gerlach and Dr.
Steve Bolin for serving on my graduate guidance committee. I am grateful for all
members of the graduate committee for their constant support, patience, encouragement,
time, effort, shared knowledge and for reviewing the manuscript, making valuable
suggestions and providing comments for improvement. I value the friendship, assistance,
time and effort provided by all the scientists and staff at ADOL. In particular, I would
like to thank Mr. Lonnie Milam, Dr. Jody Mays, Ms. Melanie Flesberg, Dr. Henry Hunt
and Mrs. Pamela Campbell for their invaluable training, help, discussion and guidance in
the laboratory. Thanks to all for this opportunity to grow professionally and personally.

I would also like to thank all my friends for all their constant help, support and
encouragement. Profound appreciation and gratitude go to my dear mother for her
unconditional love, patience, support and encouragement. I am forever grateful for my
dear parents for providing me with the best education and opportunities. I am also
appreciative to my late grandfather, sisters Dana, Doaa, and Areej, brothers Saleh and

Ehab and their families for all their support and encouragement. I love you all so much.

vi

TABLE OF CONTENTS

LIST OF TABLES .............................................................................
LISTOFFIGURES .................
KEY TO SYMBOLS AND ABBREVIATIONS ..........................................
INTRODUCTION .............................................................................

CHAPTER 1: LITERATURE REVIEW ...................................................
I. Retroviridae ................................................................................

A. Introduction .............................................................................

B. Morphology .............................................................................
C. Components of Retroviruses ..........................................................
D. Classification of Retroviruses ........................................................
E. Seven Genera of Retroviruses ........................................................
II. Diversity in Retroviruses ................................................................
A. Introduction .............................................................................
B. General Principles ........................................................................
C. Mutations ................................................................................
D. Recombination ..........................................................................
E. Rearrangements .........................................................................
F. Role of Selective Forces ................................................................
G. Evolution and Retroviruses .............................................................
III. Avian Leukosis-Sarcoma Viruses and Diseases ......................................
A. Introduction .............................................................................
B. Etiology ..................................................................................
C. Epizootiology ...........................................................................
D. Pathobiology .............................................................................
E. Immunology .................................................................................
F. Genetic Resistance .......................................................................
G. Diagnosis .................................................................................
H. Intervention Strategies ...................................................................

CHAPTER 2: Characterization of various isolates of a naturally occurring
recombinant avian leukosis virus using biological assays and polymerase chain
reaction ..........................................................................................
Abstract .........................................................................................
Introduction .....................................................................................
Materials and Methods .......................................................................
A. Origin of Viruses .......................................................................
B. Biological Assays ......................................................................
C. p27 ELISA Assay ......................................................................

vii

ix

xi

XV

OLA-kw WU)

ll
16
16
17
19
21
24
24
27
28
28
29

41
45

57
59
61
64

75
75
76
80
8O
81
82

D. Virus Neutralization ................................................................... 83

B. DNA Isolation ............................................................................ 84
F. Oligonucleotide Primers ................................................................ 84
G. Polymerase Chain Reaction (PCR) .................................................. 85
Results ......................................................................................... 86
A. Host range of AP 115 isolates ........................................................ 86
B. Antigenicity of AF 115 isolates ...................................................... 87
C. PCR amplification of DNA from AF 115-infected line 0 CEFS ................. 87
D. PCR reactivity of AF 115 isolates in 72 infected CEF S ............................ 92
E. PCR reactivity of AF 115 isolates in DF -1/J infected cells ....................... 93
Discussion ...................................................................................... 95

CHAPTER 3: Comparison of proviral DNA sequences of seven isolates of
naturally occurring recombinant avian leukosis virus and representative ALVS. 135

Abstract ......................................................................................... 135
Introduction ..................................................................................... 137
Materials and Methods ....................................................................... 140
A. Viruses and Cells ....................................................................... 140
B. DNA Extraction ........................................................................ 141
C. Amplification of Proviral DNA ...................................................... 141
D. Subcloning of PCR Products ......................................................... 142
E. Sequence Determination ................................................................ 143
F. Analysis of Sequence Data ............................................................. 143
Results ......................................................................................... 144
A. Analysis of sequence data ............................................................ 144
B. Sequence comparisons among AF 115 isolates ..................................... 145
C. Sequence comparisons of AF 115 isolates and ALV subgroups ................. 150
D. Sequence comparisons of AF 115 isolates and blasted ALV strains. . . . . 154
Discussion ...................................................................................... 1 58
CHAPTER 4: Conclusions and future directions .......................................... 209
Introduction ................................................................................... 209
Chapter 2 Conclusions ...................................................................... 210
Chapter 2 Limitations ........................................................................ 210
Chapter 3 Conclusions ...................................................................... 211
Chapter 3 Limitations ........................................................................ 214
Results Comparison ......................................................................... 214
Recommendations ........................................................................... 21 7
Future Studies ................................................................................ 219
Summary ....................................................................................... 221
REFERENCES ................................................................................. 224

viii

LIST OF TABLES

Table 2.1. Reverse PCR primers designed to detect U3 or US common sequences
of LTR of subgroup B ALV ..................................................................

Table 2.2. Forward PCR primers for different ALV subgroups............... . .
Table 2.3. Reverse PCR primers for different ALV subgroups .........................
Table 2.4. Forward and reverse primer combinations and their characteristics .......
Table 2.5. PCR reaction buffer systems ....................................................
Table 2.6. PCR cycles used with different primer sets ...................................

Table 2.7. Infectivity or host range Of AF 115 isolates in cells of different
phenotypes using virologic p27 ELISA .....................................................

Table 2.8. Antigenic relationships of AF 115 isolates to ALV subgroups B and I
using virus neutralization test ................................................................

Table 2.9. PCR detection of ALV sequences in DNA of AF 115-infected line 0
CEFS using all-subgroup primer pairs ......................................................

Table 2.10. PCR detection of ALV sequences from recombinant ALV-B/J in
DNA of AF 115-infected line 0 CEFS ......................................................

Table 2.11. PCR detection of subgroup B ALV-like sequences in genomic DNA
of AF 115-infected line 0 CEFS ..............................................................

Table 2.12. PCR detection of subgroup J ALV-like sequences in genomic DNA of
AF 115-infected line 0 CEF S .................................................................

Table 2.13. PCR detection of ALV sequences in genomic DNA of AF 115-
infected 72 (C/ABDE) CEFS ..................................................................

Table 2.14. PCR detection of ALV sequences in genomic DNA of AF 115-
infected DF -1/J (C/EJ) cells ..................................................................

Table 3.1. Forward (F) and reverse (R) PCR primers used for amplification of
proviral DNA of AF 115 isolates ............................................................

Table 3.2. Relevant accession numbers for viral sequences used in analyses. . . .

ix

104

105

106

107

108

109

110

111

112 _

113

114

115

116

117

175

176

Table 3.3. Sequence distances of gp85 amino acids of seven AF 115 isolates and
the recombinant ALV-B/J (AF 115-4) by ClustalW (slow/accurate, Gonnet). . . .

Table 3.4. Sequence identity among gp37 amino acids of seven AP 115 isolates

and the recombinant ALV-B/J (AF 115-4) by ClustalW (slow/accurate, Gonnet). . ..

Table 3.5. Percentage Identity and divergence of nucleotide sequences of 3’ UTR
and LTR of all AF 115 isolates and ALV-B/J (AF 115-4) by ClustalW
(slow/accurate, IUB) ...........................................................................

Table 3.6. Sequence distances of amino acid sequences of SU domains of AF 115
isolates and representatives of ALV subgroups by ClustalW (slow/accurate,
Gonnet) ..........................................................................................

Table 3.7. Percentage identity and divergence of amino acid sequences of TM
domains of AF 115 isolates and different ALV subgroups by ClustalW
(Slow/accurate, Gonnet) .......................................................................

Table 3.8. Sequence distances comparison among nucleotide sequences of 3’ UTR
of all AP 115 isolates and HPRS-103, a subgroup J ALV by ClustalW
(slow/accurate, IUB) ...........................................................................

Table 3.9. Comparisons of percentage identity and divergence among nucleotide
sequences of 3’ LTR of all AF 115 isolates and HPRS-103 by ClustalW
(slow/accurate, IUB) ...................................................... q .....................

Table 3.10. Sequence similarity among amino acids of gp85 regions of AF 115
isolates and associated NCBI published viral strains by ClustalW (Slow/accurate,
Gonnet) ..........................................................................................

Table 3.11. Comparisons of sequence distances among amino acids of gp37 of AF
115 isolates and highly similar published viruses by ClustalW (Slow/accurate,
Gonnet) ..........................................................................................

Table 3.12. Similarity and divergence among nucleotide sequences of 3’ UTR and
LTR of all AF 115 isolates and GenBank blasted sequences by ClustalW
(slow/accurate, IUB) .................................................................. . .........

178

181

185

188

191

I94

197

200

203

207

LIST OF FIGURES

Figure 1.1. Schematic diagram of a retrovirus virion (Avian leukosis virus
particle) ..........................................................................................

Figure 1.2. Key features of viral genomic RNA and proviral DNA of ALV
genome ..................................................................................................................

Figure 1.3. Representative unrooted pol neighbor joining (NJ) dendrogram (500
bootstraps consensus) of the seven retroviral genera: Alpharetrovirus,
Betaretrovz’rus, Gammaretrovirus, Deltaretrovirus, Epsilonretrovirus, Lentivirus
and Spumavirus .................................................................................

Figure 1.4. Two models of homologous genetic recombination in retroviruses
during minus-strand synthesis ................................................................

Figure 1.5. Replication of ALV genome through the process of reverse
transcription ....................................................................................

Figure 1.6. The life cycle of retroviruses including ALSVS .............................

Figure 2.1. Detection of ALV in proviral DNA prepared from line 0 CEFS
infected with AF 115 isolates by PCR using all-ALV primers RS-7J & REVSP
6683 ..............................................................................................

Figure 2.2. Analysis of PCR amplification products from line 0 CEFS inoculated
with AF 115 isolates utilizing recombinant ALV-B/J-specific primer pair SRB-F
& R8 .............................................................................................

Figure 2.3. Agarose gel showing the PCR products obtained by employing SRB-F
& 82 primer set on DNA samples extracted from AF 115 isolates-infected line 0
CEFS .............................................................................................

Figure 2.4. PCR amplification of DNA isolated from AF 115 isolates-infected line
0 CEFS utilizing primer combination specific for subgroup B ALV sequences
(ARK 4836 & SRB-R) ........................................................................

Figure 2.5. PCR for specific detection of subgroup B ALV with line 0 CEFS

inoculated with AP 115 isolates using primer pair specific for subgroup B ALV
(SRB-F & B LTR 8.3) .........................................................................

xi

69

7O

71

72

73

74

118

119

120

121

122

 

Figure 2.6. PCR reaction of genomic DNA from line 0 CEFS inoculated with AP
115 isolates and amplified with primers specific for subgroup B ALV (SRB-F & B
LTR 7.3) .........................................................................................

Figure 2.7. Specific PCR detection of subgroup J ALV in DNA extracted from
line 0 CEFS infected with AF 115 isolates utilizing ALV-J-specific primer set RS-
6] & S2 ..........................................................................................

Figure 2.8. Analysis of PCR carried out on proviral DNA extracted from line 0
CEFS inoculated with AF 115 isolates and amplified using primer pair specific for
ALV-J sequences (RS-7J & R8) .............................................................

Figure 2.9. PCR analysis of DNA isolated from AF 115 isolates propagated in line
0 CEFS and amplified by ALV-J sequence-specific primer combination RS-7J &
S2 ................................................................................................

Figure 2.10. Ethidium bromide —Stained agarose gel with PCR products of DNA
prepared from AF 115-infected line 0 CEFS Obtained by amplification with ALV-J
sequence—specific primers RS-7J & R5 .....................................................

Figure 2.11. PCR amplification from isolated proviral DNA samples of 72 CEFS
inoculated with AF 115 isolates employing primer pair ARK 4836 & REVSP
6683, which can detect all ALV subgroups ................................................

Figure 2.12. Genomic DNA from AF 115 isolates-infected DF-l/J cells using all-
ALV primer set ARK 4836 & REVSP 6683 ...............................................

Figure 2.13. PCR analysis of DNA extracted from DF-l/J cells inoculated with
AP 115 isolates utilizing ALV-B Specific primers ARK 4836 & SRB-R ..............

Figure 2.14. Specific PCR amplification of subgroup J ALV in DNA isolated
from DF-l/ J cells infected with AF 115 isolates with ALV-J-specific primer set
H5 & H7 .........................................................................................

Figure 2.15. Detection of ALV-J sequences in proviral DNA isolated from AP

115 isolates-infected DF-l/J cells through PCR analysis using primer combination
F5 & R8 ..........................................................................................

Figure 2.16. PCR analysis done to detect recombinant ALV-B/J sequences in
genomic DNA prepared from DF-l/J cells incubated with AF 115 isolates
employing primers SRB-F & R8 ............................................................

Figure 2.17. DNA molecular weight marker as visualized by ethidiUm bromide
staining ..........................................................................................

xii

123

124

125

126

127

128

129

130

131

132

133

134

Figure 3.1. Sequence alignment of SU domains of seven AF 115 isolates and the
recombinant ALV-B/J (AF 115—4) ........................................................... 177

Figure 3.2. Phylogenetic tree of gpSS of seven AF 115 isolates and the
recombinant ALV-B/J (AF 115-4) ........................................................... 179

Figure 3.3. Sequence alignment of TM domains of seven AF 115 isolates and the
recombinant ALV—B/J (AF 115-4) ........................................................... 180

Figure 3.4. Cladogram of gp37 of seven AF 115 isolates and the recombinant
ALV-B/J (AF 115-4) ........................................................................... 182

Figure 3.5. Sequence alignment of 3’ UTR of seven AF 115 isolates and the
recombinant ALV-B/J (AF 115—4) ........................................................... 183

Figure 3.6. Sequence alignment of 3’ LTR of seven AF 115 isolates and the
recombinant ALV-B/J (AF 115-4) ........................................................... 184

Figure 3.7. Phylogeny relationship among 3’ UTR and LTR of seven AF 115
isolates and ALV-B/J (AF 115-4) ............................................................ 186

Figure 3.8. Sequence alignment of gp85 of AF 115 isolates and representatives of
various ALV subgroups ....................................................................... 187

t Figure 3.9. Cladogram of gp85 of AF 115 isolates and prototypes of classical
ALV subgroups ................................................................................. 189

Figure 3.10. Sequence alignment of gp37 of AF 115 isolates and viral prototypes
of ALV subgroups .............................................................................. 190

Figure 3.11. Diagram of the phylogenetic relationships among gp37 of AF 115
isolates and different ALV subgroups ...................................................... 192

Figure 3.12. Sequence alignment of 3’ UTR of AF 115 isolates and HPRS-103
(ALV-J) .......................................................................................... 193

Figure 3.13. Phylogenetic tree of the 3’ UTR of all AF 115 isolates and subgroup
J ALV isolate HPRS-103 ..................................................................... 195

Figure 3.14. Sequence alignment of 3’ LTR of all AF 115 isolates with ALV-J
HPRS-103 ....................................................................................... 196

Figure 3.15. Cladogram of the 3’ LTR of all AF 115 isolates and subgroup J ALV
isolate HPRS-103 .............................................................................. 198

xiii

Figure 3.16. Sequence alignment of SU domains of AF 115 isolates and similar
blasted viral strains .............................................................................

Figure 3.17. Phylogenetic tree of SU domains of AF 115 isolates and related
blasted ALV strains from GenBank .........................................................

Figure 3.18. Sequence alignment of TM regions of AF 115 isolates and blasted
NCBI viral isolates .............................................................................

Figure 3.19. Phylogenetic relationship diagram among TM regions of AF 115
isolates and GenBank ALV viral strains ....................................................

Figure 3.20. Sequence alignment of 3’ UTR of AF 115 isolates and blasted viral
sequences from NCBI .........................................................................

Figure 3.21. Sequence alignment of 3’ LTR of all AF 115 isolates with NCBI
published viruses ...............................................................................

Figure 3.22. Cladogram of 3’ UTR and LTR of all AF 115 isolates and similar
blasted viral strains from NCBI ..............................................................

Figure 4.1. Schematic representation of DNA proviruses of AP 115 isolates and
homology to known ALV subgroups and strains ..........................................

* Images in this dissertation are presented in color.

xiv

199

201

202

204

205

206

208

223

%

+SSRNA

966

A l/EBP
ABI

ADl
ADOL
AE/E
AE/J
AE/J,AEV,E
AEV
AEV-ES4
AF 1 15
AIDS
ALSV

ALV

KEY TO SYMBOLS AND ABBREVIATIONS

Percentage

and

plus sense Single-stranded RNA

registered trademark

degree centi grade

microgram

microliter

Acute transforming avian leukosis virus subgroup J strain 966

Leucine zipper Al/CCAAT enhancer binding protein

Applied Biosystems Inc.

Reverse PCR primer for ALV-A to ALV-E

Avian Disease and Oncology Laboratory

subgroup A gp85 and subgroup E gp37 and LTR

subgroup A SU, subgroup E TM, subgroup J 3’ UTR and LTR
ALV-A SU, ALV-E TM, R & U5, ALV-J 3’ UTR & AEV U3
avian erythroblastosis virus

Avian erythroblastosis virus strain ES4

Aly Fadly 115 isolates of avian leukosis virus

acquired immunodeficiency syndrome

avian leukosis/sarcoma group of retroviruses

avian leukosis virus

XV

alv6

ALV-A

ALV-A/J

ALV-B

ALV-B/J

ALV-BE/J

ALV-C

ALV-D

ALV-E

ALV-J

AMAV-2

AMC 29

AMV

anti-Hcl

anti-RAV-Z

ARK 4836

ARS

ART-CH

ASVS

B LTR

BAI-A AMV

BBL

bet

Transgenic chicken line avian leukosis virus 6

avian leukosis virus subgroup A

avian leukosis virus with subgroup A env and subgroup J LTR
avian leukosis virus subgroup B

avian leukosis virus with subgroup B env and subgroup J LTR
ALV with ALV-B SU, ALV-E TM & ALV-J 3’ UTR & LTR
avian leukosis virus subgroup C

avian leukosis virus subgroup D

avian leukosis virus subgroup E

avian leukosis virus subgroup J

Avian myeloblastosis-associated virus 2 subgroup B

avian myelocytomatosis virus strain 29

avian myeloblastosis virus

antibodies to He] (American prototype of subgroup J ALV)
Antibodies to RAV-2 (RouS-associated virus 2 or ALV-B)
Arkansas 4836 forward PCR primer for all ALVS

Agricultura Research Service

avian retrotransposon from chicken genome

avian sarcoma viruses

subgroup B ALV long terminal repeat reverse primer

Strain BAI-A of avian myeloblastosis virus

B-F/B-L region of chicken major histocompatibility complex

cellular or viral oncogene bet

xvi

B-F
BFV
B-haplotype
BH-RSV
BIV

B-L
BLAST
BLV
Blym-l
bp

bZip
C/A
C/ABDE
C/AE
C/E
C/EBP
C/EJ
C/O

CA
CAEV
CARl
c-bic

CEFS

bursal-F chicken MHC class 1

bovine foamy virus

bursal-haplotype or chicken major histocompatibility complex
Bryan’s high titer strain of Rous sarcoma virus

bovine immunodeficiency virus

bursal-L chicken MHC class II

Basic Local Alignment Search Tool

bovine leukemia virus

A transforming gene or oncogene in Burkitt lymphoma

base pair

Basic leucine zipper DNA binding protein or transcription factor
chickens or chicken cells resistant to ALV subgroup A infection
chickens or chicken cells resistant to ALVS A, B, D & E
chickens or chicken cells resistant to infection by ALV E & A
chickens or chicken cells susceptible to all exogenous ALVS
CCAAT/ enhancer binding protein

chickens or chicken cells resistant to ALV subgroups E and J
chickens or chicken cells susceptible to infection by all ALVS
capSid

caprine arthritis encephalitis virus

Chicken arginase 1 of TNFR family

Cellular oncogene or proto-oncogene B-cell integration cluster

chicken embryo fibroblasts

xvii

 

 

ChiRVl
CLAV
CMI
CMII
C02
COFAL
c-onc
CR1

CS

CT] 0
C-terminal
CTL

D
dATP
DCPAH
dCTP
DEAE
DF- 1
DF-l/J
dGTP
DNA
dNTP

DRl

Chicken retrovirus 1

chicken infectious anemia virus

cell mediated immunity

avian carcinoma/myelocytoma virus strain CMII
carbon dioxide

complement-fixation test for avian leukosis virus
cellular oncogenes

chicken repeat 1

calf serum

avian sarcoma virus strain CT] 0

Carboxyl-terminal

cytotoxic T lymphocyte

aspartic acid

Deoxyadenosine triphosphate

Diagnostic Center for population and Animal Health
Deoxycytidine triphosphate

Diethylaminoethyl

Douglas Foster-1, a spontaneously immortalized CEF cell line
Genetically engineered DF -1 cell line resistant to ALV-J
deoxyguanosine triphosphate

deoxyribonucleic acid

deoxyribonucleotide triphosphate

direct repeat 1

xviii

ds
dTTP
E/B
E26
Ea-B
EAV
EAV-E5 1
EAV-HP
EB
EBP
EcoR I
EFI
EFIII
EFV
EGF
EIAV
ELISA
env
erbA
erbB
ES4
ev

ev/J 4.1 Rb

double stranded

Deoxythymidine triphosphate
subgroup E env (RAV-O) and subgroup B LTR (RSV-Pr-B)
avian erythroblastosis virus strain E26
erythrocyte antigen B locus
endogenous avian virus

endogenous avian virus element E51
endogenous avian virus element HP
erythroblastosis

enhancer binding protein

E. coli restriction enzyme I

enhancer factor I

enhancer factor 111

equine foamy virus

epidermal growth factor

equine infectious anemia virus
Enzyme-linked immunosorbent assay
envelope

erythroblastosis virus oncogene A
erythroblastosis virus oncogene B
avian erythroblastosis virus strain ES4
endogenous viral loci

endogenous avian virus element ev/J 4.1 Rb

xix

F5
FeLV
FF V
FIV

FP

FuSV
gag
gp37

gp85
gPr92env

gsa
H5

H7

Hcl

HCl

HI

HIV
HPRS-103

hr

forward primer

fragment 1 (DRl)

Fragment 2 (XSR or E element)

Forward 5 PCR primer

feline leukemia virus

feline foamy virus

feline immunodeficiency virus

fusion peptide

Fujinami avian sarcoma viral oncogene homolo g
Fujinami sarcoma virus

group Specific antigen

glycoprotein 37 or TM

glycoprotein 85 or SU

Glycoprotein 92 KDa of envelope

group specific antigen

Hemagglutinin 5 forward PCR primer for all ALVS
Hemagglutinin 7 reverse PCR primer for ALV-J
USA prototype of ALV-J strain Hcl
Hydrochloric acid

humoral immunity

Human immunodeficiency virus

United Kingdom prototype of ALV-J strain HPRS-103

hypervariable region

XX

 

 

 

HTLV

IBD
ICTV

16

IV
I SRV
kb

KCl

L/ S
LDLR
LL
LM
LNGV
LR-9
LTR
MA

MAV

Human T-lyrnphotropic virus

intrabdominal

intracistemal A-type particle

infectious bursal disease

International Committee on Taxonomy of Viruses
Independent locus for resistance to ALV-E or TVE
immuno globulin

integrase

' intravenous

J aagsiekte Sheep retrovirus

kilobase

Potassium chloride

Leader region

lysine

Leukosis/ Sarcoma group

low-density lipoprotein receptor

lymphoid leukosis

Leibowitz’s L-l S-McCoy’S 5A tissue culture medium
Langur virus

Avian leukosis virus strain LR-9 or leukemia retrovirus 9
long terminal repeat

matrix

myeloblastosis-associated virus

xxi

 

 

 

 

MAV- 1
MAV-2
MAV-2 (O)
MDV
MgClz
MHZ
MH2E2 1
MHC
ML

mL

ml

MLV

mM

M-MLV

MPMV
mRN A
myb

myc

Nab

NC

myeloblastosis-associated virus 1 subgroup A
myeloblastosis-associated virus 2 subgroup B
MAV-2 virus with osteopetrosis potential
Marek's disease virus

Magnesium chloride

Avian carcinoma virus strain MH2

avian carcinoma virus strain MH2E2]
major histocompatibility complex
myeloid leukosis

milliliter

milliliter

mmine leukemia virus

millimeter

millimolar

Moloney murine leukaemia virus

mouse mammary tumour virus
Mason-Pfizer monkey virus

messenger RNA

myeloblastosis oncogene
myelocytomatosis oncogene

asparagine

neutralizing antibody

nucleocapsid

xxii

 

 

NCBI
NCT
nef

ng

NJ

NK
N-linked
NP

NS

NSS

nt
N-terminal
NTRE-4
NWS
NXS/T
ORF

pl 0

pl 2

pl 5

pl 9

p27

p32

p68

National Center for biotechnology Information
asparagine, cysteine and threonine amino acids
negative factor gene of HIV

nanograrn

neighbor joining

natural killer

asparagine-linked

non-producer

non-synonymous nucleotide substitutions
asparagine and two serine amino acids
nucleotide

Amine-terminal

recombianat Rous sarcoma virus strain NTRE-4
asparagine, tryptophan and serine amino acids
aspargine, any amino acid, serine or threonine amino acids
open reading frame

protein 10 KDa or additional gag protein
protein 12 KDa or NC

protein 15 KDa or PR

protein 19 KDa or MA

protein 27 KDa or CA

protein 32 KDa or IN

protein 68 KDa or RT

xxiii

 

 

PAS

PBS

PCR
PDRC-3249
PF U

pH

PHV

PM

pmoles

pol

PPT

PR

Prl 80GAG-PR-POL
Pr76GAG-PR
Pr-B

Pr-C

PRE

pro
Pr-RSV-C
PTC-l 00
PU

QT6

Polyadenylation signal

primer binding sequence

polymerase chain reaction

Poultry Diagnostic and Research Center 3249 isolate of ALV
archaeon mococcus furiosus DNA polymerase
Power of Hydrogen

Perch hyperplasia virus

phenotypic mixing

picomoles

polymerase

polypurine tract

protease enzyme

polyprotein 180 KDa gag-pr-pol

Polyprotein 76 KDa gag-pr

Prague strain subgroup B Rous sarcoma virus
Prague strain subgroup C Rous sarcoma virus
pentanucleotide repeat element

protease

Prague strain of Rous sarcoma virus subgroup C

Programmable Thermal Controler 100
ciS-acting element or gene in the E-element of some ALSV
Quail transformed Japanese fibrosarcoma cell line 6

avian erythroblastosis virus strain R

xxiv

 

 

R allele
R primer
R region
R5

R8

RAV
RAV-0
RAV-l
RAV-2
RAV-49
RAV-50
RAV-60
RAV-7
REV

rev
REVSP 6683
RIF
RNA

‘ RNase H
ros
RPL12
R-RS-l

RS-6J

resistant allele

reverse primer

repeat region

Reverse 5 PCR primer for ALV-J

Reverse 8 PCR primer for ALV-J
Rous-associated virus

Rous-associated virus 0 subgroup E
Rous—associated virus 1 subgroup A
Rous-associated virus 2 subgroup B
Rous-associated virus 49 subgroup C
Rous-associated virus 50 subgroup D
Rous-associated virus 60 subgroup E
Rous-associated virus 7 subgroup C

Avian reticuloendotheliosis virus

Regulator of virion gene of HIV

reverse PCR primer for all ALVS

resistance inducing factor

ribonucleic acid

RNA endonuclease or ribonuclease H
Oncogene encoding a receptor-type protein tyrosine kinase
Regional Poultry Laboratory 12 strain of avian leukosis virus
Regression of Rous sarcoma 1 gene in chickens

6J forward PCR primer for ALV-J

XXV

 

 

RS-7J

RSA

RS-A

RSV

RSV-A

RSV-NTRE-7 td

RSV-Pr-C

RSV-SR-B

RSV-SR-D

RSV-td PR2257

RT

rTM

RT-PCR

S allele

S

81

S2

sag

SBl

SD0501

SDS

SFV

SIV

7] forward PCR primer for all ALVS

Rous sarcoma virus subgroup A

Rous sarcoma virus subgroup A

Rous sarcoma virus

Rous sarcoma virus subgroup A

Rous sarcoma virus strain NTRE-7 transformation defective
Rous sarcoma virus Prague strain subgroup C

Rous sarcoma virus Schmidt-Ruppin subgroup B

Rous sarcoma virus Schmidt-Ruppin subgroup D

RSV transformation defective Poultry Research 2257 strain
reverse transcriptase enzyme

redundant transmembrane region insert

reverse transcriptase-Polymerase Chain Reaction
susceptible allele Synonymous nucleotide substitutions
Synonymous nucleotide substitutions

Smith 1 forward PCR primer for ALV-J

Smith 2 reverse PCR primer for ALV-J

S‘uperanti gen

Marek’s Disease virus serotype 2 strain 881

Chinese endogenous ALV strain SD0501

Sodium Dodecyl Sulfate

simian foamy virus

Simian immunodeficiency virus

xxvi

SMRV
SnRV
SNV
SPF
SRB-F
SRB-R
src

SS
STLV
SU
T/BD
TAE
Taq
taS/bel- 1
tat
TCIDSO
TE

Th- 1
TM

TM
TMB
TNF R

tRNA

Squirrel monkey retrovirus

Snakehead retrovirus

spleen necrosis virus

specific-pathogen free

Forward PCR primer for ALV-B

Reverse PCR primer for ALV-B

sarcoma gene of Rous sarcoma virus encoding tyrosine kinase
Single stranded

Simian T-lymphotropic virus

surface

Turkey cells resistant to infection by ALV-B and ALV-D
Tris-Acetate-EDTA (ethylenediaminetetraacitic acid)
Thermus aquaticus thermostable DNA polymerase
Transcriptional transactivator gene of foamy viruses
Trans-Activator of Transcription gene of HIV

tissue culture infectious dose 50

Tris-EDTA (ethylenediaminetetraacitic acid)
Thyrnocyte 1, a T-lymphocyte antigen locus in chicken
transmembrane

trademark

3, 3', 5, 5'-tetramethyl benzidine

tumor necrosis factor receptor

transfer RNA

xxvii

TVA

TVC
TVE
TymS_90
U3

U5
UD2
UD-J 1
UR2
US
USDA
UTR
VBP

vif

WDSV
WEHV

XSR

tumor virus A locus

tumor virus B locus

tumor virus C locus

tumor virus E locus

recombinant avian leukosis virus strain TymS_90
unique 3’ region

unique 5’ region

avian leukosis virus subgroup J strain UD2
avian leukosis virus subgroup J strain UD-Jl
avian sarcoma virus strain UR2

United States

United States Department of agriculture
untranslated region

vitellogenin gene binding protein in chicken
Viral infectivity factor gene of HIV

virus neutralization

viral oncogene

Viral protein R gene of HIV

Viral protein U gene of HIV

variable region

Walleye dermal sarcoma virus

Walleye epidermal hyperplasia virus

Exogenous virus-specific region or E element

xxviii

Y73 avian sarcoma virus strain Y73

YB-l Y-box binding protein 1
yes Yamaguchi sarcoma oncogene
‘I’ Psi element, an encapsidation signal

xxix

 

 

INTRODUCTION

Avian leukosis virus (ALV) is an important poultry pathogen resulting in
economic losses due to low productivity and tumor mortality (F adly and Payne, 2003). It
causes a variety of transmissible benign and malignant neoplasms in mature and
semimature chickens. According to the International Committee on Taxonomy of Viruses
(ICTV), ALV is a member of the family Retroviridae, subfamily Orthoretrovirinae and
genus Alpharetrovirus (Linial et al., 2006). There are six subgroups of ALV in chickens,
five exogenous (subgroups A, B, C, D and J) and one endogenous (subgroup E). Like
other retroviruses, ALV is known for its high mutation and recombination rates
constituting a constant threat to the poultry industry.

Genetic recombination has been reported between endogenous and exogenous
ALVS leading to the generation of oncogenic mutants (Crittenden et al., 1980; Tsichlis
and Coffin, 1980; Weiss et al., 1973). For example, ALV-J is a recombinant virus
between endogenous (EAV-HP or ev/J) and undetermined exogenous ALV (Bai et al.,
1995b). This emerging recombinant ALV is being controlled by efficient diagnosis and
eradication of infected broiler breeder flocks. In addition, recombinant ALV isolates were
experimentally generated with subgroup A gpSS, subgroup E gp37and subgroup J LTR
through propagation of ALV-J in alv6 (C/AE) CEF S (Lupiani et al., 2000).

Limited information is available about natural recombination among different
subgroups of ALV and its impact on pathogenesis in chicken. Recombinant ALVS with
genomic regions belonging to different subgroups may present new phenotypes and

altered characteristics such as host range, antigenicity and pathogenicity. Genetic

diversity in ALV including mutations and recombination may confound its current field
diagnosis by standard virological, serological and molecular methods. Such negative
consequences may affect current efforts for prevention and control of this important virus
infection.

The studies in this dissertation mainly focus on molecular and biological
characterization of seven remaining isolates of a naturally occurring recombinant avian
leukosis virus associated with myeloid leukosis in commercial white leghorn layers. The
first chapter provides a review of the literature on family Retroviridae, diversity in
retroviruses and avian leukosis—sarcoma viruses and diseases. The consequences of
retroviral diversity including mutations and recombination are discussed. The second
chapter describes the characterization of seven remaining isolates of a naturally occurring
recombinant ALV by biological assays and polymerase chain reaction. Comparisons
_ among the different isolates, a naturally occuning recombinant ALV and representative
subgroups of ALV are demonstrated. Genetic diversity in different genomic regions of
the various recombinant ALV isolates is evaluated using different PCR primer
combinations. The (third chapter illustrates the molecular characterization of these isolates
by DNA sequencing. Sequence analyses of different genomic regions of these isolates
were compared with the naturally occurring recombinant ALV-B/J, representative ALV
subgroups and highly related ALV strains through NCBI blast search. In the fourth and
final chapter, conclusions, significant findings, limitations, discrepancies, future studies

and recommendations are discussed.

CHAPTER 1

LITERATURE REVIEW

1. RETROVIRIDAE
A. Introduction

Members of the family Retroviridae are animal enveloped viruses known as
retroviruses, which contain two identical single stranded ribonucleic acid (RNA)
molecules. In general, retroviruses share characteristics with other deoxyribonucleic acid
(DNA) and RNA viruses as well as with cellular movable genetic elements including
retrotransposons and retroposons (Temin, 1992). Some of these characteristics are similar
genomic organization and fundamental replication strategy such as reverse transcription
and transcription. In fact, retroviruses replicate efficiently in living cells through a DNA
intermediate and are considered successful obligate parasites.

Retroviruses can be divided into Simple or complex viruses with common basic
genomic features. Both Simple and complex retroviruses contain 5’ repeat (R) region,
unique 5’ (U 5) region, group specific antigen (gag), protease (pro), polymerase (pol), and
envelope (env) genes and unique 3’ (U3) region and 3’ repeat (R) region. Complex
retroviruses have larger genomes since they have additional regulatory genes; sometimes
between pol and env, or env and 3’ long terminal repeat (LTR) or both (Temin, 1992).

Retroviruses have significant agricultural impact on production of farm animals
and devastating medical and economic consequences in humans. In fact, retroviruses
have been linked to various disease conditions such as malignancies, neurological

disorders and immunodeficiencies in many species (Coffin, 1992). On the other hand,

.m/

they have been used in biotechnology and molecular biology to study other viruses and

the roles of various genes. In addition, retroviruses are being studied for their role and use

in somatic gene and cancer therapies.

B. Morphology

The morphology of retroviruses was studied in transmission electron micrographs
and has been useful in their previous classification. Every retrovirus virion consists of an
envelope, a nucleocapsid (or core) and a nucleoid. There are small densely dispersed
surface projections (8 nm long) or glycoprotein spikes, which cover the surface evenly. A
s chematic illustration of the known structure of the retrovirus virion, Specifically avian
l eukosis virus (ALV) is shown in Figure 1.1 (Fadly and Payne, 2003). In fact, retrovirus

particles range in size from 80 nm to 130 nm. Four distinct types of retrovirus particles
exi st and they are based on mode of assembly, location and Shape of the nucleocapsid in
mature virions and the appearance of the surface glycoproteins (Coffin, 1992).
Type A particles are strictly intracellular, either intracytoplasmic or intracistemal.
They consist of ring-shaped nucleoid surrounded by a membrane. On the other hand, B-
type virions assemble through budding of A partciles. Mature virions have eccentrically
beated doughnut Shaped cores and prominent surface projections. C particles have no
knOWn cytoplasmic intermediates and form directly on the cell surface. They mature into
cer‘131‘ally located condensed spherical cores with visible, but less prominent surface
proj ections. The morphology of type D virions is intermediate between B and C virions
with elongated or cylindrical dense cores. Virions of Lentivirus and Spumavirus have

”liq 11c morphologies and are surrounded by envelopes. While spumaviruses contains

prominent spikes on the surface, lentiviruses are Slightly pleomorphic and Spherical with
small or barely visible surface projections and evenly dispersed Spikes (Gifford and
Tristem, 2003). Nucleocapsids of lentiviruses are isometric and nucleoids are concentric

and Shaped like a rod, truncated cone or ice cream cone. On the other hand, spumaviruses

have central but uncondensed cores (Goff, 2007).

C‘. Components of Retroviruses

1 - Retroviral Virion
Although retroviruses exhibit differences in morphology and biology, they share

similar set of virion components. Typically, a retrovirus virion is composed of 2%
nucleic acids (RNA), 60% proteins, 35% lipids, and 3% carbohydrates (Donovan and

F u] ler, 2004). All retroviruses are surrounded by a lipid bilayer envelope consisting
mainly of phospholipids. This envelope is formed through budding from the target cell
pl asma membrane. Moreover, retroviral genome is made up of two identical plus sense
Singl e-stranded RNA molecules (+ SS RNA) non-covalently joined near their 5’ ends.
Each RNA monomer is 7-10 kb in length and has a poly (A) tract at the 3’ terminus and a
methylated nucleotide cap at the 5’ terminus. The retroviral particle contains nucleic

acids (RNA and DNA) from the host cell such as transfer RNA (tRNA). This host tRNA

is associated with the 5’ end of viral RNA and serves as a primer for the synthesis of

pt‘()\’1‘ial DNA by the viral reverse transcriptase enzyme (RT).

2' l{GP-.troviral Genome and Major Viral Genes

The genome of all retroviruses generally consists of Similarly arranged terminal
noncoding regions and diverse internal coding regions. The internal coding regions
encode structural and non-Structural virion proteins as well as regulatory proteins in

complex retroviruses (Coffin, 1992). The retroviral genome usually contains three open
reading frames (ORF) encoding structural and nonstructural proteins, which are essential
for the virus life cycle. The first ORF is in the group specific antigen (gag) gene, which
codes for retrovirus structural proteins. The polymerase (pol) gene is in the second ORF
and codes for reverse transcriptase and integrase enzymes. A third ORF is that of the
envelope (env) gene, which codes for retroviral coat proteins, surface and transmembrane

g1 ycoproteins. Nevertheless, the protease (pro) gene coding for protease enzyme iS found

in a separate ORF or in the ORF of gag or pol.
In each retroviral RNA strand, the order of the structural genes is gag-pol-env

fi‘om the 5’ end to the 3’ end. Key features of retroviral genome are illustrated in Figure

1 -2 , showing genomic organization of RNA and DNA forms of ALV (Fadly and Payne,

2003). In addition to the previously described genomic features, highly oncogenic

retroviruses have oncogenes either in the pol or env. For example, Rous sarcoma virus

(RS V) has a v-src gene at the end of ORF of env. Moreover, complex retroviruses usually
have additional genes, which regulate retroviral genome expression and other accessory

fun etions. Human immunodeficiency virus (HIV) genome contains tat gene between p01

and env encoding transactivator protein.

On both sides of the retroviral genome, important ciS-acting sequences play
essential roles in the process of reverse transcription (Coffin, 1992). The primer binding

sequence (PBS) is found downstream of U5 region in the 5’ end of retroviral genome and

is complementary to the 3’ end of the tRN A primer. An untranslated leader (L) region is
downstream of PB and upstream of gag at the 5’ end of the genomic RNA. It usually
includes a specific packaging signal and the splice donor site for generation of

subgenomic mRNA (messenger RNA), responsible for env expression. Polypurine tract

(PPT) is an AG-rich sequence and is the primer for plus-strand DNA synthesis during the

process of reverse transcription.

3 - Proviral DNA and Long Terminal Repeats
The retroviral RT enzyme reverse transcribe genomic RNA into proviral DNA,

Which is then covalently integrated into the infected host cell DNA. The proviral DNA is
usually longer than the genomic RNA due to the duplication in the repeated (R) and
unique (U5 and U3) sequences during the process of reverse transcription. These
duplicated terminal sequences are called long terminal repeats (LTRS), which are
Ilon'lcoding regions essential for viral replication in cis. They are usually composed of
three regions in the following order U3-R-U5 (Coffin, 1992). U3 region contains

enhancer, promoter and regulatory sequences for transcription initiation. On the other

hand, R region contains signals for cleavage and poly (A) addition required for transcripts
pro Cessing. In addition, R region determine the 5’ and 3’ end of retroviral genomes and
all I'ZI1RNA and ensure correct end-to-end transfer of the growing chain during reverse

transcription. Figure 1.2 shows the differences in the terminal sequences in genomic

RN‘A and proviral DNA (Fadly and Payne, 2003).

4' M ajor Viral Encoded Proteins

GAG and ENV are retroviral structural proteins encoded by gag and env genes
respectively. On the other hand, POL is non-structural protein encoded by pol gene. The
virion core of all retroviruses is generally formed of three common GAG proteins;

matrix, capsid and nucleocapsid (Coffin, 1992). The matrix (MA) protein joins the

retroviral core with the envelope and is involved in the virus assembly and budding. The

capsid (CA) protein constitutes the major structural element of the core shell or capsid. In
many retroviruses, CA antigen is mostly used in retroviral immunoassays for detection of
the virus. The nucleocapsid (NC) is a basic protein made of cysteine and histidine
aminoacids, resembling “zinc finger” and closely associated with genomic RNA. It is
O fien needed for correct packaging and it promotes RNA-RNA duplex formation for
genome dirnerization and genome-primer association. In some retroviruses, additional
Small core proteins may be present between MA and CA, CA and NC, and downstream
0 f NC (Donovan and Fuller, 2004). In most cases, these are encoded by gag and their
filnction is poorly understood.

Two ENV proteins are noncovalently linked glycoproteins and needed for specific
ho 81: cell receptor recognition and entry. Surface (SU) protein iS a knob-like glycoprotein,
Whi ch is exposed on the surface to the external environment. It directly binds cellular
rec eptors during infection and is recognized by neutralizing antibodies. The host ranges
are encoded within SU and are useful in the classification of closely associated retrovirus
SIl’eeies into subgroups (Fadly and Payne, 2003). The other glycoprotein, transmembrane
(TM), is a spike-like structure anchoring SU to retroviral envelope. It also has an amino-
tetTl‘rlinal hydrophobic region, which mediates fusion of retroviral envelope with the host

cel 1 membrane (Coffin, 1992).

Retroviral enzymes such as protease, integrase and reverse transcriptase are

essential for replication, but are less abundant in the virion than structural proteins. The

protease enzyme (PR) is encoded by pro while the reverse transcriptase (RT) and the

integrase (IN) are encoded by pol. PR is usually inactive until activated through
dimerization after virion budding. It is responsible for all the proteolytic cleavages and
generation of mature proteins during virion maturation (Coffin, 1992). Highly conserved
alnong retroviruses, RT uses genomic single stranded RNA as a template to synthesize
the retroviral double stranded DNA. It has active sites, which can function as RNA- and
DNA-directed DNA polymerases, as well as RNA endonuclease or ribonuclease H
(RN ase H). The RNase H function allows the degradation of retroviral RNA template and
primers during proviral DNA synthesis. Similarly, IN has multiple functions, cleavage
311d ligation of newly (synthesized linear proviral DNA. It recognizes the ends of the two
1 inear proviral DNA, removes two nucleotides from the 3' end of each strand, and cleaves

the DNA target. Afterwards, it joins the trimmed proviral DNA end to target host cell

DNA at random Sites.

D. Classification of Retroviruses

Similar to all viruses, taxonomical classification of retroviruses is developed and
re\fr'ewed by a Subcommittee of the International Committee on Taxonomy of Viruses
(ICTV). It was reported that retroviral classification is largely based on genome structure,
Illlcleic acid sequence and sequence similarity within the pol gene (Coffin et al., 1997).
S i Ilce retroviruses comprise a diverse group of viruses, they can be described by several

013 teria other than their evolutionary relationships. These include the type of diseases they

induce, tropism for certain host Species or target cell types, virion morphology, simple
versus complex lifestyles, mechanism of transmission, serology and biochemical features

(Donovan and Fuller, 2004).

Previously, retroviruses were classified under the family Retroviridae, which was

subdivided into three subfamilies; Oncovirinae, Lentivirinae and Spumavirinae.
However, this taxononrical classification of retroviruses is no longer appropriate.
Following recent ICTV reclassification, Retroviridae is currently subdivided into two
subfamilies, Orthoretrovirinae and Spumaretrovirinae in addition to unclassified
retroviridae (Linial et al., 2006). Each subfamily is further subdivided into different
genera and Species, which contain mostly exogenous retroviruses. Within
Orthoretrovirinae, there are Six genera namely Alpharetrovirus, Betaretrovirus,

Ga mmaretrovirus, Deltaretrovirus, Epsilonretrovirus and Lentivirus. On the other hand,
there is only one genus, Spumavirus, in the subfamily Spumaretrovz'rinae (Fauquet et a1. ,
2 005). The unclassified retroviridae contains endogenous and other unclassified
exogenous retroviruses from different Species including humans, primates, amphibians,
fi Sh, avian and others. Moreover, alpharetroviruses, betaretroviruses, gammaretroviruses

arid epsilonretroviruses have Simple lifestyles while lentiviruses, deltaretroviruses and

Splimaviruses have complex lifestyles (Weiss, 2006).

Retroviruses can be exogenous or endogenous and they can infect somatic and
g‘el‘rnline cells. When retroviruses infect gerrnline cells, they are transmitted to the next
gel'leration and are termed endogenous retroviruses. Endogenous retroviruses can persist
in the host genome for a long time. However, they are only infectious for a short time

afier integration since they acquire knockout mutations during host DNA replication.

10

Moreover, endogenous retroviruses can be partially excised from the genome by a

process known as recombinational deletion. Gifford and Tristern (2003) reported that

endogenous retroviruses are involved in viral ecology, evolution and host disease.

Classification of endogenous retroviruses has been developed separately fi'om that
of exogenous retroviruses. In general, endogenous forms of exogenous retroviruses are
grouped within the seven recognized genera. Based on their relatedness to exogenous
genera, endogenous retroviruses are classified into three classes (J cm at al., 2005).

Classes 1, II and III contain endogenous retroviruses most similar to Gammaretrovz’rus,

Betaretrovirus and Spumavz'rus respectively (Figure 1.3).

E Seven Genera of Retroviruses

l - Alpharetrovirus

Exogenous and endogenous retroviruses of Alpharetrovirus have only been
identified in wild and domestic birds. Members of this genus have C-type retroviral
morphology and simple genome organization. Within Alpharetrovirus, viruses are
01 assified into species according to differences in the genome, gene product sequences,
natural host range and nature of incorporated oncogenes. Examples of species belonging
to this genus are avian leukosis virus (ALV), avian myeloblastosis virus (AMV), avian
rIlb’elocytomatosis virus (AMC 29) and Rous sarcoma virus (RSV). Members of the avian
l e1ll<osis/sarcoma group of retroviruses (ALSV) are alpharetroviruses. ALV is a Simple

Slow transforming retrovirus and is considered the type species of this genus.

2‘ Betaretrovirus

ll

Viruses in Betaretrovirus have only been found in mammals and they mostly have

simple genomic structures. These retroviruses have type-B or type-C morphology and do
not possess viral oncogenes. Two former retroviral genera form this genus and these
include the mammalian B-type murine exogenous and endogenous retroviruses and D-
type retroviruses from primates and sheep (Gifford and Tristem, 2003). The mouse
mammary tumour virus (MMTV), J aagsiekte sheep retrovirus (J SRV), Langur virus
( LNGV), Mason-Pfizer monkey virus (MPMV), and Squirrel monkey retrovirus (SMRV)
are species in this genus. The prototype of betaretroviruses is MMTV representing
endogenous and exogenous retroviruses of mice. These murine retroviruses have an
accessory gene encoding a superanti gen (sag) and widely divergent and distinct env
genes. Moreover, they have the ability to induce mammary carcinomas by activating
cellular oncogenes (Linial et al., 2006). Murine endogenous retroviruses called

intracistemal A-type particle (IAP) are deleted or truncated in the mouse genomes and

Sometimes lack env genes (Goff, 2007).

3 - Gammaretrovirus

Gammaretrovirus is considered a unique genus because it includes the highest

nlllliber of known members. Initially, gammaretroviruses have been associated with
Sairczomas and leukemias in mice. However, retroviruses within this genus belong to many
Vertebrate classes including mammals, avian, reptiles and amphabians. Similar to
alpharetroviruses, gammaretroviruses are simple endogenous and exogenous retroviruses,

reli>lication competent or defective viruses with C-type morphology (Goff, 2007). In

12

mammals, replication competent gammaretroviruses lack oncogenes and replication
defective viruses have acquired a variety of cell-derived oncogenes fiom their hosts.
Gammaretroviruses are subdivided into mammalian, reptilian and avian virus
groups (Linial et al., 2006). Differentiation into species is based on genome sequence,
presence of oncogene, antigenic properties, natural host range and pathogenicity.
Examples include murine leukemia virus (MLV), avian reticuloendotheliosis virus (REV)
and feline leukemia virus (F eLV). MLV is the type species of Gammaretrovz’rus and is
used in gene therapy and cancer mutagenesis studies. Endogenous forms of these viruses
are widespread in the genomes of mammals, birds, reptiles and amphabians (Martin et
al., 1999). Yet, ICTV recognizes only a small number of endogenous retroviruses closely

related to their exogenous counterparts (F auquet et al., 2005).

4. Deltaretrovirus

Members of Deltaretrovims are oncogenes-free exogenous retroviruses with
complex genomic structure and C-type morphology (Goff, 2007). No endogenous
counterparts of Deltaretroviruses exist, but exogenous retroviruses are horizontally
transmitted and rarely found in few mammals such as primates and cattle.
Deltaretroviruses are classified into species based on genome sequence, absence of viral
oncogene, antigenic properties, natural host range and pathogenicity (Linial et al., 2006).

Bovine leukemia virus (BLV) is the prototype of Deltaretrovirus and other species
include Human T-lymphotropic virus (HTLV) and Simian T-lymphotropic virus (STLV).
In the host, these retroviruses are indefinitely persistent often causing slow or chronic

infections, characterized by long incubation period.

13

5. Epsilonretrovirus

Epsilonretrovirus contains complex exogenous viruses characterized by C-type
morphology in fish and reptiles. Duplicated viral homologues of the host cyclin D gene,
which may regulate the cell cycle, are the only known accessory gene products found in
these retroviruses (Fauquet et al., 2005). Classification into species is based on genome
and gene product sequences, viral oncogene and natural host range (Linial et al., 2006).
Walleye dermal sarcoma virus (WDSV) is a fish retrovirus and the prototype of the
genus. Other retroviruses in this genus include Walleye epidermal hyperplasia viruses

(WEHV) type land 2, Snakehead retrovirus (SnRV) and Perch hyperplasia virus (PHV).

6. Lentivirus
Lentivirus mainly includes exogenous complex mammalian viruses characterized
by unique virion morphology. Endogenous retroviruses show low level of homology to
their exogenous counterparts. Lentiviruses are often associated with anemia, cancer,
immune dysfunction and central nervous system disorders (Temin, 1992). Infection with
these retroviruses cause slow or chronic pattern of disease and long incubation period and
involve diverse organ systems (Gifford and Tristem, 2003). On the other hand,
1 entiviruses can be useful in gene therapy research experiments through introducing a
gene product into in vitro systems or animal models.
Lentivirus is subdivided into primate, ovine/caprine, equine, feline and bovine
l‘3111:ivirus groups reflecting the vertebrate hosts they infect. Species of lentiviruses are

‘31 assified according to genome and gene product sequences, antigenic properties, natural

14

host range and pathogenicity (Linial et al., 2006). Examples of lentivirus species are
bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), feline
immunodeficiency virus (F IV), caprine arthritis encephalitis virus (CAEV) and simian
immunodeficiency virus (SIV).

The type species of the genus is human immunodeficiency virus 1 (HIV -1 ), a
complex retrovirus containing additional regulatory genes (vif, vpr, vpu, tat, rev, and nef).
These accessory genes control transcription, RNA processing, virion assembly, gene
expression and other replication functions (Goff, 2007). Within the primate lentivirus
group, HIV-1 shows sequence divergence of more than 50% when compared to HIV -2,
which contain a vpx gene (Linial et al., 2006). However, these viruses result in
progressive decrease in CD4 T cell count, increase viral load and various life threatening

opportunistic infections, characteristics of acquired immunodeficiency syndrome (AIDS).

7. Spumavirus
The genus Spumavirus contains complex exogenous retroviruses with unique
virion morpholog. These retroviruses are widespread in mammals, but they are not
associated with any disease condition. While spumaviruses are non-oncogenic and non-
pathogenic, they establish persistent infections in many animal species. In addition, they
contain at least two accessory genes known as tas/bel-I, which encodes a transcriptional
transactivator and bet downstream of env. Although there are no endogenous equivalents,
vertebrate genomes commonly contain distinctly related viruses.
Species are differentiated according to their genomes, gene product sequences and

na-t"..1ral host ranges (Linial eta1., 2006). Examples within Spumavirus include simian

15

foamy virus (SFV), bovine foamy virus (BFV), equine foamy virus (EFV), and feline
foamy virus (F FV). The prototype example of spumaviruses is SF V, which is closely
related to HIV. SFV was named after the foamy bubbles appearing under the microscope

when infected cells fuse and form syncythia or giant cells.

11. DIVERSITY IN RETROVIRUSES
A. Introduction
Various species of viruses including RNA viruses and retroviruses mutate at a
very high rate (Svarovskaia et al., 2003). These mutations are manifested as variations in
nucleotide sequences, which enable viral propulations to evade host immune responses
and develop resistance to antiretroviral drugs. Different steps of replication cycle
contribute to genetic variability in retroviruses such as reverse transcription, DNA
integration, RNA transcription and virion assembly. However, the major factor in
retroviral variation is the low proofreading activity of reverse transcriptase (RT) enzyme
resulting in polymerization errors during the process of reverse transcription.
In retroviruses, genetic diversity is usually manifest by acquisition of cellular
genes and alteration of host range and considerable variations in incidence, severity and
symptoms of diseases. Therefore, studying retroviral genetic diversity is crucial to
understand the association between retrovirus variation and disease progression.
Mutations usually occur during the early and late stages of replication and result in subtle
genetic variability. On the other hand, recombination causes major genetic changes and
usl-lally takes place during the early stage of replication (Katz and Skalka, 1990).

Moreover, the relatively high rates of mutation and recombination of retroviruses

16

influence virus diversity, evolution, virulence, pathogenesis and the development of
effective antiviral drugs and vaccines (Mansky, 1998).

Retroviral diversity may result in the emergence of new strains (mutants or
recombinants) and subgroups of viruses. Genetic diversity arises when retroviruses are
passaged in tissue culture or animals (Katz and Skalka 1990). For example, Rous
sarcoma virus (RSV) was the first retrovirus to exhibit genetic variations and result in
different varieties of tumors in chickens such as rapidly growing and late-developing
tumors (Duran-Reynals, 1942; Rous and Murphy, 1913). On the other hand, it was
reported that naturally occurring strains of viruses are more likely to cause severe illness
than others (Roizman, 1996).

Evolution of retroviruses is influenced by selection of particular variants with
specific types of mutations in the surface and transmembrane envelope proteins
(Fleischmann Jr., 1996). Selection for these mutations is based on the interaction between
virus load, host immune responses and disease progression (Mansky, 1998). Thus, to
understand the effect of retroviral diversity on immune escape, drug resistance and
disease progression, we must investigate the genetic nature of variants produced by

different selective forces.

B. General Principles
Retroviruses are known to exhibit high rates of mutation resulting in their rapid
evolution (Coffin et al., 1980; Gojobori and Yokoyama, 1985, 1987; Hahn et al., 1986;
Monk et a1, 1992; O’Rear and Temin, 1982). These genetic variations lead to changes in

the function of virion envelope proteins and the production of new retroviral serotypes

17

with altered virulence (Fleischmann Jr., 1996). In addition, mutations can lead to epitope
deletion, failure of antigen processing, loss of major histocompatibility complex (MHC)
class I binding, impaired recognition by the T cell receptor resulting in viral persistence
and avoidance of the immune system.

In retroviruses, replication generates large numbers of genetically diverse
populations known as swarms or quasispecies (Payne, 2001 ). Thus, an individual virus is
made up of heterogeneous population of similar genomic variants, where the wild-type
sequence can only be defined by consensus (Katz and Skalka, 1990). Selective forces
generate mutations, but the genome of mutant population can not be defined since
retroviral population exists in a dynamic equilibrium. A mutation fiequency of 10'3 to 10'
4 misincorporations per genome per replication cycle results in a significant number of
mutant genomes even in biologically cloned retroviral stocks (Katz and Skalka, 1990).

During the evolution of retroviruses, cellular movable genetic elements
(transposons) evolved into endogenous retroviruses permenantly present in the host
genome. On the other hand, various mutations over time have created degenerate proviral
forms of exogenous retroviruses, which are unable to produce infectious virions (Best et
al., 1997; Lower, 1999). In Eukaryotes, endogenous retroelements are present in multiple
copies and constitute more than 10% of the host genome. In addition, several classes of
endogenous retrovirus-related elements exist in the genome of chickens (Fadly and

Payne, 2003). It is important to note that endogenous retroviruses can cause mutational
Changes in exogenous retroviruses through recombination.
Different species of retroviruses are compared through analyses of their genetic

structure and nucleotide sequences. Evolutionary trees are constructed based on

18

nucleotide or inferred amino acid sequences of different retroviral genes (Page and
Holmes, 1998). Sequence data are analyzed and trees are developed by computer
programs using various mathematical models of sequence variations. Evolutionary trees
include cladograrn, additive trees and ultrametric or dendrugram (Payne, 2001).
Cladograms are the most basic trees depicting relative recency of common ancestry.
Additive trees describe amount of evolutionary change represented by length of the
branches. Ultrametric trees (or dendrograrns) are special kinds of additive trees where the
tips of the branches are at an equal distance from the root. They express evolutionary
time indirectly by the degree of sequence divergence. Phylogenic trees show evolutionary
and epidemiological relationships among retroviruses and may reflect mutations and

recombination in retroviral genes over time.

C. Mutations
Mutations in retroviruses are very common and result in pennenant changes in the

genetic material. In RSV, the frequency of mutation in viral genomes is estimated to be

10'4 - 10'5 mutation per incorporating nucleotide per viral replication cycle (Preston and

Dougherty, 1996). Most mutations are deleterious and are removed; others are mainly

advantageous and are selected and some of the neutral ones become fixed by genetic drift

(Page and Holmes, 1998). Mutations that do not interfere with normal fimctions are
inherted and persist in a virus population. Viral mutations have been used by virologists
in developing attenuated live viral vaccines despite the possibility of back-mutations or
rewersions (Fleischmann Jr., 1996). On the other hand, conserved sequences or motifs can

lo: usefirl for developing new vaccines or antiretroviral drugs (Jacobs et al., 2008).

19

Nucleotide substitutions and rearrangement such as simple deletions, deletions
with insertions and duplications of nucleotides in retroviral genomes represent point
mutations (Payne, 2001). The main source of point mutations is the error-prone
replication due to the lack of proofreading activity (activity of 3’-5’ exonuclease) of RT.
Nucleotide substitutions (A to G) are the most common point mutations, 80% of which
are transitions whereas the remaining 20% are transversions (Figlerowicz et al., 2003).
Transition is the exchange of pyrimidine into pyrimidine or purine into purine. On the
other hand, transversion is the exchange of the pyrimidine into purine and vice versa.

Nucleoide subsitutions are more common during transcription of an infectious
avian retrovirus than replication of defective endogenous retrovirus (Payne, 2001). BLV
and HTLV replicate in the proviral forms through clonal expansion of infected cells and
have fewer mutations than otherretroviruses, which replicate by reverse transcription
such as HIV (Wain-Hobson, 1996). Moreover, substitution mutations occur in the
variable regions of gp85 SU sequence of the env gene of exogenous ALVs (Bai et al.,
1995a; Bova et al., 1986; Venugopal et al., 1998). These mutations may influence
antigenic determinants, pathogenicity, host range or target cell specificity. The env gene
of ALVs shows the highest diversity compared to gag and pol genes; while LTR
sequences are relatively variable (Payne, 2001).

. Various molecular approaches have been used for investigating retrovirus
mutations and variations (Mansky, 1998). Molecular cloning and analysis of restriction
enzyme digestion patterns of proviruses of spleen necrosis virus (SNV) allowed the study
of their biological and molecular properties (O'Rear et al., 1980). The polymerase chain

reaction (PCR) was used to isolate HIV and SIV variants and analyse variations in the SU

20

region of their env and resulting amino acid changes by DNA sequencing (Burns and
Desrosiers, 1991; Johnson et al., 1991; Overbaugh et al., 1991). In addition, DNA
sequence analysis was used to study the association among different Moloney murine

leukaemia virus (M-MLV) strains (Weiss et al., 1985).

D. Recombination

Many investigators reported that the high level of retroviral recombination is
attributed to the diploid nature of retroviruses (Coffin et al., 1997; Hu and Temin, 1990a
and b; Nichol, 1996; Zhang and Ma, 2003). Recombination usually takes place when two
related parental RNA genomes exchange genetic materials during reverse transcription.
On the other hand, it can result from physical interaction between retroviral and host
genomes. If the host genes code for growth factors and growth factor receptors, the
recombinant retroviruses may be oncogenic, carrying cellular oncogenes.

Recombination contributes to retroviral diversity and its frequency exceeded 10%
of the progeny (Alevy and Vogt, 1978; Blair, 1977; Kawai and Hanafirsa, 1972; Linial
and Brown, 1979; Vogt, 1971; Wyke and Beamand, 1979). Recombination is mainly
responsible for emergence of new viral strains and species with new viral phenotypes and
altered host range and virulence. On the other hand, it allows the introduction of foreign
genes coding for a specific immunogen, normal human or animal genes into retroviral

genomes. These recombinant viruses can be used experimentally as vaccines or can be
useful for gene therapy of a wide range of diseases (Fleischmann Jr., 1996).

Recombination was first observed after cell co-infection with two genetically

d‘r stinct avian tumor viruses (V o gt, 1971). It was observed that murine endogenous

21

 

retroviruses recombine and form pathogenic strains (Stoye et al., 1991). Recombination
between exogenous and endogenous avian retroviruses generates viruses with new host
ranges, encoded in the env SU (Rein, 1982; Weiss et al., 1973) and altered cell-specific
transcription in the LTR (Boral et al, 1989; Celander and Haseltine, 1984; Golemis et al.,
1990). This results in a more efficient replication of these pathogenic viruses and
activation of cellular oncogenes causing host cell transformation and tumor formation.

The novel ALV subgroup J may have resulted from either a single or multiple
recombination events between the endogenous and exogenous ALVs (Venugopal, 1999).
Three retroviruses ADOL 5701A, ADOL 5701AA, and ADOL 6803A are recombinants
between exogenous subgroup J ALV and a defective endogenous ALV expressing
subgroup A envelope (Lupiani et al., 2000). In addition, a naturally occurring ALV
isolate was molecularly characterized as a recombinant with a subgroup B gp85 region
and a subgroup E gp37 region and subgroup J LTR (Lupiani et al., 2006).

Genetic recombination in retroviruses can be either homologous or non-
homologous. Homologous recombination involves two similar RNA molecules
possessing long region of homology (Payne, 2001; Figlerowicz et al., 2003). It can occur
by intramolecular (more efficient) or intermolecular template switching (Hu et al., 1997).
Forced-copy choice and strand displacement-assimilation are two models propoed to
define homologous recombination in retroviruses (Coffin, 1979; Hunter, 1978; Katz and
Skalka, 1990; Skalka et al., 1982).

i The forced copy-choice model (Figure 1.4) proposes that recombination occurs
when RT jumps and switchs retroviral RNA templates during minus strand DNA

synthesis (Coffin, 1979). On the other hand, the strand displacement-assimilation (minus-

22

 

strand exchange) model proposes that recombination occurs when plus strand DNA
synthesis takes place on two templates (J unghans et al., 1982). Each model predicts the
nature of the recombinant product, a homoduplex for the forced copy-choice and a
heteroduplex for the strand-assimilation model.

In 1979, Wyke and Beamand reported that homologous recombination frequently
occur between exogenous ALSVS under experimental conditions. In the field,
homologous recombination between exogenous and endogenous avian retroviruses is
possible. For example, ALV-J is shown to have gag and pol genes closely related to those
of exogenous ALSV and an env gene nearly identical to that of EAV-HP, a member of
the EAV family of endogenous retroviruses (Bai et al., 1995b; Smith et al., 1999).
Moreover, recombination between two types of endogenous retroviruses is also possible
as suggested by the high identity of the R and U5 regions of the LTRs of EAV-HP and
ART-CH (Sacco et al., 2000).

Non-homologous recombination takes place between unrelated sequences in the
same or distinct retroviral genomes (Cheslock, 2000; Figlerowicz et al., 2003; Zhang and
Temin, 1994). In addition, non-homologous recombination occurs between retroviral
sequences and non-homologous or unrelated host sequences. This results in transduction
of cellular genes and the formation of highly oncogenic retroviruses. For example, acute
avian leukemia and sarcoma retroviruses such as RSV, FuSV, UR2, Y73, AEV-ES4,
AMV and MC29 have acquired cellular oncogenes (src, fps, ros, yes, erbA and erbB, myb
and myc) and enhanced oncogenicity (Payne, 2001). The mechanisms of non-homologous

and homologous recombinations are similar (Mansky, 1998). However, non-homologous

23

 

recombination occurs at about 0.1-1% the rate of homologous recombination (Zhang and

Temin, 1993).

E. Rearrangements

Deletions and duplications are two types of rearragement in retroviral genomes. In
retroviruses, small deletions in the order of several nucleotides occur due to template
slippage or mispriming at direct repeats. Large deletions up to thousands of nucleotides
result fiom homologous recombination between direct repeats (Bizub et al., 1984). In
fact, homologous recombination is the most fi'equent source of genetic rearrangements in
retroviruses (N egroni and Buc, 2001). Yet, non-homologous recombination in the same
retroviral genome allows more extensive genomic rearrangements such as deletions,
duplications and inversions (N egorni and Buc, 2001; Pathak and Temin, 1990a and b).

Large deletions affecting structural and enzyme genes generate replication-
defective mutants (Katz and Skalka, 1990). Moreover, Voynow and Coffin (1985)
observed deletions in RSV by a pathway independent of homologous recombination.
Deletions are also possible due to the spurious endonucleolytic action of ASLV IN near
the internal border (US) of the upstream LTR (Olsen and Swanstrom, 1985). Duplications
of retroviral sequences usually result from polymerization errors during reverse

transcription and from non-homologous recombination (Coffin, 1986).
F. Role of Selective Forces

Various models of molecular evolution have been proposed to explain the

behavior of mutating retroviral populations. In the founder effect model, few (founder)

24

 

individuals that carry some of the genetic variations of the parent population are
responsible for the establishment of a new population (Payne, 2001). Muller’s ratchet
model states that accummalating deleterious mutations lead to the loss of fitness and
survival of retroviral population (Maynard Smith, 1998). Red Queen hypothesis proposes
that as species evolve in a population, change will always occur even in a constant
physical environment (Maynard Smith, 1998). In the competitive exclusion model, one
competing species will always overcome and eliminate other species in a population
(Payne, 2001).

Selection of viral variations often takes place in four stages generating infectious,
replicating, non-neutralizable and drug-resistant viral variants (Figlerowicz et al., 2003).
During the first stage, viral particles must be infectious to enter a cell. These viruses
replicate and generate a population of diversified viral particles during the second stage
of selection. The third stage of selection is that of the host immune responses, where
immune escape mutants are generated. In the fourth stage of selection, the selective
forces of antiviral therapy generate replicating and spreadable drug-resistant mutants.

Replication is a potential selective force generating retroviral diversity. Selection
for more efficient replication increases retroviral pathogenicity through the action of viral
gene products or activation of cellular oncogenes (Stoye and Coffin, 1985; Teich et al.,
1982 and 1985). Tissue-specific replication in retroviruses has been linked to LTR, 5’
leader, gag and env and possibly other genomic regions (Robinson et al., 1985). In
murine pathogenic viruses, sequence changes in the LTR increase the activity of the

transcriptional promoter and enhancer in the U3 region (Boral et al., 1989; Celander and

25

 

Haseltine, 1984; Golemis et al., 1990). Replication in new hosts influence evolutionary
and recombination rates as well as selection profiles (Poss et al., 2008).

Selective forces target retroviral envelopes and generate genotypic and
phenotypic changes. For example, selection for tissue-specific replication results in
mutations in the receptor binding region (SU protein of env), expanded host range and
increased pathogenicity (Domer and Coffin, 1986; Dormer et al., 1985). Furthermore, the
ability of retroviruses to replicate in different host species is a selective pressure to cause
changes in the env sequences and the recognition of new receptors (Rein, 1982; Stoye
and Coffin, 1985). Selective proliferation of transformed host cells causes oncogenes
mutations and promotes the capture and activation of cellular oncogenes.

It is well documented that retroviral env gene products are targets for selective
forces of neutralizing or humoral immune response (Ho et al., 1988; Katz and Skalka,
1990; Skinner et al., 1988; Weiss et al., 1986). Moreover, retroviral diversity reflects
selection of env variants that can evade the neutralizing immune system. On the other
hand, the neutralizing immune response is not a selective force in congenitally ASLV-
infected chicken due to immunological tolerance (Teich et al., 1985). RT-inhibitor drugs
are potential selective forces resulting in variations in pol gene and eventually generating
drug- resistant retroviruses (Larder and Kemp, 1989).

Natural selection of variant viruses can be detected by comparing the numbers of
non-synonymous (NS) and synonymous (S) nucleotide substitutions within a gene
(Payne, 2001). NS substitutions alter the antigenicity or other properties of the viral
proteins since they involve changes in the first or second bases in a codon, which will

modify the amino acid product. On the other hand, S substitutions do not change the

26

 

amino acid produced since they entail a change to the third base of the codon. Positive
natural selection is said to be operating on viral variants when the number of NS codons
produced as a result of mutation exceeds the number of S codons (or the NS/S ratio is
>1). Analysis of env sequences has allowed the detection of the survival and selection of

ALV and HIV immune escape mutants (Mansky, 1998; Venugopal et al., 1998).

G. Evolution and Retroviruses

For decades, scientists have investigated the origin of retroviruses and the
relationship between cellular moveable genetic elements, endogenous and exogenous
retroviruses. Retroviruses are believed to have evolved from ancient transposable genetic
elements based on RT (pol) sequences from various retroviruses and retrovirus-like
elements (retrotransposons) of different species (Doolittle et al., 1989 and 1990; Temin,
1985). These transposable elements insert DNA sequences into non-homologous genomic
DNA with the help of the enzyme reverse transcriptase in the process of DNA
transposition. Furthermore, they are important mutagens and source of genetic variation.

Retroviruses have evolved over time as either exogenous infectious agents or
endogenous proviruses (Katz and SKalka, 1990). The retroviral genome is influenced by
opposing selective forces for passive replication as endogenous proviruses versus
efficient replication as exogenous retroviruses. These selective forces generate variations
in clustered areas of functional or non-essential regions of retroviral genomes. Germline
infection provides a selective advantage over the course of evolution since the host
survival enSures the survival of the endogenous provirus. On the other hand, selection for

high proliferative rate of exogenous retroviruses is not advantageous if it kills one host

27

 

before the virus is transmitted to another. Segments of these endogenous entities are
perpetuated through recombination with exogenous retroviruses.

Homologous recombination is a highly frequent event shaping retroviral evolution
(Casci, 2000; Schierup and Hein, 20003 and b). Around 10% of infectious strains of
human immunodeficiency virus (HIV) are the result of recombination among different
viral subtypes (McCutchan, 2000; Peeters and Sharp, 2000). Moreover, pathogenic
retroviral strains in mice are generated from recombination among endogenous
retroviruses (Stoye et al., 1991). On the other hand, non-homologous recombination is
effective at several turning points of retroviral evolution such as the capture of oncogenes
by transduction of cellular sequences and the generation of gene duplications (Goldfarb
and Weinberg, 1981; Swain and Coffin, 1992; Swanstrom et al., 1983).

Mutations in retroviruses contribute to the emergence of new virus strains and
new disease expressions. Retroviral replication generates new variants through point
mutations, rearrangements and recombination. Studies on mutations of many different
retroviruses have contributed to understanding how avian retroviruses mutate, how
mutants may be selected to become dominant forms, and the disease consequences in the

host (Payne, 2001).

III. AVIAN LEUKOSIS-SARCOMA VIRUSES AND DISEASES
A. Introduction

The viruses in the avian leukosis-sarcoma (L/ S) group are alpharetroviruses
associated with various neoplastic diseases and production problems in birds mainly

chickens. Avian leukosis virus (ALV) is a member of this group and most commonly

28

 

causes lymphoid leukosis (LL) and myeloid leukosis (ML) in field flocks. Infection and
diseases induced by ALV result in millions of US. dollars of economical losses each year

due to tumor mortality and reduced productivity (Fadly and Payne, 2003).

B. Etiology
1. Classification

According to the new classification of the International Committee on Taxonomy
of Viruses (ICTV), the avian leukosis-sarcoma (L/S) group of viruses (ALSV) belongs to
the family Retroviridae, subfamily Orthoretrovirz'nae and genus Alpharetroviruses
(F auquet et al., 2005). Members of this group share common physical, chemical and
molecular characteristics and group-specific anti gen (F adly and Payne, 2003). Under the
new taxonomy, the type species of the genus is avian leukosis virus (ALV). Other species
include Rous sarcoma virus (RSV) and other replication defective viruses such as avian
erythroblastosis virus (AEV) and avian myeloblastosis virus (AMV), carrying oncogenes.

ALSVs are classified into several categories depending on their virological,
immunological and pathological characteristics including their modes of transmission.
Within a species like ALV, viruses are grouped based on envelope glycoprotein into
subgroups, which include viral types depending on antigenic variations and strains
depending on oncogenic spectrum in various host tissues. Most viral strains are
subgroup-pathotype combinations containing mixtures of viruses (Payne, 1987) and
inducing a variety of tumors, which often overlap with other strains.

Based on the rate of tumor induction in susceptible chickens, strains of ALSV can

also be placed into 2 major classes, acute and slow transforming viruses (Fadly and

29

 

Payne, 2003). Acute transforming viruses carry viral oncogenes and cause acute
leukemia, leukosis and solid tumors usually sarcomas within few days or weeks (Enrietto
and Wyke, 1983; Graf and Beug, 1978; Moscovici and Gazzolo, 1987). The type of
neoplasm produced by a certain virus strain depends on the target cell transformed by the
viral oncogene (v-onc). These viruses are genetically or/and replication defective,
requiring a helper leukosis virus for replication. Examples include Rous sarcoma virus
(RSV), MC29, 966 and MH2 (Fadly and Payne, 2003).

Conversely, slow transforming viruses lack oncogenes and induce transformation
by promoter insertion or another mechanism near a host proto-oncogene (Weiss et al.,
1985). They are replication competent and usually induce various leukoses, leukemias
and sarcomas weeks or months after infection (Coffin et al., 1997; Cooper, 1982).
Examples include Rous-associated virus 1 (RAV-1, subgroup A ALV), RAV-2 (subgroup
B ALV), RAV-49 (subgroup C ALV), RAV-50 (subgroup D ALV), and ADOL-Hcl
(subgroup J ALV).

The mode of transmission and the nature of replication of ALSVS are used in their
classification into exogenous and endogenous viruses. Exogenous ALVs constitute free
infectious viral particles, which spread through vertical and horizontal routes (Payne,
1987). On the other hand, endogenous ALVS comprise incomplete to complete viral
particles making up part of their host genomes or free infectious entities respectively
(Rovigatti and Astrin, 1983). Therefore, they can be transmitted genetically, horizontally

and vertically (Robinson and Eisenman, 1984; Rubin et al., 1961; Smith et al., 1986).

2. Subgroups

3O

There are six ASLV envelope subgroups (A, B, C, D, E and J) in chickens based
on the diversity in the viral envelope glycoproteins. Subgroups A, B, C, D and J are all
exogenous, while subgroup E is endogenous. Moreover, Subgroups F, G, H and I are
endogenous ALVS present in the genomes of pheasants, partridge and quail (Payne,

1 992). The viral subgroup is determined by assays of antigenicity, viral interference
patterns and host range (Weiss et al., 1985).
Antigenicity is associated with viral envelope antigens neutralized by known
subgroup-specific antibodies detected by viral and serum neutralization tests respectively.
In general, viruses belonging to the same subgroup cross-neutralize each other to varying
d egrees, but do not cross-neutralize viruses from other subgroups. Moreover, viruses tend
to neutralize homologous viruses more strongly than heterologous viruses within the
Same subgroup (Chubb and Biggs, 1968). Subgroups B and J viruses appear to be more
11 eterogeneous than other ALV subgroups. On the other hand, some subgroup J viruses do
no t always cross-neutralize or cross-neutralize in one direction (F adly and Smith, 1999;
F ad 1y et al., 2000; Venugopal et al., 1998). Moreover, there is a partial cross-
I16:1._‘.l.1:ralization between subgroups B and D viruses (Payne, 1992).
Viral interference pattern is established in susceptible avian embryo fibroblast
cul tures by infection with unknown virus and challenge with different RSV subgroups.
Comparison of RSV foci reduction between infected and uninfected controls indicates the
Presence of viral interference.Host range is defined by viral SU protein interaction with a
Speci fic host cell receptor. Host range is determined by virus growth in embryo
fibroblasts from various avian species or transformed cell lines (Payne et al., 1992b). The

hOSt range of ALV is investigated in chicken embryo fibroblasts (CEFS) belonging to

31

distinct genetic lines and different phenotypes. Viral interference pattern and host range
are more reliable for subgroup classification than antigenicity assays, which are used for
strain classification (Fadly and Payne, 2003).

Furthermore, several strains of ALSVS are genetically defective, lacking an

envelope gene and thereby have no subgroup designation. Nevertheless, they require the

complement function of the helper leukosis virus present in the viral mixture for

replication. Thus, they take on the designation of the helper leukosis virus envelope
subgroup such as BH-RSV (RAV-1 ), a Bryan’s high titer strain of RSV grown with

Rous-associated virus 1 (subgroup A).

3 - Endogenous Leukosis Viruses
The normal chicken genome contains various classes of endogenous retroviruses
inherited in a Mendelian fashion (Crittenden, 1991; F adly and Payne, 2003). These
fanlilies include the endogenous viral (ev) loci, the moderately repetitive elements EAV
(endogenous avian virus) and ART-CH (avian retrotransposon from chicken genome) and
fine liighly repetitive elements CR1 (chicken repeat 1). The prototype virus of subgroup E,
Rous-associated virus (RAV-0), was spontaneously produced in line 7 chicken embryo
cell 8 (V ogt and Friis, 1971). Endogenous ALSVS rarely cause tumors apparently due to
g the: Weak promoter activity of their LTRs (Crittenden et al. 1979; Fadly and Payne, 2003;
MOtta et al., 1975). They can be beneficial and stimulate immunity or detrimental and
induce tolerance to tumor virus antigens (F adly and Payne, 2003).
The ev loci family consists of complete or defective proviral DNA sequences of

sub4gl‘0up E ALV integrated in the chicken genome (Sacco et al., 2000). They are found

32

on certain chromosomes (Tereba et al., 1981) of somatic and germ line cells and are
transmitted genetically by both sexes (Astrin et al., 1979; Crittenden et al., 1977; Payne
and Chubb, 1968). More than 30 ev loci have been documented in different chicken
strains (Sabour et al., 1992). On average, there are about 5 ev loci in each bird with
variable phenotypic expression (Rovigatti and Astrin, 1983). The ev loci have low copy
numbers segregated in the population and restrictly distributed in various species of

Gallus (Sacco et al., 2000).

Some ev loci produce defective non-infectious viral particles (ev3, ev6 and ev9)
phenotypically expressed. Others generate complete infectious virions (ev2, ev7, ev10,
e1.) 1 1, ev12, ev14, ev18 and ev21) spontaneously produced as subgroup E ALVS. For
ex ample, ev21 encodes a complete virus known as EV21 and induces immunological
to I erance and increases the susceptibility of chicks to exogenous ALV (Bacon et al.,

1 9 8 8; Harris et al., 1984; Smith and Fadly, 1988; Smith et al., 1991). Moreover, it
Yen ders White Leghorn chickens more susceptible to ALV infections resulting in reduced
egg production, increased incidence of viremia and subsequently higher tumor mortality
(B acon et al., 1988; Harris et al., 1984; Smith and Fadly, 1988). Nevertheless, certain ev

1OCi (evl, ev4, ev5, ev8, and evl 7) do not produce viral products since they are
“an scriptionally silent (Coffin, 1982).

The protein expression of some ev loci will give false positive reactions and can
interfere with the interpretation of diagnostic tests such as enzyme-linked immunosorbent
assay (ELISA) and complement-fixation test for ALV (COFAL). Line 0 chickens are
deVOid 0f any ev loci (Astrin et al., 1979; Crittenden and Fadly, 1985) and provide useful

research tools for chicken experiments and biological assays (line 0 CEFS) since they are

33

resistant to infection with subgroup E ALV (C/E). Many ev loci are associated with
decreased productivity, reduced immunity and disease resistance to varying degrees
(Crittenden, 1991). For example, chicken cells expressing envelope proteins of some ev
genes are dominantly resistant to infection by subgroup E ALV by blocking virus
receptors on cells (Payne et al., 1971; Robinson et al., 1981). Moreover, the presence of
ev2 or ev3 protects chickens from a unique non-neoplastic syndrome caused by infection
\PVIth ALV subgroup A (Crittenden et al., 1982 and 1984).
The EAV class is a heterogeneous group of highly diverged retroviral elements
found in chickens and other species of Gallus (Sacco et al., 2000). EAV-0 elements were
fi rst discovered in line 0 chickens and have typical proviral structure with deletions in the
env region (Boyce-Jacino et al., 1989). There are about 50 copies of EAV in a normal
chi cken genome. Howeve, EAVs do not produce infectious virions, but they express RT
activity especially in live virus vaccines and cell cultures (Hussain et al., 2001; Robertson
et al., 1997; Tsang et al., 1999; Weissmahr et al., 1997). EAV-HP is believed to be the
Dr-i gin of the env gene of subgroup J ALV (Bai et al., 1995a and b; Benson et al., 1998a
md b; Ruis et al., 1999; Smith et al., 1999). In addition, ALV J and EAV-0 element ev/J
4- - 1 Rb might share the same receptor (Denesvre et al., 2003)

The ART-CH family belongs to the defective retrotransposon class, requiring the
use of a helper virus to complement its replication. There are approximately 50 copies per
haI>IOid genome, each copy is composed of fimctional LTRs and short regions of
hoITIIOlogy to the ALV-related gene sequences (Nikiforov and Gudkov, 1994). The 5’ end

seq‘Jences of ART-CH highly resemble those of EAV-HP (Sacco et al., 2000).

34

The CR1 class consists of short interspersed repetitive DNA sequences, which
belong to the non-LTR class of retrotransposons possessing RT sequences (Stumph et al.,
1984). CR1 sequences are highly numerous, about 7000 to 20,000 repeats per haploid
genome (Crittenden, 1991). They have conserved 3' ends and variable 5' truncations and
are not functionally expressed. CR1 sequences are identified in several avian and

reptilian species representing ancient and primitive sequences preceding the evolution of
birds and reptiles (Crittenden, 1991; Vandergon and Reitrnan, 1994).

Recently, an endogenous retrovirus ChiRVl was identified in the chicken genome
(Borysenko et al., 2008). It clusters with murine leukemia virus (MLV)-related viruses,
but is not related to exogenous MLV-related retroviruses infecting chicken. It is 9133 bp

1 ong and contains 90% identical LTRs, gag, pol and env genes. It is tanscriptionally

active but genetically defective; thus, it is unlikely to produce viral particles.

4 - Virus Structure and Composition

ALSVs have an outer membrane, an intermediate membrane and an inner
centrally located electron-dense core with a diameter of 35-45 mm (Coffin, 1996). ALVs
are approximately 80-120 nm in diameter and have a type C morphology (F adly and

P ayrre, 2003). On the outer membrane, each transmembrane glycoprotein (projection or
spike) is 7 nm long and 8 nm diameter with a 6 nm knob like surface glycoprotein at the
tip (Beard, 1963). ALVS have a buoyant density of 1.15-1.17 g/cm3 in sucrose, a
Characteristic of type C viruses (Bauer, 1974) and a sedimentation rate of about 603-708
repreSenting the diploid viral genome. The virions are susceptible to heat, detergent and

forth aldehyde (Fadly and Payne, 2003)-

35

Each ALSV particle is composed of 30-35% lipid, 60-65% protein (of which 5-
7% is glycoprotein), 2.2% RNA and small amounts of DNA of host cell origin (Bauer,
1974; Beard, 1963). Since the virion envelope is derived from host outer cell membrane,
it is a bilayer made mainly of phospholipids (Bauer, 1974; Bolognesi, 1974). The viral

proteins are encoded by gag/pro, pol and env genes present in the retroviral genome
(Weiss et al., 1985; F adly and Payne, 2003). Four non-glycosylated proteins are encoded
by gag namely capsid (CA or p27), matrix (MA or p19), nucleocapsid (NC or p12)
proteins and an additional gag protein (p10). CA is the major group specific antigen
(gsa); MA lines the inner surface of the virion envelope; NC is a basic protein associated
with genomic RNA; and p10 is located between MA and CA. The protease (PR or p15) is
an enzyme encoded by pro gene, which lies between the gag and pol genes. The pol gene

encodes the enzymes, reverse transcriptase (RT or p68) and integrase (IN or p32). The

en 12 gene encodes 2 glycoproteins (Coffin, 1992), the surface envelope protein (SU or

gp 85) and the transmembrane protein (TM or gp3 7).
The ALV genome is a dimer of linear single stranded plus-sense RNA, 7.8 kb in

length (Coffin, 1992). The retroviral RNA consists of a 5' cap and 3' poly (A) tail
gen erated by the host transcriptional machinery. The viral genome is surrounded by 2
repeate regions (R) made of 20-nucleotides (nt), one immediately after the 5' cap and one
immediately before the 3’ poly (A) tail (Coffin, 1992). Furthermore, an 80-nt unique 5’
secIllence (U5) lies downstream of 5‘ R and contains one of the attachment (att) sites

reqllired for proviral integration. The U5 region precedes a primer binding site (PBS), an

1 8‘nt sequence, which hybridizes to the host tRNA and initiates minus-strand DNA

SyIjltl'lesis during reverse transcription (Coffin, 1992). Downstream of the PBS is the Psi

36

element (‘1’), a major encapsidation signal for the viral RNA. The genes coding for
structural proteins i.e. gag/pro-pol-env follow the ‘1’ element Downstream of the
structural genes is a short polypurine tract (PPT), a run of at least nine A and G residues
and the initiation site for the plus-strand DNA synthesis (Coffin, 1992). Subsequent to the
PPT lies a 200-nt unique 3' sequence (U3), containing a number of cis-acting elements
:for viral gene expression and one of the att sites required for proviral DNA integration.
Exogenous ALV subgroups (A, B, C and D) share 96-97% sequence homology
with subgroup J ALV in the gag/pro and pol, but not in the env. On the other hand, the
transmembrane envelope glycoproteins of ALV A to E are homologous and highly
rel ated while that of ALV J is very different (Melder et al., 2003). Moreover, ALV-J
share 97%, 95% and 75% sequence homology in the env gene with EAV-HP, ev/J and
EAV-E51 respectively (Benson et al., 1998a; Smith et al., 1999). Downstream to the
gp37 of the env, the 3' noncoding region or untranslated (3’ UTR) of ALV-J consists of a
redundant transmembrane region insert (rTM), a direct repeat (DR1) element and an B
el ement (XSR). rTM of ALV-J is more homologous (97%) to other exogenous ALVS
than to other ALV-J viruses. In addition, ALV J contains a single copy of the DR1
e1 enent found 3’ to the rTM, but avian sarcoma viruses contain two copies flanking the

src gene. Downstream of DR1 , a copy of the E element is found in replication-competent

RS Vs, but not in replication competent ALVs (Bai et al., 1995a).

5 ° Antigenic Variation

Antigenic variation has been documented in retroviruses from avian, animal and

human sources. In ALV, antigenic variation can be divided into 2 categories namely

37

subgroup-specific and type-specific based on the use of host cell receptors. ALVS use
different host cell receptors when subgroup-specific antigenic variations occur while they
use the same receptor when type-specific antigenic variations arise. In ALV infections,
subgroup-specific and type-specific antigenic variations have been reported (Fadly and
Smith, 1999; Venugopal et al., 1998). In general, subgroup-specific variation results in
the development of a novel ALV subgroup (Fadly and Payne, 2003). Moreover, type-
specific antigenic variation has been observed to be much higher in infections with
subgroup J than with other ALV subgroups (Silva et al., 2000; Venugopal et al., 1998).
Sequence variations in gp85 (SU) and gp37 (TM) of the env influence both virus-
h 0 st cell receptor interactions and neutralizing antibody (Nab) responses. The SU protein
interacts with the host receptor while the TM protein anchors the SU protein to the viral
enVelope membrane and is responsible for the fusion of viral and host membranes
( Coffin, 1992). The gp85 contains 2 hypervariable and 3 less variable regions organized
as vrI -vr2-hr1 -hr2-vr3. The gpSS protein is more variable than the gp37 protein, which is
composed of fusion peptide and membrane anchor domains. Clusters hr] and hr2
determine the specificity of receptor binding and host range in all ALV subgroups
(Domer et al., 1985; Domer and Coffin, 1986). In addition, hr2 and W3 regions of ALV J
an v gene may be targets for immune selection since they exhibited high sequence
variations along with very high NS/S ratios (V enugopal et al., 1998). The env of ALV-J
di fl”7531‘s more extensively from that of other subgroups since it contains regions of higher
Seql-lence variability designated as hypervariable regions 1-4 (Silva et al., 2000). The vr3

domain is responsible for receptor recognition specificity but not receptor binding

38

affinity. On the other hand, WI and vr2 do not seem to be crucial for receptor specificity
or binding affinity (Domer et al., 1985).

Commercial ELISA kits have been widely used to identify and eradicate ALV in
chickens (V enugopal et al., 1997). They can also be used to monitor new emerging
viruses including recombinants. However, they have limited value due to the high level
of antigenic variation in the env (Silva et al., 2000; Venugopal, 1999; Venugopal et al.,

J 998). Moreover, polymerase chain reaction (PCR) based tests have been used to monitor
flocks for known and new ALV subgroups and strains using different primers specific for
the variable regions in the env and LTR (F adly and Payne, 2003; Silva et al., 2000; Smith
et al., 1998a and b). Nevertheless, they are not entirely reliable since the primers can not
be specific and generic at the same time. This may result in false negatives if mutations
aI-i se in proviral DNA sites, where primers are designed to anneal. Therefore, constant
monitoring of the specificity and sensitivity of all diagnostic methods is indispensable for

th e successful prevention, control and eradication of ALV.

6 - Virus Replication
ALSVs including ALVs adsorb to the host cell membrane in a nonspecific
manner even in cells resistant to infection (Piraino, 1967). However, they enter the host
cell S based on the specific interaction of their envelope glycoproteins (SU and TM) with
the host cell membrane receptors. In fact, different ALV subgroups have different SU
g1 y COproteins and enter the infected cells through exploiting receptors of other molecules.
F 01’ EXample, the receptor for ALV subgroup A is genetically related to the human low-

densi ty lipoprotein receptor, LDLR (Bates et al., 1993; Young et al., 1993). Moreover,

39

Subgroups B and D ALV share the same receptor CARI , a member of the tumor necrosis
factor receptor (TNFR) family (Brojatch et al., 1996). On the other hand, the receptor for
ALV-E is almost identical to CARI , only differing in amino acids at residue 62 (Adkins
et al., 2000). The receptor for subgroup C ALSV is related to mammalian butyrophilins,
members of the immunoglobulin superfarnily (Elleder et al., 2005). In addition, Chai and
B ates (2006) identified the receptor for ALV J to be a Na+/H+ exchanger type 1 in

chickens similar to its mammalian version, which is a housekeeping protein, regulating

i ntracellular pH and cell volume.
After SU glycoprotein binds the appropriate receptor, a conformational change in

the virion envelope glycoprtoteins occur leading to viral envelope fusion with host cell
membrane (Gilbert et al., 1995). Viral entry into the host cell is facilitated via endosomes
where the virion core is released into the cytoplasm. The early phase of the retroviral life
Cycle continues with the process of reverse transcription. RNA: DNA hybrid is formed
“/11 en the plus-strand viral RNA is reverse transcribed into a minus-strand viral DNA by
RT .. While the RN ase H activity of RT facilitates the cleavage of RNA from the hybrid,
tlie RT DNA polymerase activity generates plus-strand DNA complimentary to the
minus-strand DNA. The end products are linear DNA duplexes flanked by LTRs on both

e=Il'lcl s. The process of reverse transcription of ALV by RT is depicted in Figure 1.5. The
linear double-stranded DNA migrates into the nucleus and is randomly integrated into the

ho St cell DNA at multiple sites by the viral enzyme integrase (Fadly and Payne, 2003).

The host cell RNA polymerase 11 carries out the transcription of proviral DNA

tel111:)late into viral RNA, which is exported back to the cytoplasm. The resulting viral

RNA is packaged as genomic RNA in newly formed virions. On the other hand, the viral

4O

RNA forms messenger RNA (mRN A), which is translated by polyribosomes into several
polyprotein precursors namely Pr180GAG-PR-POL, Pr76GAG-PR and gPr92env (Fadly
and Payne, 2003). Viral and cellular proteases cleave these precursors to yield the viral
proteins and glycoproteins (Luciw and Leung, 1992). The viral proteins localize directly
at the host cell plasma membrane and crescent-shaped structures develop into immature
vijions, which bud off from the cell. The resultant immature virions have large, open
spherical cores and undergo maturation to become infectious (Vo gt, 1996). The viral
protease (PR) cleaves GAG polyprotein into three major proteins, CA, MA, NC and the
mature particles are characterized by centrally located condensed cores with small

surface projections (Coffin, 1992). The retroviral life cycle is depicted in Figure 1.6
( Coffin et al., 1997).

CT. Epizootiology

l - Virus Infection Incidence

Infection of chickens with ALSVS is associated with delayed sexual maturity,

I‘<E=c1‘l..rced egg production and quality, reduced body weight and increase in mortality

( Spencer et al., 1984). It is an economic problem to layer and broiler poultry breeders in
no st areas of the world. In spite of the eradication efforts implemented by many primary
b1‘eeding companies, ALV is still widespread in commercial poultry flocks. Egg-type and
r1'l'E:«‘£3.1-type chickens, pheasants, partridges and quail are naturally susceptible to ALV

in fections; however, other avian and mammalian species can be experimentally exposed

(Ger-1m et al., 1996; Kumanishi et al., 1973; Nehyba et al., 1990; Salter et al., 1999;

Tr ej balova et al., 1999; Vogt, 1965). In fact, all chicken flocks are infected with ALV by

41

sexual maturity except those genetically resistant or virus-free through successful
eradication programs (Fadly and Payne, 2003). ALV infections were reported to be more
prevalent in egg-type chickens compared to meat-type chickens.
ALV-A is the most frequent encountered ALV subgroup in field outbreaks in
many countries, followed by subgroup B ALV (Calnek, 1968). Subgroups C and D are
extremely rare in the USA but have been isolated from commercial flocks in Finland
( S andelin and Estola, 1974). Most chicken lines and some species of galliform birds
contain endogenous retroviral genes (Crittenden, 1981 and 1991; Dimcheff et al., 2000;
Robinson, 1978; Smith, 1987a). The susceptibility to infection with exogenous ALVs
increases upon congenital infection with subgroup E ALVs such as EV21 and RAV-0
( Clittenden et al., 1987; Smith and Fadly, 1988). Fadly and Smith (1999) estimated the
incidence of ALV-J infections in meat-type breeders to be 87%. ALV subgroups F and G
h ave been isolated from pheasants, while H and I from partridges and quails respectively.
F lmhermore, the incidence rates of infection with recombinant ALVS belonging to
di fferent ALV subgroups have been reported to be high in natural and experimental

settings (Gingerich et al., 2002; Lupiani et al., 2003 and 2006).

2- - Disease Incidence

ALV subgroups A, B, C and D usually induce a variety of neoplasms;
Predominantly lymphoid leukosis; less commonly erythroblastosis, myeloblastosis and
n"lb’elocytomatosis and occasionally hemangiomas, nephroblastomas, osteopetrosis and
hi Stiocytic sarcomas (Fadly and Payne, 2003; Perek, 1960; Purchase, 1987). On the other

hatld, endogenous ALVS (subgroups B) such as RAV-0 cause little or no oncogenicity in

42

chickens (Crittenden et al., 1979; Fadly and Payne, 2003; Motta et al., 1975). In the late
1980s, subgroup J ALV was first isolated and associated with myelocytomatosis in meat-
type chickens (Payne et al., 1991). It resulted in significant economic losses for broiler
breeders and poultry industry in the US and abroad (Fadly, 1998; F adly and Smith, 1997
and 1999; Payne et al., 1992a). In addition, high incidence rates of tumors mostly LL
have been observed in chickens naturally or experimentally infected with recombinant
A IVs (Gingerich et al., 2002; Lupiani et al., 2003 and 2006).

Although lymphoid leukosis (LL) is the most common neoplasm encountered in
infected flocks, its incidence varies from to l or 2% up to 20%. Extensive losses are rare,
but can occasionally be up to 30%, which have been reported in commercial layers and
breeder flocks (Fadly and Payne, 2003; Jordan, 1990; Payne and Venugopal, 2000;

Pmchase et al., 1972). In fact, the incidence of LL is influenced by chicken genetics,
in fectious bursal disease (IBD), and vaccinations with serotype 2 MDV (SBl). Following
ALV exposure at hatch, the incidence of LL is inversely correlated with the widespread
i ncidence of IBD (Purchase and Cheville, 1975) and positively correlated with serotype 2
MDV (SB-1) vaccination (Bacon et al., 1989; F adly and Witter, 1993).

Before ALV-J isolation, the incidence of myelocytomatosis was infrequent to rare
in commercial flocks. Nevertheless, the incidence of myelocytomatosis induced by ALV-
I Strain HPRS-103 was estimated to be around 27% in broilers (Payne et al., 1992a).

F adl y and Smith (1999) found losses due to tumor mortality in commercial meat-type
breeders to be 1.5% higher per week. In chickens infected with ALV-J, the incidence of
erythroblastosis, hemangiomas, nephroblastomas and connective tissue tumors varies and

can be seen either alone or along with other neoplastic conditions. Complete eradication

43

of subgroup J ALV is a very difficult due to its persistence, sporadic isolation fi'om ALV-
free broiler-breeder flocks and mixed infections with chicken anemia virus, MDV and

other poultry pathogens (Cui et al., 2003b).

Many solid tumors belonging to avian leukosis complex occur naturally in field or

experimental infections. These include connective tissue tumors, nephromas,
nephroblastoma, other epithelial, endothelial and neural tumors (Fadly and Payne, 2003).
For example, hemangiomas (25%) and renal tumors (19%) are frequently found in
broilers infected with ALV (Campbell and Appleby, 1966). Moreover, sporadic cases of

O steopetrosis occur more fiequently in males than females and some have been associated

with LL (Fadly and Payne, 2003).

3 - Transmission

ALV is naturally transmitted either horizontally (contact), vertically (congenital)

C) :- genetically. Exogenous and complete infectious endogenous ALVS are transmitted by
Contact among birds through feces, saliva, skin and fomites (Burmester and Gentry, 1954;
Cri ttenden et al., 1987; Spencer et al., 1977). For example, chickens infected in ovo or
duri mg the first 2 weeks shed ALV in saliva and meconium and spread it by contact to
hatch-mates and pen-mates (Burmester, 1956; Burmester et al., 1960). In fact, horizontal
Wan smission maintains high levels of infection within the flock, thereby increasing the
P0 S Sibility of vertical transmission. ALV-J is known to be more efficient in horizontal

trEII‘LSInission than other ALV subgroups while endogenous ALVs are less efficient

(P ayrje, 1987; Robinson and Eisenman, 1984).

44

The rate of vertical transmission is variable, but it sustains the infection between
generations. Congenitally infected chicks contribute to the early ALV horizontal
transmission and increased disease incidence in the flock (Fadly and Payne, 2003; Weyl
and Dougherty, 1977). In fact, the efficiency of congenital transmission of ALV is mainly
influenced by the fiequency and amount of virus shed in the albumen, the presence of
N abs in the yolk and the thermo sensitivity of the virus (Fadly and Payne, 2003). In
addition, the infection status and age of the dam are the two main factors responsible for
efficient congenital transmission of ALV. Viremic dams transmit ALV to progeny at high
1 evels, while infected non-Viremic dams spread it at intermittent and lower levels. The
Shedding of ALV into eggs is less consistant and at lower proportions in older (2-3 years)
than younger (<18 months) dams (Burmester and Waters, 1956).

Conversely, the sire does not influence ALV vertical transmission since the male
germinal cells can not be infected (Distefano and Dougherty, 1966 and 1968). For
example, spermatozoa were not infected and genetic transmission through semen was not

Observed when testes were directly inoculated with RAV-1 (Afanassieff et al., 1996).
M oreover, Smith and F adly (1994) suggested that the sire venereally transmits the virus
to the dam and does not directly influence the rate of congenital transmission. In some
A IV-shedding males, semen production was not reduced but abnormal spermatozoa and
I‘fi‘rduced fertility were found (Segura et al., 1988). However, the quality and quantity of

Semen production were not affected by infection with ALV-J (Hudson et al., 2002).

D. Pathobiology

l - Introduction

45

The natural host for ALSVS is the domestic chicken, but there is a wide host range
under experimental conditions. Members of L/ S group are commonly associated with
more than one type of neoplasm, a predominant type characteristic of a certain viral strain
in addition to other tumors. Most chicken neoplastic diseases usually have nonspecific

clinical signs such as inappetance, weakness, diarrhea, dehydration and emaciation
(Fadly and Payne, 2003). Birds with LL sometimes have abdominal enlargement, pale
shriveled and cyanotic combs (Kahn, 2005). In myelocytomatosis, some chickens have
protuberances on the head, thorax and shanks, hemorrhage in the orbits of the eyes and
blindness. In addition, hemorrhage from the feather follicles may occasionally occur in
erythroblastoisis and myeloblastosis while hemangiomas are characterized by blood
blisters, which may burst and cause hemorrhage in the skin. Renal tumors sporadically
cause paralysis while osteopetrosis result in stunted pale limping birds.
The incubation period for diseases induced by ALSVS depends on virus strain and
d cse, route of and age at exposure and host genetic makeup (F adly and Payne, 2003).
A cute transforming viruses such as RSV induce tumors (4-7 weeks) more rapidly than
s 1 (3w transforming viruses (14—20 weeks) such as ALV (Beard, 1963; Fadly and Smith,
1 9 99; Kung and Maihle, 1987; Payne et al., 1993). Moreover, certain laboratory
recombinant viruses cause LL within 5-7 weeks (Kanter et al., 1988), while naturally
0c curring recombinant ALV induced LL within 30 weeks postinoculation (Lupiani et al.,
2 006; Mays et al., 2006). The incubation period of ALSVS is inversely proportional to
the virus dose and host age at exposure (Beard, 1963; Burmester et al., 1959b and 1960;

Eel-(e11 et al., 1954; Franklin and Martin, 1980). Intravenous (IV) inoculation of birds

46

induces earlier tumors and mortality than intrabdominal (IA) inoculation (Burmester et

al., 1959a and 1960; Fadly and Payne, 2003; Mladenov et al., 1967).

2. Pathogenicity

There are many factors influencing ALSV pathogenicity in chickens such as virus
subgroup, strain, dose, and route of exposure, host sex, age at exposure and genotype,
environmental factors and stress. ALV subgroup affects tumor incidence and type, which

mainly depend on LTR (Payne, 1987) and envelope respectively (Brown and Robinson,
1 988; Chesters et al., 2002; Jaffredo et al., 1993). Commonly, all ALV subgroups
predominantly cause tumors of lymphoid lineage except subgroup J, which mainly results
in tumors of myeloid lineage. Conventionally, most viral strains were named after the
predominant tumor they induce at high doses such as RSV (sarcomas), AMV
(myeloblastosis) and AEV (erythroblastosis). However, viral strains within a certain ALV
subgroup have varying capacities to induce LL, myeloid leukosis (ML) or other tumors.
The inoculated viral dose greatly influences the type of tumors produced. For
ex ample, high doses of certain ALV—A strains predominantly induce erythroblastosis
C B mmester eta1., 1959a) whereas low doses predominantly induce LL (Purchase, 1987).
In general, the route of exposure to ALV determines the effective viral dose and the type
0 f target cell exposed to the virus. For instance, subcutaneous or intramuscular
inoculations with ALV primarily induce sarcomas and LL while intravenous inoculation
I'11()stly induces erythroblastosis and myeloblastosis (Fredrickson et al., 1965).

Furthermore, resistance to tumor development is acquired more quickly when exposure to

47

the virus is through the natural fecal-oral routes than parenteral routes due to the low
effective viral dose (Burmester et al., 1959a).

The gender of the host has an important effect on the incidence of certain tumors.

Male chickens are less susceptible to LL than female and castrated male chickens
probably due to the protective nature of testosterone, which results in the regression of
target cells (Burmester and Nelson, 1945; Cooper et al., 1968). On the other hand, males
appear to be more susceptible to osteopetrosis than females (Smith, 1987a). ALV-J
induces myelocytomatosis, several types of leukemias and sarcomas, which are
independent of the sex of the host.

The host age at exposure influences both the course of infection and disease
incidence (Piraino et al., 1963; Rubin and Vogt, 1962; Rubin et al., 1962). Younger hosts
(up to 2 weeks) tend to be more susceptible to viral persistence and tumor mortality than

01 der hosts. This is certainly true in infections with subgroup J ALV and certain ALV
Strains such as RPL12 (Burmester et al., 1960; Witter et al., 2000).

The genotype of the host has a strong influence on resistance to cell infection and
transformation. Genetic resistance to viral infection is dictated by the absence of specific
Viral receptors on surface of host cells. Commercial chicken lines are characterized by a
Variable frequency of alleles encoding cellular resistance to infection (Crittenden and
Mona, 1969; Motta et al., 1973). Onthe other hand, resistance to cell transformation
depends on many alleles and varies among chicken lines (Purchase, 198 7). For example,
1 in e 6 chickens are resistance to tumor formation while line 7 is susceptible.

Several environmental factors have an effect on the incidence of certain tumor

typ 68. For example, factors that deplete bursa of Fabricius may reduce LL incidence but

48

may increase the incidence of osteopetrosis. Certain management practices such as
manual vent sexing and lack of biosecurity increase the rate of virus exposure and tumor
incidence. Environmental factors inducing stress increase tumor incidence by negatively

influencing the immune system of the host (Fadly et al., 1989).

3. Viral and Cellular Oncogenes

Tumors in chickens can result from the direct transcription of viral oncogenes in
acute transforming ALSV viruses. On the other hand, they can be induced by activation
and overexpression of cellular oncogenes through insertional mutagenesis of slow
transforming viruses. Viral oncogenes (v-onc) are usually expressed at high levels while
cellular oncogenes (c-onc) are expressed at low levels. Viral oncogenes can encode
growth factors, growth factor receptors, transcription factors or various proteins of signal
transduction cascades. These proteins normally function in normal cell growth,
proliferation and differentiation (Gordon, 1985; Lewin, 1994). Viral oncogenes are
associated with cellular oncogenes through transduction subsequent to proviral DNA
integration into the host DNA. This integration sometimes leads to rearrangement of viral
geneome and the consequent incorporation of a cellular oncogene.

Some examples of v-oncs in avian retroviruses include src, myc, myb and erb.
Rous sarcoma virus and its associated v-src were the first to be identified and

Characterized (Bishop and Varmus, 1982). In fact, c-SRC protein is membrane associated
IIon-receptor or intracellular tyrosine kinase (Lewin, 1994). It is mainly involved in the

regulation of cell proliferation, migration, differentiation and survival (Brown and

49

Cooper, 1996). On the other hand, v-SRC protein causes unrestricted cell proliferation
and leads to tumor formation.

The v-myc was originally identified in the avian myelocytomatosis virus MC29
associated with myelocytomatosis in chickens. MC29 is a transforming defective

retrovirus lacking part of gag, all of pol and most of env (Enrietto and Hayman, 1987;
Reddy et al., 1983; Sheiness et al., 1978). The porotoncogene c-myc is a highly regulated
gene, which belongs to the myc family of nuclear DNA-binding/transcription factor genes
(Sheiness et al., 1980). The c-MYC protein functions as a transcription factor, interacts
with other proteins and targets genes that regulate and control gene expression and cell
growth (Dang, 1999). Altered expression of disrupted c-myc has been associated with LL
upon infection with ALV-A or B mainly in layers and ML in broilers infected with ALV-
J. In addition, overexpression of v-MYC causes uncontrolled cell proliferation and blocks
terminal differentiation of hematopoietic cells (Lee and Reddy, 1999).

Avian myeloblastosis virus (AMV) and avian E26 leukemia virus contain the
oncogene v-myb responsible for transformation of myeloid cells (Duesberg et al., 1980;
Introna et al., 1990). The protooncogene c-myb encodes a transcription factor, which is
involved in cell cycle regulation via control of intracellular calcium concentration
(Introna et al., 1994; Oh and Reddy, 1999). Moreover, c-myb is highly expressed on all

immature cells of hematopoietic and lymphoid systems and its expression is down
regulated during differentiation (Duprey and Boettiger, 1985; Golay et al., 1997).
Truncation of N-terminal of c-myb is highly transforming while C-terminal truncation is

Weakly transforming (Lipsick and Wang, 1999). Overactivation of myb causes LL upon

50

infection with ALV subgroups A or B mainly in egg type chickens (Kanter et al., 1988;
Pizer and Humphries, 1989).

Avian erythroblastosis virus (AEV) contains v-erbA and v—erbB, which are
responsible for full transformation of erythroblasts (Lewin, 1991). The v-erbA encodes a
nuclear protein belonging to the superfamily of nuclear hormone receptors. The v-erbB
encodes a truncated form of the epidermal grth factor (EGF) receptor with a
constitutively activate tyrosine kinase function (Kris et al., 1985). The cellular homologs
of v-erb and v-erbB encode the nuclear receptor for thyroid hormone (Sap et al., 1986;
Weinberger et al., 1986) and the epidermal growth factor receptor respectively (Sawyers
et al., 1991). Slow transforming ALV strains induce the activation of the cellular
oncogenes erbA and erbB by LTR insertion (F adly and Payne, 2003). This causes
maturation arrest, proliferation and transformation of erythroblasts and results in

erythroblastosis (erythroid leukosis) in chickens.

4. Nonneoplastic Conditions
Infection of chickens with ALSV results in several nonneoplastic conditions and
many neoplastic diseases. Some of the nonneoplastic conditions are caused by certain
strains of ALSV whereas others are more generic. At an early age, subclirrical infection
Of chickens with high doses of ALV is characterized by absence of clinical signs, low
Viral loads at fewer sites in the body and detectable antibody response (Arshad eta1.,
1 997). In addition, infection with subgroup J ALV sometimes causes depressed body

Weight, reduced growth rate, stunting, non-uniform flock size in broiler breeders and

51

 

meat-type chickens probably due to low feed consumption, conversion and efficiency
(Crittenden et al., 1983; Gavora, 1987; Gavora et al., 1982; Stedman and Brown, 1999).

In ALV infections, the productivity of egg-laying chickens is negatively affected
and observed as reduced rate, size, quality (softer shells), number, fertility and
hatchability of eggs, delayed sexual maturity and age at first egg in addition to
nonneoplastic mortality (Fadly and Payne, 2003; Gavora et al., 1980). In roosters
shedding ALV, the semen quality and fertility are reduced but the semen quantity is not
affected (Segura et al., 198 8). On the other hand, ALV-J infection does not influence the
quality or quantity of semen production (Hudson et al., 2002). Congenital infection with
subgroup J ALV sometimes resulted in depressed body weight in broilers due to
decreased serum thyroxine T4 levels (Brown et al., 2000).

Other nonneoplastic conditions in experimental ALV infection include immune,
circulatory and nervous system disorders. Early exposure to RAV-1 (Rous-associated
virus subgroup A), myeloblastosis-associated virus or avian osteopetrosis virus MAV-2
(O), RAV-60 (subgroup E) and ALV subgroups B and D results in anemia, hepatitis,
immunodepression, ascites and wasting in chickens (Crittenden et al., 1982; Cummins
and Smith, 1988, Smith and Van Eldik, 1978). Irnmunodepression is exhibited as atrophy
of lymphoid organs and tissues, lower antibody production and reduced mitogen-induced

lyrnphoblastic response (Smith, 1987b).

Infection with RAV-1 (subgroup A) sometimes causes myocarditis and chronic

circulatory syndrome in adult chickens (Gilka and Spencer, 1990). Moreover, chickens
itloculated with RAV-7 (subgroup C) can show neurological signs of infection such as

a~taxia, lethargy and imbalance. Moreover, RAV-7 results in stunting, atrophy of the

52

 

lymphoid organs, obesity (high cholesterol and triglycerides), hypothyroidism (lower
thyroid hormones) and increased insulin levels (Carter and Smith, 1983; Carter et al.,
1983; Fadly and Payne, 2003). Congenital infection with subgroup J ALV is sometimes

associated with abnormal feather development in broilers (Landman et al., 2001).

5. Neoplastic Diseases

The various neoplastic diseases induced by members of the US group depend on
many factors. These factors are the site of integration of slow transforming ALVs in the
host’s genome, the type of oncogene carried by acute transforming ALVs and ALV
envelope glycoproteins. Generally, ALV infection can cause different kinds of benign or
malignant tumors affecting lymphoid, myeloid, epithelial (carcinoma) or endothelial
(sarcomas) cells and tissues.

L/S associated neoplastic diseases include lymphoid leukosis (LL),
myelcytomatosis (myeloid leukosis, ML), myeloblastosis, erythroblastosis, connective
tissue tumors such as fibroma and fibrosarcoma, osteoma and osteosarcoma, myxoma
and myxosarcoma, chondroma and chondrosarcoma, renal tumors including nephroma
and nephroblastoms, adenoma and carcinoma, hemangioma, hemangiosarcoma,
osteopetrosis and other rare neoplasms (Fadly and Payne, 2003). The most commonly

Occurring neoplasms associated with ALV-related viruses. in chickens are LL, ML,
myeloblastosis and erythroblastosis.

Lymphoid leukosis is usually observed in chickens older than 14 weeks of age,

peaking at sexual maturity (Fadly and Payne, 2003). Thus, it is a significant problem for

broiler breeders and layers, but not commercial broilers. LL is usually caused by slow

53

transforming ALV strains such as subgroups A and B. Although subgroup J ALVS are
seldom associated with LL (Payne, 2000), bursal transformation was detected in infected
white leghoms (Williams et al., 2004).

Clinical signs of LL are non-specific such as emaciation, pale or cyanotic comb
inappetance, enlarged abdomen and organ dysfimction leading to death. LL is often
associated with gross or/and microscopic lesions in the bursa of Fabricius and other
visceral organs. The principle bursal cells for ALV infection are the medullary
macrophages, which transmit infection to lymphoid cells (Gilka and Spencer, 1987). The
target cells for transformation are bursal lymphocytes or stem cells expressing surface
IgM (Purchase and Gilrnour, 1975).

ALV provirus is often inserted next to a cellular oncogene in infected B
lymphocytes commonly c-myc and rarely c-myb (Kanter et al., 1988; Pizer and
Humphries, 1989). This insertion allows the enhancer elements in the U3 region to drive
the transcription, activation and overexpression of cellular oncogenes. The altered
expression of the oncogene products causes proliferation of B cells and maturation arrest
of transformed B cells blocking intraclonal Ig class switch from IgM to IgG (Crittenden
and Kung, 1984). Progression of the transformed follicle is mediated by additional
genetic changes and other cellular oncogenes such as Blym-I (Goubin et al., 1983) and c-
bic (Clurman and Hayward, 1989).

In chickens infected in ovo or at hatch, hyperplasia or neoplastic transformation of

individual bursal follicles can be observed as early as 4 weeks in experimental studies
(Nieman et al., 1980). By 7 weeks, these chickens have one or more abnormal, altered or

ti‘ansformed bursal follicles (Cooper et al., 1968). However, most of the hyperplastic and

54

neoplastic follicles regress due to anti-tumor immune response (Purchase, 1987). After 12
weeks, transformed B cells in the clonal bursal tumors metastasize to different viscera
such as the liver (mostly), spleen, kidney, lung, gonad, heart, bone marrow, digestive
tract and mesentery leading to terminal disease (Fadly and Payne, 2003).

Myelocytomatosis occur naturally in immature susceptible chickens upon
infection by acute or slow transforming strains of ALV (Purchase, 1987). The incubation
period is usually variable depending on ALV strain and is often shorter than LL, but
longer than erythroblastosis and myeloblastosis. Acute transforming ALV strains cause
myelocytomas within 4-6 weeks (Chester et al., 2001), while slow transforming ALV
strains such as HPRS-103 have a latent period of about 9-20 weeks in meat-type chickens
(Payne, 1998). Acute transforming strains of ALV such as MC29, CMII and 966 carry v-
myc and induce neoplastic transformation of the target myelocyte (Enrietto and Hayman,
1987; Moscovici and Gazzolo, 1987). On the other hand, slow transforming strains of
ALV such as HPRS-103 and ADOL-He] induce transformation of the target cell by
insertional activation of c-myc (Chester et al., 2001; Fadly and Payne, 2003).

Clinical signs include bony protuberances on the skull, ribs and other periosteal
surfaces. Gross lesions are characterized by moderate to massive enlargement of the liver
and myelocytomas of the bones in the skull, sternum and pelvis (Mladenov et al., 1967).

Gross and microscopic lesions are usually observed in the bones and some internal
Organs. The target cell for infection and transformation is the myeloid stem cell in the
bone marrow. The neoplastic myelocytes arise from the bone marrow stem cell,
proliferate and infiltrate the bone marrow sinusoids. Tumors are generally found in the

bvones, liver, spleen, ovary, thymus, oral cavity, trachea and eye (Fadly and Payne, 2003).

55

Natural field cases of myeloblastosis are rare and are generally seen in adult
chickens prior to 24 weeks of age (Purchase, 1987). Nevertheless, acute transforming
strains of ALV such as E26 and BAI-A AMV can cause the disease within 10 days
(F adly and Payne, 2003). Moreover, ALV subgroups A, B, and J are usually associated
with myeloblastosis. The target cell is the myeloblast in the bone marrow and the v-myb
oncogene of AMV or insertional activation of c-myb is responsible for its transformation
(Enrietto and Hayman, 1987).

Differentiation of the transformed cell is arrested and proliferation occurs in the
bone marrow. This is followed rapidly by leukemia and infiltration of different parts of
the body such as the liver, spleen, kidney and other organs (Purchase, 1987). Clinical
signs are similar to LL in addition to severe leukemia, thrombocytopenia and anemia
(Payne, 1992). Gross lesions are characterized by massive enlargement of the liver,
mottling of the visceral organs and presence of solid, yellowish-gray tumor nodules in the
bone marrow (Purchase, 1987).

Erythroblastosis usually occurs between 12-24 weeks of age in susceptible
chickens. It is not restricted to a certain subgroup, but its incidence in ALV-J infections is
variable. However, erythroblastosis incidence is high in chickens experimentally infected
with acute transforming stains of ALV-J such as strain 966 (Venugopal et al., 2000). In
slow transforming ALV infections, the incidence of erythroblastosis depends on the

proviral LTR insertional activation of a cellular oncogene c-erbB (Fung et al., 1983;
Kurth and Bauer, 1972). On the other hand, some acute transforming viruses possess the

Viral oncogene V-erbA or v—erbB such as avian erythroblastosis viruses (AEV) isolates

56

ES4 and R. These ALV strains induce erythroblastosis and mortality after a short
incubation period of 7-14 days (Graf and Beug, 1978).

The target cells for transformation are the erythroblasts in the bone marrow
sinusoids (Purchase, 198 7). The altered expressions of the cellular and viral oncogenes
lead to proliferation, maturation arrest and transformation of the target cells. The
transformed erythroblasts enter the circulating blood causing erythroblastic leukemia and
metastasize to visceral organs forming solid tumors in the liver and spleen. Moreover,
erythroblasts continue to accumulate in different organs until death of the host (Ponten
and Thorell, 1957).

Clinical signs of erythroblastosis are similar to LL in addition to anemia. The
most characteristic gross lesions are diffuse enlargement of the liver, spleen and
sometimes the kidneys. These organs are usually soft and cherry red to mahogany in
color due to infiltration of neoplastic erythroblasts (Fadly and Payne, 2003). The affected
birds are anemic and have petechial hemorrhages in various visceral organs (Jordan,
1990). The bone marrow is replaced by proliferating erythroblasts and is hyperplastic,

semi-solid and red in color.

E. Immunology
1. Humoral Immunity
Humoral immune response plays vital role in protection against ALSV infection
tl‘rrough reducing virus replication and neoplasia. The source of passive immunity in
Chicks is maternal antibodies (IgG), which are passed from the hen through the egg yolk.

I t lasts 3-4 weeks after hatch and delays the onset of ALV infection, diminishes the levels

57

of viremia and shedding, and eventually reduces the incidence of tumors (Burmester,
1955; Fadly, 1988; Witter et al., 1966).

Most chicks develop transient viremia and neutralizing antibodies (N abs), which
persist for life after infection with ALV (Rubin et al., 1962; Solomon et al., 1966). The
age of the bird at exposure to ALV is inversely proportional to the duration of viremia
and the delay in development of Nab. When birds are infected before or 2 weeks post
hatch, they develop permenant viremia and no Nabs and are referred to as tolerant
viremics. In fact, seroconverted and tolerant virernic chickens are both susceptible to
tumor mortality. However, the incidence of tumors in tolerant Viremic birds is likely to be
higher than in immune birds.

After chickens are infected with ALV, antibodies are raised against different viral
proteins and have different roles in protection against the virus. For example, Nabs are
directed against virus envelope glycoproteins namely SU (gp85) and TM (gp37). They
are highly specific for each subgroup as well as strains within each subgroup (Meyers,
1976; Qualtiere and Meyers, 1983; Vogt and Ishizaki, 1965). They restrict the amount of
ALV circulating and shedding in birds and thus limit tumor formation. Antibodies
produced against gsa (p27) are not likely to participate in virus neutralization and have no
effect on tumor growth (Roth et al., 1971; Sigel et al., 1971). Moreover, antibodies
against RT have been detected in ALV infected and uninfected chickens (Graevskaya et
al., 1982). Chickens with LL usually have high titers of IgM, which do not protect

against ALV (Cooper et al., 1974; Smith et al., 1980).

2. Cell-Mediated Immunity

58

 

 

In ALSV infections, cell-mediated immunity (CMI) is thought to be directed
against virus antigens and tumors. Cytotoxic T lymphocytes (CTL) against viral envelope
antigens were observed in some ALV infections (Bauer et al., 1976; Kurth and Bauer,
1972; Schat, 1987). Moreover, studies on regressing avian sarcomas demonstrated that T
cells are important in CMI against ASV-induced tumors (Perry et al., 1978; Wainberg et
al., 1979). Chicken MHC class 1 amino acid residues were shown to be essential in
antigen presentation to ALV-induced CTL responses (Fulton et al., 1995; Thacker et al.,
1995). In addition to T cells, natural killer (N K) cells and macrophages were implicated

in anti-tumor immunity (Qureshi and Taylor, 1993; Wainberg et al., 1983 and 1987).

F. Genetic Resistance
1. Cellular Resistance to Infection

Cellular resistance to virus infection is one of two levels of genetic resistance to
ALSV-induced tumors. In fact, cellular responses to infection by ALSV subgroups are
controlled by independent autosomal loci, which are inherited in a simple Mendelian
fashion (Crittenden, 1975). In 1994, the Poultry Committee of the United States
Department of Agriculture (USDA) National Animal Genome Research Program has
adopted a new nomenclature system for genes controlling cellular susceptibility to
ALSVs (F adly and Payne, 2003). The new gene designations are tumor virus followed by
the subgroup such as TVA, TVB, TVC and controversial TVE (Adkins et al., 2000; Bates
et al., 1998; Crittenden, 1991; Crittenden et al., 1973; Pani, 1974 and 1976).

In chickens, TVA and TVC loci are genetically linked and TVB locus controls

responses to subgroups B and D ALVS (Crittenden and Motta, 1975; Pani, 1975; Payne

59

and Pani, 1971 ). Suceptibility to ALV subgroup E depends on the interaction between
genes at two loci, TVE and 1°. Moreover, I" is a dominant resistance gene, which acts
epistatically and blocks susceptibility by TVE locus (Payne and Pani, 1971). However,
some studies suggested that I“ is an ev locus encoding ENV glycoproteins, which block
ALV subgroup E receptor (Robinson et al., 1981). Genetic resistance to cellular infection
by subgroup J ALV has not yet been documented in chickens. Nevertheless, several avian
species are known to be resistant to ALV J infection such as pheasants, Japanese quail,
guinea fowl, ducks and geese (Payne et al., 1991 and 1992b).

Alleles for susceptibility encode subgroup-specific virus receptors on host cell
surface. In susceptible cells, the subgroup-specific virus receptors interact with viral
envelope glycoproteins and allow viral penetration and cell infection. Alternatively, no
subgroup-specific virus receptors are expressed by resistance alleles. At each locus,
susceptibilty alleles (S) are always dominant over resistance (R) alleles (Crittenden,
1968). Host cells or birds can be resistant to infection by one or several members of ALV
subgroups. The phenotype is designated according to the cell or bird species (C for
chicken) and the subgroup to which they are resistant U).

The phenotype C/A denotes a cell or chicken resistant to ALV subgroup A
infection and susceptible for the other subgroups. On the other hand, the C/O phenotype
denotes cells or chickens susceptible to all ALV subgroups. Currently, there are no
chickens or chicken cells naturally resistant to subgroup J ALV infection. However, a
new genetically engineered cell line, DF-l/J (C/EJ) was developed to be resistant to

subgroup J and E ALV infection (Hunt et al., 1999). These cells and chickens can be

60

considered useful diagnostic tools for screening, isolation and identification of field and

laboratory isolates of ALSV.

2. Resistance to Tumor Formation

The second level of genetic resistance to ALSV-induced tumors is that to tumor
induction and formation. Genetically resistant birds can not be infected or develop tumors
when exposed to a certain ALSV subgroup (Crittenden, 1975; Crittenden and Okazaki,
1966). Regression of Rous sarcoma is controlled by R-RS-I, a dominant gene located in
the BBL region within the chicken MHC complex or B-haplotype (Collins et al., 197 7 ;
Gebriel et al., 1979; Schierman et al., 1977). MHC and non-MHC genes in the B-L/B-F
regions or closely linked regions may be responsible for Rous sarcoma regression
(Auclair et al., 1995; Taylor J r., 2004). It is believed that these genes induce tumor
regression through immune recognition of a tumor-specific antigen or by influencing
viral replication. Genetic resistance to LL tumor development has been associated with
MHC locus of B erythrocyte antigen (Ea-B), non-MHC lymphocyte locus (Th-1) and
immunoglobulin genes (Bacon et al., 1981, 1985 and 1986). Nevertheless, the basis for
resistance to LL tumor formation seems to be the inherent inability of the bursal target

cells to be infected or transformed (Purchase et al., 1977).

G. Diagnosis
1. Virus Isolation and Identification
ALV infections are detected by assays for virus isolation in infected commercial,

pathogen-free and breeder flocks. ALV detection tests can also identify and classify new

61

isolates and test vaccine safety. There are various sample materials suitable for ALV
detection. The most common ones are blood, plasma, serum, meconium, cloacal and
vaginal swabs, oral washings, egg albumin, embryos and tumors (Crittenden and Smith,
1984; Fadly, 2000; Fadly and Witter, 1998; Fadly et al., 1981a; Spencer et al., 1977).
ALV can also be isolated from saliva, feces (Burmester, 1956), feather pulp (Spencer et
al., 1983) and semen (Segura et al., 1988).

Most ALV diagnostic tests depend on the detection of specific gene products or
proviral DNA or viral RNA sequences. Currently, virus isolation is carried out in cell
culture, which is fast, economical and suitable for large-scale use. Since most ALV
strains do not produce cytopathic changes in cell cultures, indirect virus detection
methods are essential. Different biological assays can be used to detect ALV p27, the
most common of which is ELISA (Crittenden et al., 1987; Fadly and Witter, 1998; Smith
et al., 1979; Tsukammoto et al., 1991). Commercial ELISA kits for p27 detection are
available, but in-house preparation is possible using commercially available anti-p27
antibodies and conjugates.

In all biological assays, using the correct chicken embryo fibroblasts (CEFs) with
specific host range is necessary. To detect any ALV, CEFS that are susceptible to
infection with all ALV subgroups (C/O) are employed. On the other hand, CEFS of
genotype C/E, which are resistant to infection with endogenous ALV are necessary to
isolate and detect exogenous ALVS. Moreover, different CEFS and cell lines that are
resistant to different ALV subgroups can be utilized to confirm the subgroup of the
isolated ALV such as alv6 (C/AE), 72 (C/ABDE) and DF -1/J (C/EJ) cell line (Crittenden

and Salter, 1990; Fadly and Payne, 2003; Hunt et al., 1999; Salter and Crittenden, 1991).

62

They have also been used to isolate and characterize recombinant ALVS fi'om field and
laboratory isolates (Gingerich et al., 2002; Lupiani et al., 2003 and 2006).

Several tests have been utilized for the detection of ALV nucleic acids such as the
widely used polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-PCR).
PCR-based methods have been reported for detection of different subgroups of ALV
isolated from contaminated vaccines, infected cell cultures, plasma, tumors, feather
follicles and eggs (Gingerich et al., 2002; Hauptli et al., 1997; Pham et al., 1999; Silva et
al., 2000 and 2007; Smith et al., 1998a and b; Van Woensel et al., 1992; Zavala et al.,
2002). Furthermore, different primers specific for different ALV subgroups were used to
identify the nature of recombinant ALVs (Lupiani et al., 2000). Most ALV sequences
used for designing PCR primers are located in the env and LTR regions (Fadly and
Payne, 2003). If mutations occur where the developed primers anneal, the desired
sequences will not be amplified, not all variant viruses will be detected and false

negatives will be reported.

2. Serology Tests

For the detection of antibodies to ALV, samples of plasma, serum or egg yolk can
be utilized (Fadly and Payne, 2003). Many cell culture assays based on the reaction
between the antibodies and ALV or RSV have been described such as ELISA (Mizuno
and Itohara, 1986; Smith et al., 1986; Tsukamoto et al., 1985 and 1991) and flow
cytometry (Hunt et al., 1999). In fact, several ELISA kits for detecting antibodies to
subgroups A, B and J are commercially available (Fadly and Payne, 2003; Lee et al.,

1986; Venugopal et al., 1997). The most commonly used virus neutralization (VN) test

63

assays for residual virus and indirectly detect antibodies to ALV (Fadly and Witter,
1998). This rrricroneutralization test has been used in many studies to detect and
quantitate antibodies to different ALV subgroups including recombinant ALVs (F adly
and Smith, 1999; Lupiani et al., 2003, Mays et al, 2005 and 2006; Venugopal et al.,

2000; Witter et al., 2000)

3. Differential Diagnosis

Differential diagnosis of lymphomas in chickens is essential for two important
reasons. First, different avian oncogenic viruses may cause similar symptoms, tumors and
lesions. For example, lymphoid tumors may be observed in Marek’s disease (MD), LL
and Reticulendotheliosis virus (REV) infections (Fadly, 1987 and 1997; Purchase and
Sharma, 1973; Wakenell, 1997). In addition, avian oncogenic viruses are widespread in
poultry even in the absence of tumors. This renders standard virological, serological and
molecular assays of little value in diagnosis of virus-induced lymphoma. However,
molecular assays such as Southern blots and PCR tests for detection of proviral DNA in
tumors can be helpful (Fadly, 1997). In most cases, differential diagnosis is based on
flock history, age, clinical signs, gross and microscopic lesions and cytological
examination of tumors including target cell characteristic and cell surface markers (Fadly

and Payne, 2003).

H. Intervention Strategies

1 . Immunization and Treatment

64

Vaccination is often used to elicite an efficient immune response and to offer
protection to chickens against ALV infections and induced lymphoma. On the other hand,
congenitally infected chicks are immunologically tolerant and thus are not good
candidates for immunization. Moreover, they are Viremic and they shed and spread ALV
to other chicks. In fact, congenital transmission of ALV is best prevented by maternal
antibodies passed fi'om the hen to the progeny. Several attempts have been made to elicit
similar immune response, but none has been promising so far. The use of replicating
retrovirus as a vaccine is not reasonable because of its integration into the host genome
and the increased chance of congenital transmission. On the other hand, Burmester
(1952) showed that viruses in inactivated ALV vaccines lost their antigenicity and their
ability to elicit an immune response.

Induction of immunity in chickens through the use of CEFS or quail QT6 cells
expressing ALV envelope glycoproteins was not successful (Chebloune et al., 1991;
Hunt et al., 1999). The efficacy of some subunit vaccines containing envelope
glycoprotein gp85 in different expression systems was not satisfactory (Lee et al., 1998;
Nazerian and Yanagida, 1995 ; Notebom et al., 1990). Nevertheless, Bennett and Wright
(198 7) reported some protection of subunit vaccines containing viral envelope
glycoproteins against RSV—induced sarcomas.

Recombinant live ALV vaccines expressing exogenous ALV env gene and LTRs
showed some protection (Robinson et al., 1985). However, recombinant viruses in these
vaccines may persist in chickens or mutate into more pathogenic or virulent forms.
Moreover, recombinant subunit vaccines made of immunogenic endogenous envelope

gp85 proteins can induce some degree of protection. On the other hand, they may result

65

in decreased Nabs production or immunological tolerance to exogenous ALV (Crittenden
et al., 1984). Therefore, recombinant ALVs expressing exogenous gp85 proteins might
be successful in controlling ALV in commercial poultry operations (F adly and Payne,

2003). Thus far, there are no treatments of virus-induced neoplasms of chickens.

2. Prevention and Control by Eradication

To date, the most effective means for controlling ALV infection in chickens is
eradication. Previously, ALV eradication was only applied to experimental and specific-
pathogen free (SPF) flocks used for vaccine production. Development of more feasible
and practical testing methods made eradication possible in broader range of flocks
including commercial flocks (Chase, 1991; Okazaki et al., 1975; Payne and Howes,
1991; Rispens et al., 1970; Spencer, 1997; Spencer and Gilka, 1982). So far, several
breeding companies have significantly reduced or eradicated ALV subgroups A, B and J
from their lines (F adly and Payne, 2003).

ALV eradication programs maily depend on the elimination of vertical and
horizontal transmission of ALV in all flocks. Therefore, eradication protocols involved
accurate ALV diagnosis in hens, hatching eggs, embryos and chicks (Fadly and Payne,
2003). Several test materials are used for ALV detection, but vaginal swabs, egg albumin
and plasma are most commonly used. Detection of ALV-infected chicks is facilitated by
testing with more than one technique such as biological assays and PCR. ALV detection
is followed by culling of ALV-positive dams, hatching and rearing ALV-free chickens in

total isolation in small groups (Spencer et al., 1977).

66

ALV-positive hens are culled by frequent testing of adequate number of samples
for viremia, antibody and shedding in eggs (albumin gsa) and embryos (Fadly and Payne,
2003). Negative dams are selected for producing ALV-free fertile hatching eggs by
vaginal swabs and albumin tests and include immune non-shedders and non-immune
non-shedders and non-immune or immune non-Viremic (Fadly et al., 198 1 a; Hughes et
al., 1963; Zander et al., 1975).

To avoid contact transmission, ALV-free chicks are hatched and reared in small
groups in isolation following biosecurity procedures (Fadly et al., 1981b; Witter and
Fadly, 2001). Groups of ALV-free chicks are selected for producing future generations
through discarding the entire lot containing ALV-positive chicks (Okazaki et al., 1979).
In general, chicks are more susceptible to infection immediately after hatching from
congenitally infected hatchmates and later fiom penmates, fomites and the environment.

Good management practices should be implemented to reduce or eliminate ALV
transmission from various sources such as other chicks and the enviromnent (Fadly and
Payne, 2003). General management procedures to be implemented are many and include
using eggs and chicks from one source, using disposable chick boxes, rearing one
chicken age group, mechanical vent sexing, MDV vaccination with separate needles and
rearing on wire-floored cages. Furthermore, all farm equipment including the ones used
‘ for incubation, hatching, and brooding of fertilized eggs and chicks should be thoroughly

fiimi gated, cleaned and disinfected with detergent after each use.

3. Selection for Genetic Resistance

67

Artifical selection for the two levels of genetic resistance is difficult in chickens
for several reasons. Cellular resistance to virus infection is mainly controlled by single
dominant susceptibility alleles to certain ALV subgroup. Alternatively, cellular resistance
alleles are recessive and more prevalent in meat-type chickens (Crittenden and Motta,
1969). Moreover, commercial chicken lines differ in the frequency of resistance alleles
and breeding for resistance mostly depends on crosses between several lines (Crittenden,
1983). On the other hand, genetic resistance to tumor development is controlled by
multiple alleles not related to ALV subgroups (Crittenden, 1975). Selection for resistance
to ALV infection may be overcome by mutations while resistance to development of
neoplasms is based on selection for host viability (Fadly and Payne, 2003).

Artifical poultry breeding is aimed toward selection for genetic resistance to the
predominant subgroup A and sometimes subgroup B ALV. For example, one primary
broiler breeder company has successfully selected ALV subgroup A-resistant chickens
(McKay and Rosales, 2000). Moreover, the search for chicken lines genetically resistant
to ALV-J infection has failed thus far. During ALV-J eradication, culling tolerant
virernic birds from the already limited broiler breeder stocks may result in the loss of
genes linked to some production traits (McKay, 1998). Crittenden suggested that the
control of ALV infections will be possible by the development of ALV resistant breeder

stocks by transgenesis (1991).

68

 

Lipid
Bilayer

Figure 1.1. Schematic diagram of a retrovirus virion (Avian leukosis virus, particle). The
viral envelope is a lipid bilayer expressing the gp37 transmembrane (TM) and the gpSS
surface (SU) glycoproteins, encoded by the env gene. Internal components encoded by
the gag/pro gene are p19 matrix (MA) protein, p27 capsid (CA) protein, p12
nucleocapsid (NC) protein, and p15 protease (PR). The pol gene encodes the reverse
transcriptase (RT) and p32 integrase (IN). The core of the particle contains 2 viral RNA
strands. Adapted from Diseases of Poultry, 11th edition (Fadly and Payne, 2003).

69

RNA VIRUS
R US gag/pro pol env U3 R

5’ CAP I..-l 1' i _ E3 AAA3’

Controlling Coding sequences

 

 

 

Controlling
sequences sequences
Reverse transcription
& integration
‘0'?!"
DNA PROVIRUS 1?
5’ LTR 3’ LTR Host

U3 R US gag/pro pol env U3 R U5 DNA

u .
‘ - . 1
P....-.—..._~... -— . no.4.) . . .—

   

 

 

 

 

 

Controlling Codrng sequences Controlling
/ Transcription
sequences , \ sequences
15v"; '3' i I Kai \‘a it
55. $135 ‘94}; “Nit-5
' t t s.
, Messenger RN As Progeny viral
Translation 2 l genomic RNA
. ,1. ~.
Structural core proteins: Reverse Envelope
p19 (MA), p10 transcriptase (RT proteins:
p27 (CA), integrase (1N) gp85 (SU),
. p12 (NC). gp37 (TM)
p15 protease (PR)

Figure 1.2. Key features of viral genomic RNA and proviral DNA of ALV genome. The
RNA genome of ALV has a methylated nucleotide CAP at the 5’ end and a poly (A) tract
(AAA) or polyadenylation at the 3’ terminus. It is surrounded by repeat (R) sequence on
both sides. It also contains unique 5’ end sequence (U5) and unique 3’ end sequence

(U 3). These three regions form the long terminal repeat (LTR) flanking the DNA
provirus. ALV also contains three genes (gag/pro, pol and env) encoding various
structural core proteins, viral enzymes and envelope glycoproteins respectively. For other
abbreviations, see Figure 1.1 and the text. Adapted from Diseases of Poultry 11th edition
(Fadly and Payne, 2003).

70

 

  
   

a Class [II
it
IEL E
.1 g g Spuma-like
i 3 u
s a' 9 0 .
§% 5; I «‘ EV
.. £- g - ff ff.
g“ it

Am Alpha

manner-1551M.

 
   

Class I
Class II

' Primate ‘ M-U ° Fish
“ Feline ' Bird ' Amphibian
' Rodent " ‘ Reptile ' Insect

Figure 1.3. Representative unrooted pol neighbour joining (NJ) dendrograrn (500
bootstraps consensus) of the seven retroviral genera: Alpharetrovirus, Betaretrovirus,
Gammaretrovirus, Deltaretrovirus, Epsilonretrovirus, Lentivirus and Spumavirus. The
endogenous retroviral classes (I, H and HI) are indicated in the periphery and most
similar to Gammaretrovirus, Betaretrovirus and Spumavirus respectively. Some
retrovirses belong to Intermediate Beta-like (IBL), Intermediate Epsilon-like (IEL) and
Errantiviruses (EV). Adapted from Retrovirology (J em et al., 2005).

71

 

A Forced copy Choice B Minus strand exchange

(nonprocessive recombination) (processive recombination)

 

 

 

5’ , __ .. , 3’ 5’ . y ,_ e 3’

5’ 3’ 5’ ' RNaseC L? T 3’
G:

4 DNASynthesis

3,

i
_..,__,e....- , , ~ I \ .. 3,
l

1 DNA Synthesis

 

 

t

RNaseH Cleavage
5’ 7 3’ . -- “g "5"“ 3’

5" *-

i l

59

5, «R3, 5, ' k

 

 

//

 

5,

 

 

 

 

 

Figure 1.4. Two models of homologous genetic recombination in retroviruses during
minus-strand synthesis. (A) Forced copy choice model or nonprocessive recombination is
believed to occur when a break in one RNA template (donor template, thin black line)
forces reverse transcriptase (RT) to continue DNA polymerization using the homologous
region of the copackaged RNA (acceptor template, thin grey line) as a template. (B)
During processive recombination or minus strand exchange, DNA synthesis (thick black
line) proceeds farther along on one of the RNA molecules (thin black line) than the other
(thin grey line). RNase H cleavage (C) of the associated donor RNA template (thin black
line) produces internal regions of single—stranded DNA which can then base pair with the
other copackaged RNA molecule (acceptor template; thin grey line). Recombination
results from a repositioning of the nascent DNA (primer strand) terminus onto the
acceptor template strand. Adapted from Journal of Virology (Brincat et al., 2002).

72

 

 

 

 

fl tRNA
5* AAA 3’
R U5 PBS PPT U3 R
US DNA synthesis
4 J; " AAA
R US PBS PPT U3 R
minus-strand R US RNase H
strong-stop
PBS PPT U3 R
First strand transfer R US
v 4. ____/ ;
PBS PPT U3 R AAA

DNA synthesis & RNase H

 

it ”err U3 R US Q

 

PBS
DNA synthesis
& RNase H PBS
PBS

Second strand transfer

U3

 

l __ n...

 

plus-strand
strong-stop
DNA

DNA synthesis

 

 

U3 R U5 PBS 1 PPT U3 R US

a , >

U3 R US PBS PPT U3 R US
LTR LTR

Figure 1.5. Replication of ALV genome through the process of reverse transcription.
Black line represents RNA while the light and dark colored lines correspond to minus-
strand and plus—strand DNAs respectively. PBS stands for primer binding sequence and
PPT corresponds to polypurine tract. For other abbreviations, see Figure 1.2 and the text
for a description of this process. Adapted from Retroviruses (Coffin et al., 1997).

73

Membrane
0 fusion and

O 0
core entry
0 O

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Adsorption . \‘Ofl
. O
to specrfic r Y Y Y Y ® 4
receptor ® /
l Reverse transcription
/ i Nuclear translocation
{:1} {LEI
__ l Integration
we 1 ifl ,
Transcription
a} 1:13 .
fl ' 1 —{:fl Splicing
\ 1// 1 /
53—54—Ca a] :1:
‘ Progeny
_ / Genorruc RNA & vrrron
Translation proteins assembly

 

 

cwweo

Release by budding

P‘/roteolytic maturation

/.

0-.

31!

-0

Figure 1.6. The life cycle of retroviruses including ALSVS. The retrovirus particle
interacts with specific host cell membrane receptors, fuses directly with the plasma
membrane and the viral core gains entry into the cyoplasm. Then, the ss RNA genome is
reverse transcribed by retroviral enzymes into ds DNA, which is transported into the
nucleus and integrated into the host DNA. The integrated viral DNA or provirus is
transcribed by the host RNA polymerase, generating mRN As and genomic RNA
progeny. The host cell machinery translates the spliced mRN As into glycoproteins and
nucleocapsid proteins. The latter assemble with genomic RNA to form progeny
nucleocapsids, which interact with the membrane-bound viral glycoproteins. Eventually
the progeny virions are released by budding from the host cell membrane followed by
proteolytic maturationof virion proteins. Adapted from Retroviruses (Coffin et al., 1997).

74

CHAPTER 2
Characterization of various isolates of a naturally occurring
recombinant avian leukosis virus using biological assays and

polymerase chain reaction

ABSTRACT

This study was conducted to evaluate the effect of natural recombination between
different ALV subgroups on the relatedness of the resulting strains or isolates. Moreover,
in an attempt to develop specific diagnostics, biological assays and polymerase chain
reaction were used to characterize seven isolates of a naturally occurring recombinant
avian leukosis virus from commercial layer flocks affected with myeloid leukosis (ML).
Biological assays of AF 115 isolates were performed to exclude the presence of a mixture
of ALV subgroups. Host range utilizing two chicken embryo fibroblasts (line 0 and 72
CEFS) and two cell lines (DF-l and DF -1/J ) was examined to propagate and identify
ALV subgroups in the seven AF 115 isolates (AF 115-1, AF 115-5, AF 115-7, AF 115-
10, AF 115-13, AF 115-14 and AF 115-16). Moreover, antigenic relationships of all
seven AF 115 isolates with known ALV subgroups were studied by virus neutralization
assays. In order to identify the nature of the ALV sequences in the AP 115 isolates, a
series of PCR reactions was performed on proviral DNA isolated from line 0 and 72 CEFS
and DF-l/J cell line infected with the isolates using different combinations of primers
specific for different ALV subgroups.

All the AF 115 isolates tested positive for ALV by p27 ELISA on line 0 (C/E)

CEFS, DF-l (C/E) and DF-l/J (C/EJ) cell lines; but tested negative on 72 (C/ABDE)

75

CEFS. Thus, the host range indicated the presence of exogenous ALV subgroups other
than subgroup J. Data from virus neutralization assays revealed antigenic relationships of
all AF 115 isolates primarily with ALV-B, suggesting that ALVs in AF 115 isolates were
of subgroup B envelope. The PCR amplification results, using different primer sets,
indicated the presence of ALV-J like and ALV-B like sequences as well as recombinant
ALV-B/J sequences in all the AF 115 isolates grown in line 0 CEFs and DF -1/J cell line.
Four ofthe seven AF 115 isolates (AF 115 -1, AF 115-13, AF 115-14 and AF 115- 16)
propagated in line 0 CEFS showed identical amplification patterns to the naturally
occurring recombinant ALV-B/J (AF 115-4) in PCR tests using different primer sets.
This implied that these four isolates may be closely related to the naturally occuning
recombinant ALV-B/J, which will be later confirmed by DNA sequencing. The
remaining three isolates (AF 115-5, AF 115-7 and AF 115-10) demonstrated different
patterns of PCR amplification and may thus be distant from the naturally occurring

recombinant ALV-B/J (AF 115-4) and the rest of the AF 115 isolates.

INTRODUCTION

Avian leukosis virus (ALV) is the one of the most common naturally occurring
avian retroviruses associated with neoplastic diseases in chickens (Fadly and Payne,
2003). Infection with ALV continues to be an economical threat to the poultry industry
due to reduced productivity, high tumor mortality and emergence of new mutant and
possibly recombinant viral strains. In chickens, ALVs are classified into six subgroups
(A, B, C, D, E and J) depending on viral envelope glycoproteins, which determine host

range, antigenicity and viral interference (Coffin, 1992). Based on their mode of

76

transmission, exogenous ALVS (subgroups A, B, C, D and J) are transmitted vertically
and horizontally; while endogenous ALV (subgroup E) is transmitted mainly genetically
and sometimes like exogenous viruses (Payne, 1998). In addition, exogenous viruses are
usually highly oncogenic while endogenous viruses are non-pathogenic.

In egg-type chickens, subgroups A and B ALVS are most commonly associated
with lymphoid leukosis (LL). On the other hand, ALV subgroup J is mainly implicated in
causing myeloid leukosis (ML) in meat-type chickens. Currently, ALV infection is
largely controlled by identification and eradication of breeder dams and progeny chicks
positive for the virus (Spencer, 1984). Yet, high level of horizontal and vertical
transmission as well as significant genetic and antigenic variations in different strains and
field isolates of ALV have made control and prevention of ALV infection more difficult
(Fadly and Smith, 1999; Hunt et al., 1999; Silva et al., 2000; Venugopal et al., 1998).
Moreover, the emergence of new mutants and recombinants may confound current field
diagnosis of ALV infection by standard biological and molecular methods. Such
limitations may have a negative impact on prevention and control of this economically
important virus infection in chickens.

Eradication programs have been established and proven effective for controlling
exogenous ALV subgroups (Payne and Howes, 1991; Spencer et al., 1977; Venugopal,
1999; Witter and F adly, 2001). In general, multiple diagnostic tests are used and repeated
to identify shedder hens and ALV-positive progeny chicks reared in isolated small groups
(Witter et al., 2000). These tests may include isolation of the virus in CEFs of different
phenotypes, detection of ALV by ELISA and PCR (Venugopal et al., 1997). Direct

ELISA measures p27 group specific antigen (gsa) levels in vaginal and cloacal swabs,

77

plasma, egg albumin and other clinical specimens (Smith et al., 1979), while indirect
ELISA measures antibodies to the gp85 envelope glycoprotein (Hunt et al., 2000). The
polymerase chain reaction (PCR) usually utilizes primers specific for different regions of
ALV genome mainly env and LTR (Silva et al., 2000; Smith et al., 1998a and b).

Chickens and CEFS with different phenotypes constitute essential diagnostic tools
in detection of known and new emerging isolates of ALSV. p27 ELISA tests have been
used in eradication programs to recognize and remove the chickens exposed to ALV and
to screen for new emerging viruses (V enugopal et al., 1997). However, the results fiom
these tests may be confounded by the high antigenic and genetic diversity in the env gene
of ALV (Silva et al., 2000; Venugopal, 1999; Venugopal et al., 1998). Moreover, the
application of polymerase chain reaction (PCR) procedures using primers specific for
different regions of the retroviral genomes have contributed to the effectiveness of ALV
eradication programs (Fadly and Payne, 2003; Silva et al., 2000; Smith et al., 1998a and
b). Nevertheless, these tests may result in false negatives when mutations arise in regions,
where primers anneal. Therefore, continuous testing and evaluation of diagnostics
especially on new ALV isolates, mutants and recombinants will aid in the present efforts
of ALV eradication.

Genetic diversity in retroviruses is mainly attributed to low fidelity of the reverse
transcriptase enzyme and the diploid nature of retroviral genome. In fact, retroviruses are
known for their high mutation and recombination rates (Elena et al., 2000; Hu and
Temin, 1990a and b; Katz and Skalka, 1990; Mansky, 1998; Nichol, 1996). Exogenous
retroviruses have been reported to recombine with other exogenous and endogenous

retroviruses or nonhomologous cellular genes (Alevy and Vogt, 1978; Blair, 197 7; Kawai

78

and Hanafusa, 1972; Linial and Brown, 1979; Rein, 1982; Svoboda et al., 1986; Weiss et
al., 1973; Wyke and Beamand, 1979). For example, subgroup J ALV has gag and pol
genes closely related to those of exogenous ALSV and an env gene nearly identical to
that in EAV-HI, a member of the EAV family of endogenous retroviruses (Bai et al.,
1995b; Benson et al., 1998a; Silva et al., 2000; Smith et al., 1999).

Three ALV isolates were characterized as recombinants with subgroup A gp85,
subgroup E gp37 and subgroup J LTR (Lupiani et al., 2000). These viruses were
experimentally induced by propagating ALV-J field samples in alv6 CEFS and they
mainly caused LL in layers (Lupiani et al., 2003). Information is currently limited on
natural recombination among different ALV subgroups and its impact on virus isolation,
diagnosis and pathogenesis in chickens. In 1997, three commercial white leghorn layer
flocks used for human vaccine production were hatched in a hatchery used for hatching
broiler breeder eggs. They were diagnosed with ML at 33 weeks of age based on gross
and microscopic lesions. This was the first reported field case of ML in commercial egg-
type chickens (Gingerich et al., 2002). In 2004, Xu and coworkers reported field cases of
ML caused by ALV-J in commercial layers from 12 farms in China.

Eight of the seventeen plasma samples (AF 115-1, AF 115-4, AF 115-5, AF 1 15-5,
AF115-7, AF 115-10, AF 115-13, AF 115-14 and AF 115-16) tested positive for ALV
using p27 ELISA on CEFS of both line 0 (C/E) and alv6 (C/AE), indicating the presence
of an exogenous ALV probably other than subgroup A (Gingerich et al., 2002). Initial
characterization of these isolates by PCR analysis of the long terminal repeat (LTR)
suggested that they were similar to subgroup J (Gingerich et al., 2002). However, further

sequence analysis of one isolate AF 1 15-4 revealed that it is a recombinant virus

79

expressing a subgroup B gp85, a subgroup E gp37 and a subgroup J LTR (Lupiani et al.,
2006). Inoculation of AF 115-4 into susceptible experimental egg-type chickens and
different parental lines of commercial white leghorn layers induced predominately LL
(Lupiani et al., 2006; Mays et al., 2006).

The aim of this study was to characterize the seven remaining AF 115 isolates of
a naturally occurring recombinant ALV by biological assays and polymerase chain
reaction. Two hypotheses were tested in this research work. 1) Closely and distinctly
related strains of recombinant ALV can arise from natural recombination between
different ALV subgroups in the same outbreak. 2) The annealing location and sequence
of PCR primers will reveal variations in different genomic regions of recombinant ALVS.
The objectives were to: 1) Compare the host range and antigenic relationship of the seven
AF 1 15 isolates with those of the naturally occurring recombinant ALV-B/J. 2) Show that
PCR analysis of DNA of different CEFS and cells infected with AF 115 isolates can
reveal the nature or subgroup 0f ALV sequences present in AF 115 isolates when
different primer combinations are used. 3) Demonstrate that PCR amplification patterns
of AF 115 isolates with different primers can indicate mutations in different genomic

regions and may reveal different viral strains.

MATERIALS AND METHODS
A. Origin of Viruses

Prototypes from various ALV subgroups maintained at Avian Disease and
Oncology Laboratory (ADOL) were used as controls in virus propagation and

identification using cell cultures, virus neutralization, p27 ELISA and PCR. These

80

included RAV-1 (subgroup A), RAV-2 (subgroup B), ADOL Hcl (subgroup J, F adly and
Smith, 1997 and 1999; Smith et al., 1998b). In fact, ADOL-Hcl is the earliest ALV J
viral strain isolated in the United States and is considered as the American prototype
ALV J (Fadly and Smith, 1999). AF 115-4 is a recombinant ALV with subgroup B gp 85,
subgroup E gp 37 and subgroup J LTR (ALV-B/J, Lupiani et al., 2006). The seven
remaining AF 115 isolates are AF 115-1, AF 115-5, AF115-5, AF115-7, AF 115-10, AF
115-13, AF 115-14 and AF 115-16 (Gingerich et al., 2002). Viral stocks of all AF 115
isolates were obtained from line 0 CEFs inoculated with the original AF 115 samples and
stored at -80° C. The virus titers of all AF 115 isolates were determined by limiting
dilution after a couple of passages in tissue culture using ADOL line 0 secondary chicken
embryo fibroblasts (CEFS) that support all ALVs except for the endogenous viruses
(Subgroup E ALVS). The titers of AF 115 isolates varied from 105'0 to 107'5 infectious
units per milliliter (TCIDSO/ml) using p27 ELISA performed on supematants of infected

cells in 12 well plates.

B. Biological Assays

Biological assays using CEFS and cell lines with different phenotypes (Fadly and
Smith, 1999; Fadly and Witter, 1998) were used to determine ALV subgroup and host
range ofthe different AP 115 isolates (AF 115-1, AF 115-5, AF 115-7, AF 115-10, AF
115-13, AF 115-14 and AF 115-16). The cells used for virus propagation were line 0
(CE) CEFS (Crittenden et al., 1987), 72 (C/ABDE) CEFS, DF-l (C/E) and DF-l/J (C/EJ)
cell lines. DF -1 (C/E) is a fibroblastoid cell line developed spontaneously fi'om high

density seeding of fibroblasts derived fi'om line 0 chicken embryos (Himly et al., 1998;

81

Maas et al., 2006). On the other hand, DF -1/J is a genetically engineered cell line
expressing envelope protein from ADOL-Hcl strain of ALV-J (Hunt at al., 1999).

All seven AF 115 samples, recombinant ALV-B/J (AF 115-4), negative
(uninfected media) and positive (appropriate prototypes of ALV subgroups) reference
virus controls were assayed for virus replication in different cells in duplicates using
virologic assays according to the procedures described earlier (Fadly and Witter, 1998).
Briefly, around 100 pl of each undiluted viral stock or control (kept at - 80° C) was added
to 0.5 x 106 secondary CEFS suspended in 4% calf serum (CS, Sigma Chemical Co, St.
Louis, MO) in 1:1 Leibowitz’s L—lS-McCoy’s 5A tissue culture medium (LM, Sigma
Chemical Co, St. Louis, MO) containing penicillin/streptomycin (Sigma Chemical Co,
St. Louis, MO), 2 ug/ml arnphotericin B and 2 pg/ml DEAE-Dextran in 35-mm tissue
culture plates. Twenty four hours later, the 4% CS LM media was replaced with 1% CS

LM media and the plates were incubated in 4% CO2 at 37° C for 7-9 days.

C. p27ELISA Assay

All the infected and uninfected cells (CEFS and cell lines) described earlier were
completely lysed with 50 ul of 0.5% tween 80 (Sigma Chemical Co, St. Louis, MO) and
two alternate cycles of freezing at - 80° C and thawing at 37° C. About 100 pl of the cell
lysate was used to test for p27 group-specific antigen (gsa) by enzyme-linked
immunosorbent assay (ELISA) (Smith et al., 1979). The p27 gsa ELISA was carried out
using rabbit anti-p27 polyclonal antibody (IgG) coated immunolon® plates (Dynatech,
Chantilly, VA), rabbit anti-p27 IgG antibody conjugated to horse-radish peroxidase

(SPAFAS, Storrs, CT) and TMB substrate (3, 3', 5, 5'—tetramethy1benzidine, BD

82

Biosciences Pharmingen, San Diego, CA). The plate was read at an absorbance of 630
nm within one hour of stopping the reaction with stop buffer (Sigma Chemical Co, St.

Louis, MO) using a MRX microplate reader (Dynex, Chantilly, VA).

D. Virus Neutralization

Virus neutalization tests were performed for typing and determining the antigenic
relationship of all seven AF 115 isolates as well as recombinant ALV-B/J (AF 115-4) to
ALV subgroups B and J as described earlier (F adly and Witter, 1998). Subgroup B ALV
(RAV-2) and ALV-J (Hcl) were included to confirm neutralization by the respective
subgroup-specific antibody controls. As a negative control, negative sera and media were
used to assess for cross contamination of samples. Specific antibodies to gpSS of ALV
subgroups B (anti-RAV-2) and J (anti-Hcl) or negative sera were diluted 1:5 in serum-
free LM media and incubated at 56° C for 30 minutes to denature the complement
factors. About 500 - 1000 ALV viral particles from each AF 115 sample, ALV-B (RAV-
2) and ALV-J (Hcl) in 50 pl LM media were incubated with 50 ul of heat-denatured 1:5
diluted antibodies or negative controls in duplicates in 96-well flat bottomed tissue
culture plates for 45 minutes at 37° C and 4% CO2.

After the incubation, about 5x104 cells of line 0 CEFS in 150 pl of 4% CS LM
media were added into each of the 96 wells and incubated in 4% CO2 at 37 ° C for a day
and later replaced by 1% CS LM media and incubated in 4% CO2 at 37° C for 7-9 days.
At the end of incubation, the cell cultures were completely lysed with 20 ul of 0.5%
tween 80 (Sigma Chemical Co, St. Louis, MO) and were subjected to two alternate cycles

of freezing at -70° C and thawing at 37° C. The cell lysates were tested for p27 gsa

83

ELISA as described earlier (Smith et al., 1979). Samples with a chromogenic reading of
l or negative on the p27 gsa ELISA read-out was considered to be positive for
neutralization by the reference antibodies and samples with a chromogenic reading of 3 2

on the p27 gsa ELISA read-out was considered to be negative.

E. DNA Isolation

After 7-9 days of virus replication in 35-mm plates of tissue culture, total
DNA was extracted from uninfected and infected cells (line 0 and 72 CEFS and
DF-l/J cells) using standard proteinase K, phenol-chloroform extraction
procedures (Maniatis et al., 1982). Briefly, the medium was removed and the cells
were scraped off the plate and pelleted by centrifugation. The pellet was treated
with lysis buffer (10 mM TRIS, 1 mM EDTA, 100 mM NaCl and 0.5% SDS) and
proteinase K (100 jig/ml) overnight at 56°C. DNA was extracted twice with
phenol-chloroform, treated with RN Ase (2 ill/ml) at 37°C for an hour,
precipitated with ice-cold absolute ethanol, dried at room temperature, dissolved

in TE buffer and stored at 4°C.

F. Oligonucleotide Primers

Most of the primer sets specific for one or different ALV subgroups were adapted
from the literature (Silva et al., 2000 and 2007; Smith et al., 1998a and b) and used for
PCR detection and identification of ALV subgroups in the AF 115 isolates. To generate
PCR primers for subgroup B LTR, assembly and alignment of available subgroup B viral

strains and isolates contained in GenBank were performed. Based upon the alignment and

84

the common sequences, PCR primers (Table 2.1) were designed based on U3 and U5
common sequences of LTR of subgroup B ALV isolates using Oligo 403 software
(National Biosciences, Plymouth, MN). The forward and reverse PCR primers adapted
from other studies, their nucleotide sequences, their armealing locations in the ALV
genome and their ALV subgroup specificities are listed in Tables 2.2 and 2.3
respectively. The PCR primer pairs and PCR cycle used, the estimated amplified PCR

product size, the ALV subgroup specificity are listed in Table 2.4.

G. Polymerase Chain Reaction (PCR)

In each PCR assay, positive and negative controls were included with the AF 115
samples. Negative controls consisted of molecular grade water and uninfected cells in
order to test for cross contamination. At least duplicate PCR reactions were carried out
for each primer set used to amplify ALV sequences in the AF 115 isolates. Each 50-ml
PCR reaction contained 1x reaction buffer (A, B or C, Table 2.5), 25 mM of each dNTP,
25 pmoles of each primer, 2.5 units Taq polymerase, and around 100 ng of template DNA
or water. PCR reaction buffer systems A and D were used with most of the primer sets
for screening under six different PCR cycles or programs. On the other hand, PCR buffer
reaction systems B and C were employed to resolve the problem with extra bands for two
primer pairs, RS-7J and R8 and SRB-F and R8 (Table 2.5). Moreover, buffer system D
was mainly used for the designed subgroup B ALV-specific PCR primer pairs and it
contained reaction buffer, dNTPs, Taq and tracking dyes.

The following PCR conditions (cycle A, Table 2.6) were used with most of the

primer pairs; following an initial template melting or preheating step at 95°C for 3 min,

85

the DNA was amplified during 30 cycles with denaturation at 95°C for 1 min, primer
annealing at 57°C for 1 min, and amplification or extension at 72°C for 2 min, followed
by a final elongation step at 72°C for 10 min and cold storage at 4°C in PCR
thermocycler, PTC-100 Programmable Thermal Controler, Peltier-effect Cycling (MJ
Research, Inc., Waltham, MA). Five other PCR cycles performed for some PCR primer
sets were similar to the previously mentioned PCR conditions except for differences in
the temperatures and times of primer annealing and extension (Table 2.6). Amplified
products were resolved by electrophoresis through 1% agarose gel in 1X TAE buffer

containing 200 ng/rnl ethidium bromide and examined under ultraviolet transillumination.

RESULTS
A. Host range of AF 115 isolates

Host range of each of the seven AF 115 isolate was determined in different CEFs
and cell lines. Line 0 (C/E) is obtained from chicken lines known to be sensitive to all
ALV subgroups except E. On the other hand, 72 (C/ABDE) is resistant to subgroup A, B,
D and E. Cell lines DF-l (C/E) is sensitive to all ALV subgroups except E, while DF-l/J
(C/EJ) is resistant to subgroups J and E ALVs. As seen in Table 2.7, no ALV was
detected in 72 CEFS inoculated with any of the seven AF 115 isolates or the recombinant
ALV-B/J (AF 115-4), while ALV was detected by p27 ELISA in line_0 CEFS, DF-l and
DF -1/J infected cells.

Results obtained from cell culture analysis indicated the presence of an exogenous
ALV subgroup (Subgroup A, B or D) and the absence of ALV-J in the AF 115 isolates.

In addition to the seven AF 115 isolates and the recombinant ALV-B/J (AF 115-4),

86

negative (uninfected media) and positive (appropriate prototypes of ALV subgroups)
virus controls were used to test the functionality of the assay and assess cross
contamination. Infectivity of different cells with the negative and positive controls were
as expected and verified the functionality of the procedures and confirmed the absence of

any cross contamination.

B. Antigenicity of AF 115 isolates

Virus neutalization tests were performed for typing the seven remaining AF 115
isolates (subgroup classification) and determining the antigenic relationship of AF 115
isolates to ALV subgroups B and J. As noticed in Table 2.8, all AF 115 isolates as well as
recombinant ALV-B/J (AF 115-4) were neutralized by ALV subgroup B gp85-specific
antibodies, but not by ALV-J gp85-specific antibodies. In other words, no ALV was
detected by p27 ELISA in line 0 CEFS inoculated with AF 115 isolates or recombinant
ALV-B/J (AF 115-4) and ALV subgroup B gp85-specific antibodies (anti-RAV-2).

On the other hand, ALV was detected in line 0 CEFs inoculated with AF 115
isolates and recombinant ALV-B/J (AF 115-4) incubated with ALV subgroup J gp85-
specific antibodies (anti-Hcl ). The functionality of the test was verified through
neutralization of subgroup B ALV (RAV-2) and ALV-J (Hcl) by the respective subgroup
gp85-specific antibodies. The absence of neutralization of all viruses used when

incubated with the negative control (negative sera and media) confirmed that no cross

contamination among samples took place.

C. PCR amplification of DNA from AF 115-infected line 0 CEFS

87

1. Detection of ALV sequences

In order to identify and confirm ALV subgroups present in the seven AF 115
isolates, a series of PCR reactions was performed using different sets of primers specific
for all ALV subgroups or certain ALV subgroups such as ALV-B, ALV-J and ALV-B/J.
In fact, it is critical to establish background or endogenous levels of PCR product, which
may be spuriously or non-specifically amplified from cells proposed for use in ALV
isolation and propagation. In each PCR run, water and uninfected line 0 CEFS were used
to assess cross contamination, background levels and proper procedure or technical
errors. As expected, there were no detectable PCR products in any of the negative
controls in any of the PCR runs performed (Figures 2.1 - 2.10).

To confirm the presence of ALVS in these isolates, DNA prepared from line 0
CEFS infected with AP 115 isolates were amplified using two all-ALV subgroup primer
pairs (ARK 4836 & REVSP 6683 and RS-7J & REVSP 6683) and primers specific for
ALV A-E (H5 & ADl ). Table 2.9 demonstrated that the three different primer sets
amplified different PCR products or DNA fragments fi'om line 0 CEFs inoculated with all
seven AF 115 isolates (AF 115-1, AF 115-5, AF 115-7, AF 115-10, AF 115-13, AF 115-
14 and AF 1 15-16). It was not expected to observe amplification in DNA of Hcl-infected
line 0 CEFS using primer pair H5 & AD]. In addition, DNA fi'om all ALV positive
controls including naturally occurring recombinant ALV-B/J (AF 115-4), RAV-2
(subgroup B ALV) and Hcl (subgroup J ALV) tested positive for the respective ALV
PCR products. Figure 2.1 represented a successful amplification of ALV sequences in all

ALV infected samples by all-ALV subgroup primer pair (RS-7] & REVSP 6683).

88

2. Detection of recombinant ALV sequences

To find out whether recombinant ALVS were present in the seven AF 115
isolates, DNA extracted from these isolates were PCR amplified using two primer
combinations (SRB-F & S2 and SRB-F & R8), which detect sequences from recombinant
ALV-B/J (AF 115-4). SRB-F was chosen since it annealed to gp 85 of env of subgroup B
ALV and $2 and R8 were selected because they annealed to LTR of subgroup J ALV.
PCR analysis of DNA from all seven AF 115 isolates-infected line 0 CEFS (SRB-F &
R8) showed a recombinant-specific fragment or an amplified product of the same size as
that fi'om the naturally occuning recombinant ALV-B/J (Table 2.10 and Figure 2.2).

As shown in Table 2.10, DNA from line 0 CEFS inoculated with four AF 115
isolates (AF 1 15-1, AF 115-13, AF 115-14 and AF 115-16) showed the presence ofthe
expected PCR products using SRB-F & S2 primer pair. On the other hand, no fragments
were obtained fiom proviral DNA of AF 115-5 and AF 1 15-10. A very faint band of PCR
product was observed in AF 115-7-infected line 0 CEFS using SRB-F & $2 primer pair
(Figure 2.3). As predicted, no amplification products of expected sizes with any of the
two PCR primer sets were detected in ALV belonging to subgroups B (RAV-2) and J
(Hcl ). However, faint non-specific band of a smaller size (0.6 Kb) was detected in DNA
of RAV-2-infected line 0 CEFS when SRB-F & R8 primers were used. Moreover, using
different buffer systems and PCR cycles did not remove or resolve the non-specific

binding in RAV-2, the naturally occurring recombinant ALV—B/J and AF 115 isolates.

3. Detection of ALV-B sequences

89

To test the presence of subgroup B ALV-like sequences in the seven AF 115
isolates, DNA prepared from these isolates were PCR amplified using different primer
combinations specific for subgroup B ALV sequences, which were already published or
newly designed. Amplification of RAV-2 (subgroup B ALV)-infected line 0 CEFS DNA
using all ALV-B-specific primer sets resulted as predicted in all cases in significant yield
of the expected size fragments. None of the primer sets produced amplification products
in Hcl-infected and uninfected line 0 CEFS (Table 2.11).

The first two primer pairs (ARK 4836 & SRB-R and SRB-F & REVSP 6683)
amplified PCR products when all seven AF 115 isolates and the naturally occurring
recombinant ALV-B/J (AF 115-4) were propagated in line 0 CEFs (Table 2.11). Figure
2.4 showed a successful PCR reaction of subgroup B (RAV-2), naturally occurring
recombinant ALV-B/J and all seven AF 115 samples with a representative primers
ARK4836 & SRB-R.

Similar PCR analysis results were observed with primer SRB-F in combination
with one of two of the newly designed primers B LTR 3.3 and B LTR 6.3. Amplified
products yielding the expected sizes were successfully obtained from proviral DNA of all
seven AF 115 isolates and the naturally occuning recombinant ALV-B/J (AF 115—4)
grown in line 0 CEFs (Table 2.11). On the other hand, three primer sets (SRB-F & B
LTR 2.3, SRB-F & B LTR 4.3 and SRB-F & B LTR 8.3) detected RAV-2 (subgroup B
ALV), but none of the seven AF 115 isolates or the naturally occurring recombinant
ALV-B/J. Figure 2.5 denoted a representative of a successful amplification of RAV-2

using subgroup B ALV-specific primers (SRB-F & B LTR 8.3).

90

Using SRB-F and B LTR 1.3 primers, amplification of DNA of AF 115-1, AF
115-5, AF 115-7, AF 115-10 and the naturally occurring recombinant ALV-B/J (AF 1 15-
4) propagated in line 0 CEFS produced a faint band of expected size. However, AF 115-
13, AF 115-14 and AF 115-16 were not amplified successfully when the same primers
were utilized (Table 2.11). PCR with SRB-F & B LTR 5.3 primers resulted in
amplification of fragments from the naturally occurring recombinant ALV-B/J (AF 115-
4) and all AF 115 isolates except AF 115—5 and AF 115-10. Moreover, when SRB-F & B
LTR 7.3 primer combination were used, faint PCR products were detected from DNA of
the naturally occurring recombinant ALV-B/J (AF 115-4) and all AF 115 isolates except

AF 115-5, AF 115-10 and AF 115-l4, which were not amplified (Figure 2.6).

4. Detection of ALV-J sequences

PCR analysis was performed to examine the presence of subgroup J ALV-like
sequences in the seven AF 115 isolates using different already published primer sets
specific for subgroup J ALV sequences. As expected, PCR using the different primer
combinations amplified fragments with the expected sizes from line 0 CEFS inoculated
with Hcl (subgroup J ALV). None of the primers produced PCR products in RAV-2-
infected and uninfected line 0 CEFS (Table 2.12).

Five primer pairs (H5 & H7, RS-6J & REVSP 6683, RS-6J & S2, RS—6J & R8
and RS-6J & R5) produced specific fragments of the expected sizes only from DNA of
Hcl -infected line 0 CEFS (Table 2.12). However, PCR failed to amplify products from
DNA of the AF 115 isolates or the naturally occurring recombinant ALV-B/J (AF 115-4).

Figure 2.7 designated positive amplification results of subgroup J ALV (Hcl) using

91

subgroup J ALV-specific primers (RS-6] & S2). Conversely, PCR products were
amplified fiom all seven AF 115 isolates and the naturally occurring recombinant ALV-
B/J (AF 115-4) when three primer sets (ARK 4836 & R8, F5 & R8 and RS-7J & R8)
were employed. Figure 2.8 referred to a successful PCR amplification of Hcl, the
naturally occurring recombinant ALV—B/J and all seven AF 115 samples using primer
pair RS-7J & R8. A faint non-specific band of smaller size (1.2 Kb) was observed in
RAV—2-infected line 0 CEFs in addition to extra faint non-specific bands in AF 115
isolates. In addition, the non-specific binding in RAV-2, the naturally occurring
recombinant ALV-B/J and AF 115 isolates did not disappear when different buffer
systems and PCR cycles were utilized.

PCR tests using primer combinations (RS-7J & S2 and S1 & 82) revealed that AF
115-1, AF 115-13, AF 1 15-14 and AF 115-16 in addition to the naturally occurring
recombinant ALV-B/J (AF 115—4) were positive. On the contrary, AF 115-5, AF 115-7
and AF 1 15-10 gave a negative PCR tests (Table 2.12). However, we were not able to
replicate the amplification results of these AF 115 isolates with S1 & S2 primers. Figure
2.9 illustrated PCR assay results of AF 115 isolates and other ALV subgroups with
primers RS-7J & S2. Furthermore, PCR amplification of all AF 115 isolates except AF
1 15-10 revealed fragments similar in sizes to those of the naturally occurring
recombinant ALV-B/J (AF 115-4) when primer sets, RS-7J & R5 and F5 & R5, were
used. Figure 2.10 displayed PCR analysis results of all seven AP 115 isolates and

representatives of ALV subgroups employing primer pair RS-7J & R5.

D. PCR reactivity of AF 115 isolates in 72 infected CEFS

92

PCR analysis was conducted on DNA extracted fiom 72 CEFS inoculated with
each of the seven AF 115 isolates, the naturally occurring recombinant ALV-B/J (AF
1 15-4), positive (Hcl) and negative (RAV-2) control ALVs. The primer combinations
utilized were either all-ALV subgroup primer pairs (ARK 4836 & REVSP 6683) or
subgroup J ALV-specific primer sets (S1 & 82, RS-6J & SZ, H5 & H7 and F5 & R8)
since it was expected that only subgroup J ALV will be able to infected these CEFS.
Since 72 CEFS are resistant to subgroup B ALV, primer specific for ALV-B were not
utilized in the PCR assays. These PCR tests were performed in order to investigate
whether the AF 115 isolates could infect 72 CEFS and if they contain mixture of ALV
subgroups as well as to confirm the biological assays results.

The results shown in Table 2.13 indicate that no PCR products or fragments were
detected in the DNA prepared from 72 CEFS infected with any of the seven AF 115
isolates, the naturally occuning recombinant ALV-B/J (AF 115-4) or RAV-2 (subgroup
B ALV) or uninfected 72 CEFS controls with any of the primer sets used. As predicted,
successful amplification of DNA of Hcl-infected 72 CEFS was observed with all the
primers employed. Figure 2.11 revealed PCR amplification results of proviral DNA of all

seven AF 115 isolates employing primer pair ARK 4836 & REVSP 6683.

E. PCR reactivity of AF 115 isolates inDF -1/J infected cells

1. Detection of ALV sequences

PCR analysis results of DNA prepared from AF 115-infected DF -1/J cells using
different primer combinations were similar to those of line 0 infected CEFs (Table 2.14).

Similarly, there were no observable PCR fragments in any of the negative controls or

93

uninfected samples including water and uninfected DF-l/J cells in any of the PCR runs
performed (Figures 2.12 - 2.16). However, ALV was not detected fiom DNA extracted
from Hcl-infected DF-l/J cells in any of the PCR reactions utilizing different primers
(Table 2.14).

In order to confirm the infection of DF -1/J cells with ALVs in the AF 115
isolates, DNA of DF -1/J cells inoculated with all seven AF 115 isolates were amplified
using an all-ALV subgroup primer pair (ARK 4836 & REVSP 6683). Figure 2.12
verified that this primer set amplified DNA fragments from DF-l/J infected with all
seven AF 115 isolates (AF 115-1, AF 115-5, AF 115-7, AF 115-10, AF115-13, AF 115-
14 and AF 115-16). Arnplifiable DNA was also observed from all ALV positive controls

including naturally occurring recombinant ALV-B/J (AF 115-4) and RAV-2 (ALV-B).

2. Detection of ALV-B sequences

To verify the existence of subgroup B ALV-like sequences in DNA prepared from
DF-l/J infected with the seven AF 115 isolates, the naturally occurring recombinant
ALV-B/J (AF 115-4) and subgroup B ALV (RAV-2), PCR assay was carried out
employing subgroup B ALV-specific primers (ARK 4836 & SRB-R). Products of
identical sizes were obtained from all seven AF 115 isolates, ALV-B/J and RAV-2 (Table

2.14 and Figure 2.13).
3. Detection of ALV-J sequences

PCR analysis was performed to study subgroup J ALV-like sequences in AF 115

isolates-infected DF-l/J using different primer pairs (81 & 82, H5 & H7 and F5 & R8).

94

Since DF -1/J is resistant to infection by subgroup J ALV, DNA from Hcl-infected line 0
CEFS were used in PCR as a positive control. PCR products were only observed in DNA
prepared from Hcl-infected line 0 CEFS when primer pairs (S1 & S2, and H5 & H7)
were used. Figure 2.14 revealed PCR amplification results of all seven AF 115 isolates
and representatives of ALV subgroups employing primer pair H5 & H7. On the other
hand, primers F5 & R8 amplified fragments from all seven AF 115 isolates and the
naturally occurring recombinant ALV-B/J (Figure 2.15). In all cases, PCR failed to

amplify DNA extracted from RAV-2-infected DF-l/J cells.

4. Detection of recombinant ALV sequences

To determine if recombinant ALV-B/J propagated in DF -1/J cells, DNA of DF-
1/J infected cells with the seven AF 115 isolates were PCR amplified using primers SRB-
F & R8. Positive PCR signals of the expected size were observed in all seven AF 115
isolates as well as the positive control, the naturally occurring recombinant ALV-B/J
(Table 2.14 and Figure 2.16). As predicted, no amplification products were detected in

the negative controls, ALV belonging to subgroups B (RAV-2) and J (Hcl).

DISCUSSION

ALV infections can be mainly controlled through the early detection and removal
of virus-shedding birds to reduce the congenital and contact infection to other birds
(Smith et al., 1998b; Witter et al., 2000). Diagnosis of ALV infections is based on assays
for virus isolation, which identify and classify new isolates. Currently, virus isolation and

propagation are carried out in cell culture, followed by indirect biological assay such as

95

p27 ELISA for ALV (Crittenden et al., 1987; Fadly and Witter, 1998). In all biological
assays, using chicken embryo fibroblasts (CEFS) with different phenotpyes helps in
characterizing the new isolates. It was shown by many investigators that CEFs resistant to
different ALV subgroups can be utilized to confirm the subgroup of the isolated ALV
(Crittenden and Salter, 1990; F adly and Payne, 2003; Hunt et al., 1999; Salter and
Crittenden, 1991).

Various approaches were used to characterize and identify ALV subgroups
present in the seven AP 115 isolates (AF 115-1, AF 115-5, AF 115-7, AF 115-10, AF
115-13, AF 115-14 and AF 115-16). In fact, host range pattern constitutes the most
reliable method for subgroup classification (Fadly and Payne, 2003). Propagation of these
isolates was investigated in line 0 CEFS (C/E), 72 CEFS (C/ABDE), DF-l (C/E) cells and
DF-l/J (C/EJ) cells to exclude the presence of a mixture of ALV subgroups. The host
range or cell culture analysis of the seven AF 115 isolates suggested that ALVs from all
isolates belonged either to ALV subgroup A, B or D, but not to J (Table 2.7).

Previous observations showed that all AF 115 isolates tested positive for ALV on
CEFS of line 0 (C/E), 15 Bl (C/O), 71 (C/A) and cell lines of DF-l (C/E) and DF-l/J
(C/EJ); but tested negative on 72 (C/ABDE) CEFS (Lupiani and Fadly, unpublished data).
Analysis of all data obtained from current and previous virus isolation and
characterization assays indicated that all AF 115 isolates may have subgroup B or D ALV
and no subgroup J ALV. The fact that subgroup D ALV is rarely found in the field
suggested that subgroup B ALV may be the ALV subgroup present in these isolates. The

subgroup of ALV present in these isolates will be later confirmed by sequence analysis.

96

Antigenicity is usually determined by virus neutralization with known subgroup-
specific antibodies (Fadly and Payne, 2003). It can also be used for strain classification,
but is less reliable than host range assay. The finding that all AF 115 isolates were
neutralized by ALV subgroup B gp85-specific antibodies, but not by ALV-J gp85-
specific antibodies suggested antigenic relationships of all AF 115 isolates primarily with
ALV-B (Table 2.8). Moreover, earlier flow cytometry results indicated that DF-l cells
infected with the different AF 115 isolates reacted with chicken polyclonal serum specific
for ALV subgroup B but not subgroups A and J specific anti-sera (Lupiani and Fadly,
unpublished data).

Analysis of the virus neutralization results in addition to previous flow cytometry
data confirmed the host range data, indicating the presence of a subgroup B exogenous
ALV and the absence of subgroup J ALV. Even though there is a partial cross-
neutralization between ALV subgroups B and D, presence of subgroup D ALV in AF 115
isolates may be excluded since it is rarely found in the field and this will later be
confirmed by DNA sequence analysis.

Molecular techniques such as PCR have been proven to be rapid, specific,
sensitive and effective in ALV detection (Garcia et al., 2003; McKay, 1998; McKay and
Rosales, 2000; Payne, 2000; Smith et al., 1998a and b). Currently, PCR technology could
be used to characterize and establish relationships among emerging ALV field isolates.
including mutants and recombinants. Available sequence data about ALV genome has
been used to design primers for detection of ALV by PCR (Bai, 1995a and b; Coffin,
1992; Silva et al., 2000 and 2007; Smith et al., 1998a and b; Van Woensel et al., 1992).

In addition, most newly designed primers anneal at env and LTR regions.

97

The continuous variations exhibited by ALV resulted in the development of new
specific reagents for the amplification and characterization of new emerging isolates
(Garcia et al., 1998; Lupiani et al., 2000 and 2006; Silva et al., 2000; Venugopal, 1999;
Venugopal et al., 1998). For example, specific primer pairs were developed for the
detection of recombinant subgroup J ALVs expressing subgroup A envelope (Lupiani et
al., 2000). However, if mutations occur in ALV genome where primers anneal, the
desired sequences will not be amplified, not all variant viruses will be detected and false
negatives will result.

The data obtained from PCR analysis of genomic DNA extracted from line 0
CEFS and DF -1/J cells inoculated with the seven AF 115 isolates using all—ALV primers
(ARK 4836 & REVSP 6683 and RS-7J & REVSP 6683) indicated the presence of ALV
smuences in these isolates. The finding that ALV sequences were detected in DNA
prepared from line 0 CEFS (Table 2.9; Figure 2.1) and DF-l/J cells (Table 2.14 and
Figure 2.12), but not 72 CEFS (Table 2.13; Figure 2.11) confirmed the host range and
antigenicity data, indicating the presence of subgroup B ALV in AP 115 isolates.

Collectively, analysis of all PCR assays conducted here revealed that there no
cross contamination among samples, background or endogenous levels of ALV in
uninfected line 0 or 72 CEFs or DF-l/J cells. This conclusion is supported by the absence
of any observable background or any detectable PCR products in any of the negative
controls used including uninfected line 0 CEFS (Table 2.9 and Figures 2.1 - 2.10), 72
CEFS (Table 2.13; Figure 2.11) and DF-l/J cells (Table 2.14; Figures 2.12-2.16).

Given that an earlier study indicated that H5 and AD] primers were designed to

detect ALV subgroup A-E, but not ALV-J (Smith et al., 1998b). In our study, we were

98

not expecting to detect PCR products fiom DNA isolated from Hcl -infected line 0 CEFS
using these primers (Table 2.9). The reasons for this discrepant result were not clear; but
they might be due to differences in the ALV-J isolate used in the earlier study (HPRS-
103). Different strains of ALV-J may have variable nucleotide sequences in the env gene
(gp 85 or SU), where ADI primer can anneal to one, but not the other.

Analysis of amplification results attained from line 0 CEFS and DF-l/J cells
infected with all seven AF 115 isolates using primer set SRB-F & R8 showed a
recombinant-specific fragment of the same size as that from the naturally occurring
recombinant ALV-B/J (Tables 2.10 and 2.14; Figures 2.2 and 2.16). The results presented
here demonstrated that primers SRB—F & R8 are specific for recombinant ALV-B/J found
in all seven AP 115 isolates propagated in line 0 CEFS and DF-l/J cells. In addition, no
spurious or non-specific amplification were observed in uninfected line 0 CEF and DF-
1/J cells respectively indicating the absence of endogenous sequences that may cross-
hybridize with these PCR primers. Therefore, these primers constitute the principle tools
for confirming the isolation and detection of recombinant ALV-B/J from field isolates as
well as experimental settings.

Although the recombinant ALV-B/J-specific primer set SRB-F & 82 detected
most of the AF 115 isolates, certain isolates such as AF 115-5, AF 115-7 and AF 115-10
were not detected, confirming molecular variation among AF 115 isolates (Table 2.10;
Figure 2.3). We hypothesize that sequence analysis of these later isolates may show
nucleotide sequence changes in their LTRs where 82 primer anneals (U5 region). Thus,

the annealing location and sequence of the primers can provide indications for subgroup

99

1..

‘ n
I1

designation of field isolates. They may also provide information about mutations in ALV
genomes and variations among various ALV strains.

All primer combinations used to amplify subgroup B ALV sequences were
specific since they amplified PCR fiagments fiom RAV-2-infected, but not Hcl -infected
or uninfected line 0 CEFS and DF-l/J cells (Tables 2.11 and 2.14). However, some of the
primer pairs were also able to detect the naturally occurring recombinant ALV-B/J (AF
115-4) and all seven AF 115 isolates since the forward and reverse primers were able to
anneal to complementary sequences in different locations of the ALV genomes of these
isolates (Tables 2.11 and 2.14). These primer sets included ARK 4836 & SRB-R, SRB-F
and REVSP 6683, SRB-F & B LTR 3.3 and SRB-F & B LTR 6.3 (Figures 2.4 and 2.13).
Other primers such as SRB-F & B LTR 1.3, SRB-F & B LTR 5.3 and SRB-F & B LTR
7.3 amplified PCR products from some of the AF 115 isolates to varying degrees since
the reverse primers were not able to anneal to LTR sequences of all AF 115 isolates
(Figure 2.6). This may be due to the presence of sequence variations in the LTR
sequences of AF 115 isolates where these primers are designed to anneal. Data from PCR
assays presented here clearly document that all AF 115 isolates contain subgroup B
ALV-like sequences, thus confirming host range and antigenicity results. However,
BLAST search of GenBank indicated that some of the primers might anneal to sequences
from ALV subgroups A, C, D and J to varying degrees. On the other hand, three primers
(SRB-F & B LTR 2.3, SRB-F & B LTR 4.3 and SRB-F & B LTR 8.3) were very specific
since they only detected DNA of RAV-2-infected line 0 CEFs (Figure 2.5). The failure of
detection of ALV sequences in AF 115 isolates by these primers may be due to the

differences in the LTR sequences of RAV-2 (ALV-B) and AF 115 isolates (ALV-B/J).

100

Analysis of PCR assays with five primer sets specific for ALV-J (H5 & H7, RS-
6J & REVSP 6683, RS-6J & S2, RS-6J & R8 and RS-6J & R5) revealed positive reaction
with DNA obtained from line 0 and 72 CEFS, but not DF-l/J cells inoculated with Hcl
(T ables 2.12-2.14; Figures 2.7 and 2.14). Since DF-l/J is resistant to infection by
subgroup J ALV, ALV was not detected fiom DNA extracted fiom Hcl-infected DF-l/J
cells in any of the PCR reactions utilizing different primers (Table 2.14). These primers
seem to be very specific to ALV-J since the forward and reverse primers anneal only to
the envelope and LTR regions of ALV-J. In addition, there were no amplifications from
DNA extracted RAV-2-infected or uninfected line 0, 72 CEFS and DF-l/J cells using all
the primer combinations for detection of ALV-J ~1ike sequences (Tables 2.14-2.15;
Figures 2.7-2.10 and 2.14). PCR products of expected sizes were found in AP 115
isolates-infected in addition to the naturally occurring recombinant ALV-B/J (AF 115-4)-
infected line 0 CEFS and DF -1/J when primer pairs ARK 4836 & R8, F5 & R8 and RS-7J
& R8 were utilized (Tables 2.12 and 2.14; Figures 2.8 and 2.15). These amplifications
were expected since two of the forward primers used (ARK4836 & RS-7J) were generic
and able to anneal to all ALV subgroups. On the other hand, F5 is a forward primer,
which usually anneals to ALV-J envelope, but it annealed to ALV-B/J and other AF 115
isolates since the 10 nucleotide at the 3’ end of the primer has 100% homology with
ALV-B envelope. Moreover, the reverse primer (R8) is specific to LTR of ALV-J and
hence the amplification of ALV-J, ALV-B/J and all isolates. These results significantly
establish that all seven AF 115 isolates contain subgroup J ALV-like sequences.

Four primer’combinations (RS-7J & S2, 81 & S2, RS-7J & R5 and F5 & R5)

resulted in variable patterns of amplifications of AF 115 isolates. This may be due to the

101

difference in the nucleotide sequences in different regions of ALV sequences in our
isolates affecting the annealing of the different primers. Moreover, four of the seven AP
115 isolates, namely AF 115 —1, AF 115-13, AF 115-14 and AF115- 16 showed identical
amplification patterns in PCR tests using these primer combinations to that of the
naturally occurring recombinant ALV-B/J (Table 2.12; Figures 2.9-2.10) in infected line
0 CEFS. This revealed that these four isolates may be identical or closely related to the
naturally occurring recombinant ALV-B/J (AF 1 15-4) which will be later confirmed by
DNA sequencing. The remaining three isolates, AF 115-5, AF 115-7 and AF 115-10
demonstrated different pattern of PCR amplification and may thus be different or
distantly related to the naturally occurring recombinant ALV-B/J (AF 115-4) and the rest
of the AF 115 isolates. While a more thorough study of the molecular makeup of these
isolates through sequence analysis is necessary, the results observed here along with our
earlier findings suggest that these isolates may display genomic diversity or nucleotide-
level differences including mutations, deletions and insertions.

There was discrepant results regarding the detection of ALV-J sequences in DNA
prepared fiom AF 115 isolates-infected line 0 CEFS, but not in that of DF-l/J cells using
primers Sl & 82 for unknown reasons. However, we noticed that $1 & 82 primers were
able to amplify DNA from four of the AP 115 isolates-infected line 0 CEFS in the first
trial, but not in later trials. All the data from PCR analysis with subgroup J ALV
sequence-specific primers (F5 & R5, RS-6J & S2, RS-7J & $2 and RS-7J & R5) were
consistent with earlier studies on AF 115 isolates inoculated into line 0 CEFS (Lupiani
and F adly, unpublished data). The only exception was amplification of all AF 115

isolates with S] & S2 primers (Gingerich et al., 2002; Lupiani and Fadly, unpublished

102

data)- It is worth mentioning that Lupiani and F adly were not able to amplify all the AP
115 isolates from the first PCR trial. The length or sequences of the primers (8] & S2),
the but fler system and amplification conditions used may have been different than the
ones utilized in our study.

J’CR analysis in conjunction with sequence analysis of these AF 115 isolates can
yield important information about identity and genomic-level natural variations within
these re «combinants. Design of future primers would be improved as more information is
accmulating about ALV diversity. These new primers may be able to detect specifically

a Wider range of new and emerging mutant and recombinant ALVS in field flocks and

exParitnental settings.

103

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106

Table 2.4. Forward and reverse primer combinations and their characteristics

\

Product Size3

 

Primer PairI ALV Subgroup2 (~kb) PCR Cycle4
m 4836 &
7
RE VSP 6683 All -.4 A and E
RS-7J &
RE vsp 6683 All 2.4 A and c
H 5 & ADI A-E 0.3 B and H
ARK 4836 &
SRB-R B 1.0 A and F
SRB—T & REVSP

6683 B 1.8 A
HS & H7 J 0.5 B and H
Si & 82 J 0.4 B and H

ARK 4836 & R8 J 2.9 A

F5 & R5 J 1.0 A

F5 & R8 J 1.5 A

RS-6J &

REVSP 6683 J 2'1 A
RS-6J & R5 J 1.9 A
RS-6J & R8 J 2.3 A

RS-6J & 82 J 2.3 A
Rs-7J & 82 J 2.5 A
Rs-7J & R5 J 2.0 A
Rs-n & R8 J 2.5 A, c, D and E
S RB-F & 82 EU 2.1 A
S 113-F & R8 EU 2.1 A, D and E
\

 

J
The PCR forward and reverse primers were chosen either from already published
2 Sequences or primers available at ADOL.
ALV subgroup specificities of the PCR primer sets were determined based on the
ealing location of each primer in the ALV genome and from already published use
3 of th ese primer combinations for certain ALV subgroups.
e PCR amplification product size was estimated according to the annealing locations
4 of pt‘imer pairs in the respective ALV subgroups.
The PCR cycle of each primer pair was chosen based on the Tm of the primers and
publ i shed procedures. Some primer sets were run in more than one cycle to resolve the

prob} ems of non-specific binding and extra bands or for better resolution and
ampl ification.

107

Table 2.5. PCR reaction buffer systems

k

 

 

Bu fier System Contents
A' 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2
and 0.01% gelatin
B2 10 mM Tris-HCI (pH 9.0), 50 mM KC1, 1.5 mM Mng,
and 0.1% Triton® X-100
C3 100 mM Tris-HCI (pH 8.3), 500 mM KCl, 15 mM MgClz
and 0.01% gelatin
D4 2X Green GoTaq® Reaction Buffer (pH 8.5), yellow and
blue dyes, 400uM dATP, 400uM dGTP, 400uM dCTP,
400uM dTTP and 3mM MgClz

 

 

‘ Tiger system A is a 10x buffer for Taq DNA polymerase (Sigma-Aldrich, St. Louis,
)

2 Buffer system B is a 10x buffer for Taq DNA polymerase in storage buffer B (Promega,
Madison, WI)
Bufi‘er system C is a 10x buffer for JumpstartTM Taq DNA polymerase (Sigma-Aldrich,
4 St- Louis, MO) '
Bu fret system D is a GoTaq® Green Master Mix contains Taq and Taq buffer, dNTPs
and tracking dyes (Promega, Madison, WI)

3

108

Table 2.6. PCR cycles used with different primer sets

x

PC R Cyclel Conditions2
A Primer annealing at 57°C for 1 min, and amplification or
extension at 72°C for 2 min
B Primer annealing at 49°C for 15 sec, and amplification or
extension at 72°C for 15 sec
C Primer annealing at 60°C for l min, and amplification or
extension at 72°C for 2.5 min
D Primer annealing at 60°C for 1 min, and amplification or
extension at 72°C for 2 min
Primer annealing at 63°C for 30 sec, and amplification or
extension at 72°C for 2 min
F Primer annealing at 60°C for 30 sec, and amplification or
extension at 72°C for 2 min
G Primer annealing at 53°C for 1 min, and amplification or
extension at 72°C for 2 min
H Primer annealing at 53°C for 15 sec, and amplification or
extension at 72°C for 15 sec

{Tl

\

The following PCR conditions were used in each cycle; following an initial template
me 1 ting or preheating step at 95°C for 3 min, 30 cycles with denaturation at 95°C for 1
min, variable primer annealing and amplification or extension temperature and time
a("'ctbrding to the specific cycle, followed by a final elongation step at 72°C for 10 min
zand cold storage at 4°C.

2% PCR conditions 1n each cycle were programmed into FTC-100 Programmable
Thleermal Controller, Peltier-effect Cycling (MJ Research, Inc., Waltham, MA).

109

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117

34567M8910111213

1 2

u .u””§-.".”§— 2.4Kb

H

 

Figure 2.1. Detection of ALV in proviral DNA prepared from line 0 CEFS infected with

AF 1 15 isolates by PCR using all-ALV primers RS-7J & REVSP 6683. Lanes and
respective samples: M = DNA molecular weight marker (Figure 2.17); 1 = uninfected
line 0 CEFS (negative control); 2 = Hcl (ALV-J, positive control); 3 = RAV-2 (ALV-B,
positive control); 4 = RAV-2 (ALV-B, positive control) DNA; 5 = Naturally occurring

recombinant ALV-B/J (AF 115-4, positive control); 6 = AF 115-1; 7 = AF 115-5; 8 = AF
115-7; 9 =AF115-10; 10 = AF115-13;11= AF 115-14; 12 = AF 115-16; 13 = water
(no DNA). The approximate 2.4 Kb hybridizing fragments of DNA correspond to all-

ALV amplified fragments and are characteristics of ALV DNA.

118

6 7M8 910111213M

12345

M

2.1 Kb

 

Figure 2.2. Analysis of PCR amplification products from line 0 CEFS inoculated with AF
1 15 isolates utilizing recombinant ALV-B/J-specific primer pair SRB-F & R8. Lanes and

respective samples: M = DNA molecular weight marker (Figure 2.17); 1 = uninfected
line 0 CEFS (negative control); 2 = Hcl (ALV-J, negative control); 3 = RAV-2 (ALV-B,
negative control); 4 = RAV-2 (ALV-B, negative control) DNA; 5 = Naturally occurring

recombinant ALV-B/J (AF 115-4, positive control); 6 = AF 115-1; 7 = AF 115-5; 8 = AF
115-’7;9=AF115-10;10=AF115-13; 11 =AF115-14; 12 =AF 115-16; 13 =water
(no DNA). Amplification products of around 2.1 Kb were obtained for DNA templates of

recombinant ALV-B/J.

119

789101112

56M

234

2.1 Kb

 

Figure 2.3. Agarose gel showing the PCR products obtained by employing SRB-F & $2
primer set on DNA samples extracted fi'om AF 115 isolates-infected line 0 CEFS. Lanes

and respective samples: M = DNA molecular weight marker (Figure 2.17); 1 =
uninfected line 0 CEFS (negative control); 2 = Hcl (ALV-J, negative control); 3 = RAV-
2 (ALV-B, negative control); 4 = Naturally occurring recombinant ALV-B/J (AF 1 15-4,

positive control); 5 = AF 115-1; 6 = AF 115-5; 7 = AF 115-7; 8 = AF 115-10; 9 = AF
11 5— 13; 10 = AF115-14;11= AF 115-16; 12 = water (no DNA). Successful
aJIIIDIification of DNA templates in various samples produced recombinant ALV-B/J-

Specific fragments of approximately 2.1 Kb.

120

8M9101112M

1.0 Kb

W «1w

 

Figure 2.4. PCR amplification of DNA isolated from AF 115 isolates-infected line 0
CEFS utilizing primer combination specific for subgroup B ALV sequences (ARK 4836
& SRB-R). Lanes and respective samples: M = DNA molecular weight marker (Figure
2.17); 1 = water (no DNA); 2 = uninfected line 0 CEFS (negative control); 3 = Hcl
(ALV-J, negative control); 4 = RAV—2 (ALV-B, positive control); 5 = Naturally
occurring recombinant ALV-B/J (AF 115-4); 6 = AF 115-1; 7 = AF 115-5; 8 = AF 115-7;
9 = AF 115-10; 10 = AF 115—13; 11 = AF 115-14; 12 = AF 115-16. The estimated 1.0 Kb

PCR products correspond to ALV-B-specific amplified fi'agments.

121

M1234M5678M9101112M

1.8 Kb

 

Figure 2.5. PCR for specific detection of subgroup B ALV with line 0 CEFs inoculated
with AF 115 isolates using primer pair specific for subgroup B ALV (SRB-F & B LTR
8.3). Lanes and respective samples: M = DNA molecular weight marker (Figure 2.17); l
= water (no DNA); 2 = uninfected line 0 CEFS (negative control); 3 = Hcl (ALV-J,
negative control); 4 = RAV-2 (ALV-B, positive control); 5 = Naturally occurring
recombinant ALV-B/J (AF 115-4); 6 = AF 115-1; 7 = AF 115-5; 8 = AF 115-7; 9 = AF
115-10; 10 = AF115-13;11= AF 115-14; 12 = AF 115-l6. Successful amplification of
DNA templates in RAV-Z-infected line 0 CEFS formed subgroup B ALV-specific
fi'agment of approximate size of 1.8 Kb.

122

M1234M5678M9101112M

1.9 Kb

 

Figure 2.6. PCR reaction of genomic DNA from line 0 CEFs inoculated with AF 115
isolates and amplified with primers specific for subgroup B ALV (SRB-F & B LTR 7.3).
Lanes and respective samples: M = DNA molecular weight marker (Figure 2.17); 1 =
water (no DNA); 2 = uninfected line 0 CEFS (negative control); 3 = Hcl (ALV-J,
negative control); 4 = RAV-2 (ALV-B, positive control); 5 = Naturally occurring
recombinant ALV-B/J (AF 115-4); 6 = AF 115-1; 7 =AF 115-5; 8 =AF 115-7; 9 = AF
115-10; 10 = AF115-13;11= AF 115-14; 12 = AF 115-16. The near 1.9 Kb DNA
fragments correspond to ALV-B like sequence-specific amplified fragments.

123

M1234567M8910111213

2.3 Kb

 

Figure 2.7. Specific PCR detection of subgroup J ALV in DNA extracted fiom line 0
CEFS infected with AF 115 isolates utilizing ALV-J-specific primer set RS-6J & $2.
Lanes and respective samples: M = DNA molecular weight marker (Figure 2.17); 1 =
uninfected line 0 CEFS (negative control); 2 = Hcl (ALV-J, positive control); 3 = RAV-2
(ALV-B, negative control); 4 = RAV-2 (ALV-B, negative control) DNA; 5 = Naturally
occurring recombinant ALV-B/J (AF 115-4); 6 = AF 115-1; 7 = AF 115-5; 8 = AF 115-7;
9 = AF 115-10; 10 = AF115-13;11= AF 115-14; 12 = AF115-16; 13 = water (no
DNA). Successful amplification of DNA templates in Hcl -infected line 0 CEFS created
subgroup J ALV-specific fragment of rough size of 2.3 Kb.

124

M11234M5678M9101112M

 

Figure 2.8. Analysis of PCR carried out on proviral DNA extracted from line 0 CEFs
inoculated with AF 115 isolates and amplified using primer pair specific for ALV-J
sequences (RS-7J & R8). Lanes and respective samples: M = DNA molecular weight
marker (Figure 2.17); 1 = water (no DNA); 2 = uninfected line 0 CEFS (negative control);
3 = RAV-2 (ALV-B, negative control); 4 = Hcl (ALV-J, positive control); 5 = Naturally '
occuning recombinant ALV-B/J (AF 115-4); 6 = AF 115-1; 7 = AF 115-5; 8 = AF 115-7;
9 = AF 115-10; 10 = AF115-l3;11= AF 115-14; 12 = AF 115-16. PCR products of
estimated sizes of 2.5 Kb correspond to ALV-J like-specific amplified fragment.

125

M123456M789101112

2.5 Kb

 

Figure 2.9. PCR analysis of DNA isolated fi'om AF 115 isolates propagated in line 0
CEFS and amplified by ALV-J sequence-specific primer combination RS-7J & SZ. Lanes
and respective samples: M = DNA molecular weight marker (Figure 2.17); 1 =
uninfected line 0 CEFS (negative control); 2 = Hcl (ALV-J, positive control); 3 = RAV-2
(ALV-B, negative control); 4 = Naturally occurring recombinant ALV-B/J (AF 1 15-4); 5
=AF115-1;6=AF115-5;7 =AF 115-7; 8 = AF 115-10; 9=AF 115-13; 10 =AF115-
14; 11 = AF 115-16; 12 = water (no DNA). The nearly 2.5 Kb bands correspond to ALV-
] like sequence-specific amplified fragments.

126

M123456M789101112

2.0 Kb

 

Figure 2.10. Ethidium bromide -stained agarose gel with PCR products of DNA
prepared from AF 115-infected line 0 CEFS obtained by amplification with ALV-J
sequence-specific primers RS-7J & R5. Lanes and respective samples: M = DNA
molecular weight marker (Figure 2.17); l = uninfected line 0 CEFS (negative control); 2
= Hcl (ALV-J, positive control); 3 = RAV-2 (ALV-B, negative control); 4 = Naturally
occurring recombinant ALV-B/J (AF 115-4); 5 = AF 115-1; 6 = AF 115-5; 7 = AF 115-7;
8 = AF 115-10; 9 = AF 115-13; 10 = AF115-14;11= AF 115-16; 12 = water (no DNA).
The roughly 2.0 Kb PCR products correspond to ALV-J like sequence-specific amplified
fragments.

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M1234M56789101112M

2.4 Kb

 

Figure 2.11. PCR amplification from isolated proviral DNA samples of 72 CEFS
inoculated with AF 115 isolates employing primer pair ARK 4836 & REVSP 6683,
which can detect all ALV subgroups. Lanes and respective samples: M = DNA molecular
weight marker (Figure 2.17); 1 = water (no DNA); 2 = uninfected 72 CEFS (negative
control); 3 = RAV-2 (ALV-B, negative control); 4 = Hcl (ALV—J, positive control); 5 =
Naturally occurring recombinant ALV-B/J (AF 115—4); 6 = AF 115-1; 7 = AF 115-5; 8 =
AF 115-7; 9 = AF 115-10; 10 = AF115-13;11= AF 115-14; 12 = AF 115-16. Amplified
product of about 2.4 Kb represents all-ALV fiagrnent.

128

M12345M6789M10111213

finammmn-pm-_ 2.4 Kb

. I '9 w ..

 

Figure 2.12. Genomic DNA from AF 115 isolates-infected DF-l/J cells using all-ALV
primer set ARK 4836 & REVSP 6683. Lanes and respective samples: M = DNA
molecular weight marker (Figure 2.17); 1 = water (no DNA); 2 = uninfected DF-l/J cells
(negative control); 3 = Hcl (ALV-J); 4 = Hcl (ALV-J propagated in line 0 CEFS); 5 =
RAV-2 (ALV-B); 6 = Naturally occurring recombinant ALV-B/J (AF 115—4); 7 = AF
115-1; 8 = AF 115-5; 9 = AF 115-7; 10 = AF115-10;11=AF115-l3;12 = AF 115-14;
13 = AF 115-16. Amplified products of around 2.4 Kb characterize all-ALV fragment.

129

M1234M5678M9101112M

a an... III-un- 1-0Kb

 

Figure 2.13. PCR analysis of DNA extracted from DF-l/J cells inoculated with AF 115
isolates utilizing ALV-B specific primers ARK 4836 & SRB-R. Lanes and respective
samples: M = DNA molecular weight marker (Figure 2.17); 1 = water (no DNA); 2 =
uninfected DF-l/J cells (negative control); 3 = Hcl (ALV-J); 4 = RAV-2 (ALV-B); 5 =
Naturally occurring recombinant ALV-B/J (AF 1 15-4); 6 = AF 115-1; 7 = AF 115-5; 8 =
AF 115-7; 9 = AF 115-10; 10 = AF115-13;11= AF115-14; 12 = AF 115-16. The
estimated 1.0 Kb PCR fiagments signify ALV-B-specific amplified fragments.

130

M123456M789101112

0.5 Kb

 

Figure 2.14. Specific PCR amplification of subgroup J ALV in DNA isolated from DF-l/
J cells infected with AP 115 isolates with ALV-J-specific primer set H5 & H7 . Lanes and
respective samples: M = DNA molecular weight marker (Figure 2.17); 1 = uninfected
DF-l/J (negative control); 2 = RAV-2 (ALV-B, negative control); 3 = Hcl (ALV-J); 4 =
Hcl (ALV-J propagated in line 0 CEFS, positive conuol) DNA; 5 = Naturally occuning
recombinant ALV-B/J (AF 115-4); 6 = AF 115-1; 7 = AF 115-5; 8 = AF 115-7; 9 = AF
115-10; 10 = AF115-13;11= AF 115-14; 12 = AF 115-16. Successful amplification of
DNA templates in Hcl-infected line 0 CEFS produced subgroup J ALV-specific fragment
of approximately 0.5 Kb.

131

M1234M5678M9101112

1.5 Kb

 

figure 2.15. Detection of ALV-J sequences in proviral DNA isolated from AF 115
isolates-infected DF-l/J cells through PCR analysis using primer combination F5 & R8.
Lanes and respective samples: M = DNA molecular weight marker (Figure 2.17); 1 =
water (no DNA); 2 = uninfected DF-l/J cells (negative control); 3 = Hcl (ALV-J); 4 =
RAV-2 (ALV-B); 5 = Naturally occurring recombinant ALV-B/J (AF 115-4); 6 = AF
115-1; 7 =AF 115-5; 8 = AF 115-7; 9 = AF 115-10; 10 =AF115-l3;11= AF115-14;
12 = AF 115-16. The estimated 1.5 Kb PCR products correspond to ALV-J sequences-
specific amplified fragments.

132

M1234567M8910111213

"53

 

Figure 2.16. PCR analysis done to detect recombinant ALV-B/J sequences in genomic
DNA prepared from DF -1/J cells incubated with AP 115 isolates employing primers
SRB-F & R8. Lanes and respective samples: M = DNA molecular weight marker (Figure
2.17); 1 = uninfected DF-l/J cells (negative control); 2 = Hcl (ALV-J); 3 = RAV-2
(ALV-B); 4 = RAV-2 (ALV-B propagated in line 0 CEFS) 5 = Naturally occurring
recombinant ALV-B/J (AF 115-4); 6 = AF 115-1; 7 = AF 115-5; 8 = AF 115-7; 9 = AF
115-10;10 = AF115-13;11= AF 115-14; 12 = AF 115-16; 13 + water (no DNA).
Successful amplification of DNA templates in AF 115 isolates-infected DF-l/J cells
formed recombinant ALV-B/J-specific fi'agments of around 2.1 Kb.

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23,130
9,416
6,557

4,361

2,322
2,027

1,500

 

600

100

 

Figure 2.17. DNA molecular weight marker as visualized by ethidium bromide staining.
It consists of 1:1 of lambda DNA-Hind III-digested and 100 bp DNA marker or ladder.
Values are in base pairs (bp).

134

CHAPTER 3
Comparison of proviral DNA sequences of seven isolates of naturally

occurring recombinant avian leukosis virus and representative ALVS

ABSTRACT

This study was performed in order to molecularly characterize seven AF 115
isolates of a naturally occurring recombinant avian leukosis virus associated with
myeloid leukosis in commercial layers. Sequence analyses of different genomic regions
of these AF 115 isolates were carried out to confirm the identity of the subgroup of these
recombinant viruses. The deduced amino acid sequences of gp85 and ng7 and the DNA
sequences of 3’ UTR and LTR of the seven isolates were compared with the naturally
occurring recombinant ALV-B/J. These sequences were also evaluated for variations and
relationship with prototypes of various ALV subgroups and related ALV strains through
NCBI blast search. Sequence alignment, % identity, divergence and phylogenetic trees
were studied to draw associations and identify important potential sites for protein
structure and retrovirus transcription. Moreover, potential conserved sequences in
different regions of these isolates were assessed for predicted stability and functions.

Six AF 115 isolates (AF 115-1, AF 115-7, AF 115-10, AF 115-13, AF115-14 &
AF 115-16) showed high sequence identity to the naturally occurring recombinant ALV-
B/J, subgroup B ALV and RAV-2-like viruses in the SU domain of the envelope. On the
other hand, the gpSS of AF 115-5 shower lower sequence identity to that of recombinant
ALV-B/J and was more similar to that of ALVS subgroup A such as those of MAV-l-like

viruses and recombinant ALVs with MAV-l-like gpSS. Moreover, the TM domains of all

135

AF 115 isolates showed similar sequences to that of ALV-B/J, subgroup E ALVS and
different ALV-E isolates and recombinant ALVS with subgroup E gp3 7. The 3’ UTR of
all AF 115 isolates showed high sequence similarity to those of recombinant ALV-B/J,
different ALV-J isolates and recombinant ALVS with subgroup J 3’ UTR containing a
DR1 region and E element. Moreover, all AF 115 isolates except AF 115-10 had a
3’LTRs, which were similar to those of ALV-B/J, ALV-J viruses, recombinant ALV-A/J.
On the other hand, AF 115-10 contained a 3’ LTR with similar sequences to that of
recombinant ALV among endogenous and different exogenous viruses. Some sequence
rearrangements including base insertions and deletions as well as mutations were
observed in the 3’ UTR and LTR of AF 115-10 exhibiting the addition and loss of
potential enhancer elements.

All AP 115 isolates contained conserved cysteine residues and potential N-linked
glycosylation sites in the SU and TM envelope glycoproteins needed for their proper
structure and function. The fusion peptides of all AP 115 isolates were similar to that of
ALV-B/J as well as other endogenous and exogenous viruses reflecting its importance in
retroviral life cycle. The putative transcription regulatory elements in the 3’ UTR and
LTR of all AF 115 isolates were mostly conserved. These included CCAAT motif, PU1
and PU2 core regions in the 3’ UTR and TATA box, PAS signal, CCAAT motif and
CCAAT-related LTR enhancer motif and two boxes of Y, CArG and PRE in the 3’ U3
region. These elements in all AF 115 isolates were similar to those of exogenous ALVS in
number and sequence. When compared with ALV-J, these isolates showed deletion in

one of two adjacent repeated motifs in the U3 region of the 3’ LTR. AF 115-10 contained

136

extra enhancer elements in the rearranged sequences (base insertions) of U3 region of the

3’ LTR and had more mutations in the enhancer elements than the rest of the isolates.

INTRODUCTION

Avian leukosis viruses (ALVS) belong to the avian leukosis-sarcoma group of
viruses (ALSVS). They are classified into exogenous subgroups A, B, C, D, J and
endogenous subgroup E in chickens (Fadly and Payne, 2003). Subgroup A is frequently
detected in commercial layers while ALV-B is occasionally encountered and both mainly
cause lymphoid leukosis (LL). Subgroups C and D are rare in field flocks and the
endogenous subgroup E is found in most commercial chickens. Since its discovery,
subgroup J has caused huge economical losses to the poultry industry and is usually
associated with myeloid leukosis (ML) in broilers (Payne, 1998).

Viral strains in ALSV are divided into acute or slow transforming viruses (Fadly
and Payne, 2003). Acute transforming viruses such as Rous sarcoma virus (RSV) and
avian myeloblastosis virus (AMV) are defective and require helper viruses for replication
(Dougherty, 1987). On the other hand, Rous associated viruses 1 (RAV-1) and 2 (RAV-
2) are slow transforming viruses and prototypes of subgroup A and B helper viruses for
RSV respectively (Hanafusa, 1965). Myeloblastosis associated virus-1 (MAV-l) is a
subgroup A helper virus for AMV (Baluda et al., 1983; Moscovici and Vogt, 1968).

Like other retroviruses, ALV is known for its genetic diversity including
mutations and recombinations (Coffin, 1992; Katz and Skalka, 1990; Mansky, 1998).
This diversity is exhibited by emergence of new mutant and recombinant strains with

altered characteristics. For example, variations in the env gene and long terminal repeat

137

(LTR) may affect virus-host receptor interaction and virus replication respectively (Boral
et al, 1989; Celander and Haseltine, 1984; Golemis et al., 1990). In addition, molecular
variations in different regions of ALV genome may lead to difficulties in diagnosis,
prevention and control of ALV infections (Fadly and Smith, 1999; Silva et al., 2000;
Venugopal et al., 1998).

Subgroup J ALV is a recombinant ALV with gag and pol genes similar to
exogenous ALSV and an env gene nearly identical to an endogenous retrovirus EAV-HI
(Bai et al., 1995b; Benson et al., 1998a; Silva et al., 2000; Smith et al., 1999; Venugopal,
1999). Three experimental recombinant ALVS with recombinant envelopes of subgroups
A and E and subgroup J LTRs were previously described (Lupiani et al., 2000). A
naturally occurring recombinant ALV (AF 115-4) containing the a subgroup B gpSS
region and a subgroup E gp37 region and a subgroup J LTR (ALV-B/J) was isolated from
commercial layer flocks affected with ML (Gingerich et al., 2002; Lupiani et al., 2006).

We previously showed that seven other isolates (AF 115-1, AF 115-5, AF 115-7,
AF 115-10, AF 115-13, AF 115-14 and AF 115-16) from the same field case were similar
to the naturally occurring recombinant ALV-B/J by biological assays and PCR.
Biological assays suggested the presence of an exogenous ALV other than subgroup J
probably having a subgroup B envelope. AF 115 isolates were PCR amplified with
different primer sets specific for ALV-J, ALV-B and ALV-B/J sequences. Moreover,
PCR data suggested that four isolates (AF 115-l, AF 115-13, AF 115-14 and AF 115-16)
were similar to ALV-B/J while AF 115-5, AF 115-7 and AF 115-10 were more diverse.

Various molecular approaches have been used for studying retroviral diversity

and characterizing new and emerging viral isolates (Mansky, 1998). Polymerase chain

138

reaction (PCR) can be used to specifically amplify different regions of ALV genome,
which can then be cloned and sequenced. DNA sequence analysis has been used to
identify, characterize and establish relationships among ALV variants (Bai et al., 1995b;
Reddy et al., 1983; Venugopal et al., 1998). Moreover, it can facilitate the study of the
genetic variations within different regions of ALV genome (Bai et al., 1998a and b; Bova
et al., 1986 and 1988; Chesters et al., 2002 and 2006; Hue et al., 2006; Zachow and
Conklin, 1992).

The main aim of this study is to molecularly characterize the seven AF 115
isolates of a naturally occurring recombinant ALV by DNA sequence analysis. Sequences
of various genomic regions of seven AF 115 isolates including env (gp85 and gp3 7),
3’UTR and LTR will be compared with prototypes of ALV subgroups. These molecular
analyses may reveal how and to what extent different ALV subgroups have contributed to
the origin and unique features of these viruses. It is also necessary to compare both
envelope SU and TM amino acid sequences and 3’ UTR and LTR DNA sequences with
related sequences through NCBI blast search. This will be carried out to understand the
molecular basis and significance of the emergence of these recombinants. Moreover, it
will more accurately establish the evolutionary relationships of AF 115 isolates with
other related viruses as well as the classical ALV subgroups.

Three general hypotheses were tested in this research work. 1) The subgroup
designation of different recombinants will be identified and confirmed by sequencing
different genomic regions of these viruses. 2) Rearrangements (deletions and insertions)
and mutations in different genomic regions of recombinant ALV can predict associations

with different ALV strains. 3) Sequences in different genomic regions of recombinant

139

ALVS can be used to speculate their stability, presence of potential regulatory elements
and recombination hotspots or boundaries among different ALV subgroups. The
objectives were to: 1) Compare the sequences of various genomic regions of seven AF
115 isolates including env (gpSS and gp37), 3’UTR and LTR with prototypes of ALV
subgroups to identify and confirm the subgroups of these recombinant viruses. 2)
Compare SU and TM amino acid sequences and 3’ UTR and LTR DNA sequences of
these isolates with similar blasted ALV strains to predict relevant associations and
relationships. 3) Compare cysteine residues and predicted N-linked glycosylation sites in
the SU and TM glycoprtoeins as well as transcription regulatory elements in the 3’ UTR
and LTR of these isolates with those of other ALVS to draw relationships and predict

stability and recombination hotspots.

MATERIALS AND METHODS
A. Viruses and Cells

All seven AF 115 isolates (AF 115-1, AF 115-5, AF 115-7, AF 115-10, AF 115-
13, AF 115-14 and AF 115-16) were propagated in line 0 CEFS as described previously
(Fadly and Witter, 1998). Briefly, around 100 pl of each undiluted viral stock (kept at -
80° C) was added to 0.5 x 106 secondary CEFS suspended in 4% calf serum (CS, Sigma
Chemical Co, St. Louis, MO) in 1:1 Leibowitz’s L-15-McCoy’s 5A tissue culture
medium (LM, Sigma Chemical Co, St. Louis, MO) containing penicillin/streptomycin
(Sigma Chemical Co, St. Louis, MO), 2 ug/ml amphotericin B and 2 rig/ml DEAE-

Dextran in 35-mm tissue culture plates. Twenty four hours later, the 4% CS LM media

140

was replaced with 1% CS LM media and the plates were incubated in 4% CO2 at 37° C

for 7-9 days.

B. DNA Extraction

Total genomic DNA was extracted from infected cells using standard proteinase
K, phenol-chloroform extraction procedures (Maniatis et al., 1982). Briefly, the medium
was removed and the cells were scraped off the plate and pelleted by centrifugation. The
pellet was treated with lysis buffer (10 mM TRIS, 1 mM EDTA, 100 mM NaCl and 0.5%
SDS) and proteinase K (.100 ug/ml) overnight at 56°C. DNA was extracted twice with
phenol-chloroform, treated with RNAse (2 ul/ml) at 37°C for an hour, precipitated with
ice-cold absolute ethanol, dried at room temperature, dissolved in TE buffer and stored at
4°C. The extracted DNA from the seven different samples was used as templates for

polymerase chain reaction (PCR).

C. Amplification of Proviral DNA

To broadly and more extensively investigate the relationship between the seven
AF 115 isolates, the naturally occurring recombinant ALV-B/J and known prototypes of
various ALV subgroups, PCR was performed on extracted DNA using primer set (RS-7]
& R8). DNA from AF 115-1 and AF 115-10 isolates were additionally amplified using
two other sets of primers (ARK 4823 & SRB-R, SRB-F & R8). This was done to ensure
that similar sequences were obtained. The sequence and annealing locations of these
primers are shown in Table 3.1. The primer sets in combination (ARK 4823 & SRB-R,

SRB-F & R8) or alone (RS-7J & R8) were expected to yield PCR amplified fragments

141

extending roughly from near the 3’ end of the polymerase gene to the 5’ end of US of
LTR. These will amplify sequences spanning all of the variable and hypervariable
regions of gp85, the relatively conserved gp37 region and 3’ UTR and LTR regions.

Three independent PCR reactions are canied out on each of the DNA samples of
line 0 CEFS inoculated with AF 115 isolates. Each SO-ml PCR reaction contained lx
reaction buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 and 0.001%
gelatin), 25 mM of each dNTP, 25 pmoles of each primer, 2.5 units Taq polymerase, and
around 100 ng of template DNA. The same PCR conditions were used with all the primer
pairs. Following an initial template melting or preheating step at 95°C for 3 min, the
DNA was amplified during 30 cycles with denaturation at 95°C for 1 min, primer
annealing at 57°C for l min, and amplification or extension at 72°C for 2 min, followed
by a final elongation step at 72°C for 10 min and cold storage at 4°C. This was carried
out in PCR thermocycler, PTC-l 00 Programmable Thermal Controler, Peltier-effect

Cycling (MJ Research, Inc., Waltham, MA).

D. Subcloning of PCR Products

Amplified PCR products were resolved by electrophoresis through 1% agarose
gel in 1X TAE buffer containing 200 ng/ml ethidium bromide and examined under
ultraviolet transillumination. PCR products were purified by agarose gel separation and
extraction from gel slices using the GeneClean®111 kit (MP Biomedicals, Solon, OH).
Then, the purified PCR fragments were blunt-ended using PFU polymerase and
subcloned into pCR®-Blunt vector (Invitrogen, Carlsbad, CA) following manufacturer’s

instructions. The ligated PCR insert into the vector was transformed using One Shot ®

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TOP] 0 competent cells (E. coli) following manufacturer’s instructions. Several
antibiotic-resistant colonies or transforrnants were selected and plasmid DNA isolated.
Restriction analysis using EcoR I was used to confirm the presence of the desired insert.
Three individual positive clones or transformants from each of three independent PCR
products of AF 115 isolates were selected for sequence analysis. Plasmid DNA was
isolated and purified using PureLinkTM Quick Plasmid Miniprep kit (Invitrogen,

Carlsbad, CA).

E. Sequence Determination

The DNA sequences of each clone were determined using several forward and
reverse primers designed with Oligo 4.0-3 software (National Biosciences, Plymouth,
MN). DNA sequencing was carried out using the BigDye terminator cycle sequencing kit
(Applied Biosystems, Foster City, CA) on an ABI PRISM® 3100 genetic analyzer
(Applied Biosystems, Foster City, CA). The sequence conti gs were constructed using

SequencherTM version 4.5 (Gene Codes Corp., Ann Arbor, MI).

F. Analysis of Sequence Data

The proviral DNA sequences of 3’ UTR and LTR and deduced amino acid
sequences of SU (gp85) and TM (gp3 7) of the envelope of all viruses were compared
using ClustalW multiple sequence alignment algorithm of MegAlign program
(DNASTAR Inc., Madison, WI). The ClustalW slow accurate method was used for
sequence analysis since it is the most accurate and effective for typical multiple

alignment when the similarities among sequences extend over a majority of their length.

143

It also leads to refinement of sequence alignments for phylogenetic analysis. The DNA
sequences and deduced amino acid sequences of all AP 115 isolates were compared
among each others, with the naturally occurring recombinant ALV-B/J (AF 115-4), with
prototypes of various ALV subgroups and with related published sequences of viral
strains from GenBank using the BLAST program from NCBI.

Phylogenetic relationships of the isolates were generated using the neighbor-
joining (NJ) distance-based method utilized in MegAlign. The sequence identity and
divergence in sequence distances between viruses were used for the construction of a
Cladogram. These phylogenetic trees (cladograrns) are rooted trees, where branch
distances correspond to sequence divergence. The length of each pair of branches
represents the distance between sequence pairs, while units at the bottom of the tree
indicate the number of substitution events. The relevant accession numbers for sequences

of viruses used in the analyses are found in Table 3.2.

RESULTS
A. Analysis of sequence data

PCR products of DNA of line 0 CEF infected with the AF115 isolates using all
primer sets were of expected sizes. To characterize the genomic structure of seven AF
115 isolates, proviral DNA sequencing of PCR products spanning the env gene (gp85 and
gp3 7) and 3’ UTR and LTR was performed and analyzed. The DNA sequences of AF
115-1 and AF 115-10 using the primer sets (ARK 4823 & SRB-R, SRB-F & R8 in

V combination or RS-7J & R8 alone) were identical.

144

B. Sequence comparisons among AF 115 isolates
1. Comparison of the envelope SU glycoprotein (gpSS)

Compared to ALV-B/J, the amino acid sequences of gp85 region of all AF 115
isolates except AF 115-5 were found to be conserved but scattered amino acid
substitutions were observed (Figure 3.1). The N- and C-terminal SU amino acid
sequences of all AF 115 isolates were conserved and identical to ALV-B/J (AF 115-4).
Conversely, the middle sequences of the isolates showed varying degree of amino acid
substitutions in conserved and variable (vrl , vr2 and W3) and hypervariable regions (hrl
and hr2). For example, AF 115-16 showed sequence diversity only in vrl . On the other
hand, AF 115-5 showed the most sequence variations in all variable and hypervariable
regions when compared with the rest of the isolates and ALV-B/J.

There were 14 conserved cysteine residues in the gp85 of all seven AF 115
isolates as well as ALV-B/J (Figure 3.1). One of the conserved cysteine residues was in
hrl and two were in hr2 in all AP 115 isolates. Moreover, the SU envelope glycoproteins
of three AF 115 isolates (AF 115-7, AF 115-13 and AF 115-16) contained 14 potential N-
linked glycosylation sites (NXS/T). The remaining four AF 115 isolates (AF 115-1, AF
115-5, AF 115-10 and AF 115-14) in addition to ALV-B/J (AF 115-4) contained 15
possible N-linked glycosylation sites (NXS/T) in the SU envelope glycoproteins (Figure
3.1). Twelve of these likely glycosylation sites were conserved in all AF 115 isolates and
ALV-B/J, one of which was in hrl (NSS).

Two of the conserved probable glycosylation sites were consecutive just upstream
of hrl of all isolates and ALV-B/J. Upstream of vrl, a possible glycosylation site (NCT)

was found in all isolates and ALV-B/J except AF 115-7, where asparagine (N) was

145

replaced by lysine (L). One potential site (NW S) was in hr2 of all isolates and ALV-B/J
except AF 115-5. Another likely glycosylation site was observed downstream of hr2 in
five AF 115 isolates and ALV-B/J. However, AF 115-13 and AF 115-16 had an amino
acid substitution of aspartic acid (D) instead of asparagine (N). Downstream of vr3 of AF
115-5, a probable glycosylation site not present in the other isolates of ALV-B/J was
observed where N substituted for serine (S).

Simple comparison of sequence distances displaying the divergence and percent
identity values among gp85 amino acid sequences of seven AF 115 isolates and ALV-B/J
are shown in Table 3.3. The SU-encoding sequences of six AF 115 isolates showed
95.7%-99.7% identity to each other and 96.3%-99.1% to naturally occurring recombinant
ALV-B/J (AF 115-4). However, AF 115-5 was a unique isolate, having very low gp85
sequence identity with the other six AF 115 isolates (79.4%——81.8%) and ALV-B/J
(79.1%). The degree of similarity ranged from 79.1% (AF 115-5 and ALV-B/J) up to the
highest similarity between isolates AF 115-10 and AF 115-14 at 99.7%. Conversely, the
highest divergence was observed when AF 115-5 was compared with the rest of the AF
115 isolates and ALV-B/J (20.9%-24.6%).

Figure 3.2 demonstrate the relationships of gp85 amino acid sequences of seven
AF 115 isolates and the naturally occurring recombinant ALV-B/J (AF 115-4). All AF
115 isolates and ALV-B/J (AF 115-4) formed a separate group with AF 115-16 clustering

the furthest and AF 115-5 being in a distinct group.

2. Comparison of the envelope TM glycoprotein gp37

146

The alignment of TM or gp37 amino acid sequences of the seven AF 115 isolates
and the naturally occurring recombinant ALV-B/J (AF 1 15-4) are shown in Figure 3.3.
Compared to ALV-B/J, all amino acid sequences of gp37 region of all AF 115 isolates
were conserved and identical to ALV-B/J. Compared to ALV-B/J. Single amino acid
substitution was observed in gp37 of four of the isolates (AF 115-7, AF 115-10, AF 115-
14 and AF 115-16). There were 9 conserved cysteine residues in the gp37 of all seven AF
115 isolates as well as ALV-B/J (Figure 3.3). Moreover, all TM envelope glycoproteins
studied contained only 3 potential conserved N-linked glycosylation sites (N XS/T ).

By analogy with the conserved sequence of fusion peptide (FP) in ALSV
subgroup A envelope, a conserved sequence was observed in the TM domain of all AF
115 isolates and ALV-B/J (Figure 3.3). Similar to ALSV-A, two conserved cysteines
flanking the internal fusion peptide were observed in all AP 115 isolates and ALV-B/J. In
addition, a conserved proline residue was observed in the middle of the predicted fusion
peptides of all AF 115 isolates and ALV-B/J.

The percentage identity and divergence among gp37 amino acid sequences of all
AF 115 isolates and ALV-B/J are shown in Table 3.4. The TM-encoding sequences of all
seven AF 115 isolates showed 99%—100% identity to each other and 99.5%-100% to
naturally occuning recombinant ALV-B/J (AF 115-4). Figure 3.4 demonstrate the
relationships of TM glycoprotein sequences of seven AF 115 isolates and the naturally
occuning recombinant ALV-B/J (AF 115-4). All AF 115 isolates clustered very close to

ALV-B/J (AF 115-4).

3. Comparison of the 3’ UTR and LTR

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Compared with the naturally occurring recombinant ALV-B/J, all AF 115 isolates
had 3’ UTR containing a single DR1 region and an E element (Figure 3.5). The DNA
sequences of 3’ UTR of all AF 115 isolates were very similar to those of ALV-B/J.
However, scattered transition and few transversion nucleotide substitutions were
observed in most isolates particularly AF 115-10. Interestingly, AF 115-10 had a 5-
nucleotide deletion and a 6—base insertion in the E element. A putative CCAAT enhancer
(ACAAT) was observed in all AF 115 isolates and ALV-B/J at the junction of DR1 and
the E element.

Three potential C/EBP binding sites with one mismatch (in bold letter) compared
to the consensus sequence were identified in all AF 115 isolates and ALV-B/J (Figure
3.5). The two 5’ potential sites (core region of PUl) were overlapping with conserved
sequences (ATGGCCAAAT and ATAGAGCAAG) in six AP 115 isolates and ALV-B/J.
On the other hand, AF 115-10 had the following respective ATGGCTAAAT and
ATAGGGTAAG sequences for these sites. The 3’ possible C/EBP binding site (core
region of PU2) had the following conserved sequence GCTGCGAAAT in all AP 115
isolates and ALV-B/J except AF 115-10 (ACTACGAAAT).

Most of the DNA sequences of 3’ LTR of all AP 115 isolates were common with
those of ALV-B/J. (Figure 3.6). However, scattered transition and transversion
substitutions were mostly observed in U3 regions of AF 115-5, AF 115-7 and AF 115-10
isolates. Distinctively, there was a 13-base insertion in the U3 region of AF 115-10
(CT'I‘ACACAATAGC). The R region was identical in all AF 115 isolates and ALV-B/J .

Several potential transcriptional regulatory sites were found in the U3 region of all AF

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115 isolates and ALV-B/J. These included TATA, PAS, Y, PRE, CCAAT, CCAAT
related LTR and CArG boxes (Figure 3.6).

A CCAAT box was identified with the consensus sequence (GCAAT) in all AF
115 isolates and ALV—B/J except AF 115-10 (GCTAT). An additional CCAAT enhancer
element (ACAAT) was observed in the insertion of AF 115-10 (CT‘TACACAATAGC).
Close by, a CCAAT LTR related enhancer motif (TTAT/CGCAAT) was located in all
AP 115 isolates and ALV-B/J. The S’CArG box had the CC(T/A)TATAAGG sequence
while the sequence for the 3’ CArG box was CCT‘TATTAGG. A highly conserved TATA
box with a consensus sequence TATTI‘AA was found in all AF 115 isolates and the
naturally occurring recombinant ALV-B/J.

Two highly conserved Y boxes (ATTGG) were situated in two distinct sites in all
AP 115 isolates and ALV-B/J. Two closely situated PRE motifs or boxes (GGTGG) were
observed in all AF 115 isolates and ALV-B/J. The 5’ PRE box sequences overlapped
with the 5’ Y box sequences. AF 115-7 had two mutations, one in the 5’ PRE box
(GGTGA) and another in the 3’ PRE motif (GATGG). Similarly, AF 115-5 and AF 115-
10 had the same mutation in the 3’ PRE box as AF 115-7. A key polyadenylation signal
(AATAAA) was found in the 3’ distal end of U3 of all AF 115 isolates and ALV-B/J.

The 3’ UTR and LTR sequences of four AF 115 isolates showed 99.1%-99.6%
similarity among each other and high identity ranging fiom 99.2% to 99.8% to the
naturally occurring recombinant ALV-B/J (AF 115-4). Yet, AF 115-5, AF 115-7 and AF
115-10 exhibited 94.2%, 94.2% and 89.6% identity with ALV-B/J respectively. They
also showed 89.4%-94.4% identity with the other AF 115 isolates and higher identity

among each other (94.2%-96.2%) in the 3’ UTR and LTR regions (Table 3.5).

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These results were supported by the locations of the different isolates in the
cladograrn (Figure 3.7). All AP 115 isolates and ALV-B/J were in the same cluster except
AF 115-10 due to its sequence mutations and rearrangements in the 3’ UTR and LTR.
Moreover, the genetic variations noticed in the LTR proviral DNA sequences of AF 115-
5 and AF 115-7 placed them in between AF 115-10 and the rest of the AP 115 isolates

and the naturally occurring recombinant ALV—B/J.

C. Sequence comparisons of AF 115 isolates and ALVsubgroups
1. Comparison of the envelope SU glycoprotein gpSS

When the seven AF 115 isolates were compared with representative viruses from
different ALV subgroups, the amino acid sequences of gp85 regions of ALV-B/J and all
AP 115 isolates except AF 115-5 were found to be very similar to RAV-2 and RSV-SR—B
(subgroup B) particularly in the variable and hypervariable regions (Figure 3.8). On the
other hand, AF 115-5 shared common sequences with RAV-1 in the vrl, vr2, vr3, hrl
and hr2 clusters. In addition, different ALV subgroups exhibited sequence variations
mostly in the five clusters (vrl, vr2, vr3, hrl and hr2). Subgroup J ALV has the most
diverse sequences in these five clusters as well as other regions of gp85 when it was
compared with the other ALV subgroups.

The14 conserved cysteine residues in the gpSS of all seven AF 115 isolates and
ALV-B/J were found to be conserved in all ALV subgroups except ALV-J. Nine
potential N-linked glycosylation sites were conserved in all AF 115 isolates, ALV-B/J
and ALV subgroups A-E. However, there were only five conserved potential N-linked

glycosylation sites (NXS/T) in all AF 115 isolates, ALV-B/J and all ALV subgroups.

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Moreover, there were 14 or 15 potential N-linked glycosylation sites in SU glycoproteins
of all AF 115 isolates and ALV-B/J. On the other hand, there were 11 potential sites in
RAV-1 (subgroup A), 14in Pr-RSV-C (subgroup C) and RSV-SR-D (subgroup D), 15 in
RAV-2 (subgroup B), 15 in ALV ev-6 (subgroup E) and 18 in HPRS-103 (ALV-J).

The deduced amino acid sequence of the surface (SU) glycoprotein (gp85) of all
AF 115 isolates exhibited the highest identity (93.1%—98.8%) to RAV-2 and RSV-SR-B
(subgroup B) except AF 115-5 (Table 3.6). The amino acid sequences of gp85 of AF
115-5 showed 89.4% similarity to RAV-1 (subgroup A), 78-78.6% to RAV-2 and RSV-
SR-B (subgroup B), 85.2% to Pr-RSV-C (subgroup C), 83.4% to RSV-SR-D (subgroup
D), 84.1% to ALV ev-6 (subgroup E) and 35.5% to HPRS-103 (subgroup J).

Six AF 115 isolates and ALV-B/J (AF 115-4) clustered close to RAV-2 and RSV-
SR-B (subgroup B) in the phylogenetic tree. However, AF 115-5 was in a separate clade
closer to RAV-1, a subgroup A ALV (Figure 3.9). All these results suggested that there
are two classes of viruses characterized by different SU domains belonging to two

different ALV subgroups.

2. Comparison of the envelope TM glycoprotein gp37
I Compared to prototypes of various ALV subgroups, the amino acid sequences of
the TM of seven AF 115 isolates and the naturally occurring recombinant ALV-B/J (AF
115—4) were very similar to all ALV subgroups except HPRS-103, a subgroup J ALV
(Figure 3.10). Few scattered amino acid substitutions were observed in gp37 of all ALV
subgroups compared with AF 115 isolates. HPRS-103 showed the highest divergence in

amino acid sequences with the other viruses.

15]

Six of the 9 conserved cysteine residues in the gp37 of all seven AF 115 isolates
and ALV-B/J were found to be conserved in all ALV subgroups. Moreover, TM envelope
glycoproteins of subgroups A (RS-A), B (RSV-SR-B) and D (RSV-SR-D) contained 9
cysteine residues. On the other hand, there were 7 cysteines in subgroups C (Pr-RSV-C)
and J (HPRS-103) and 8 such residues in ALV-E (ALV ev-6). The 3 potential N-linked
glycosylation sites (NXS/T) conserved in the gp37 of all AF 115 isolates and ALV-B/J
were common to all other ALV subgroups except ALV-J (HPRS-103).

The conserved sequence of the internal fusion peptide (F P) in the TM domain of
all AP 115 isolates and ALV-B/J was identical to ev-6 and RSV-SR-D (Figure 3.10).
However, there was one amino acid substitution in PP of RS-A, RSV-SR-B and Pr-RSV-
C; but four residue changes in HPRS-103. The two cysteines flanking the internal fusion
peptide and the central proline were shared by all AF 115 isolates and ALV subgroups.

The TM-encoding amino acid sequences of all seven AF 115 isolates were
93.7%—95.1% identical with RS-A, 94.1%—95.1% with RSV-SR-B, 95.5%—96% with Pr-
RSV-C, 94.7%—95.6% with RSV-SR-D, 95.1%—98.5% with ev-6, and 56.9%—57.4%
with HPRS-103 (Table 3.7). All seven AP 115 isolates and ALV-B/J (AF 115-4) were
grouped closer to subgroup E (Figure 3.11). As expected, subgroup J ALV was in a
separate cluster than the rest of the ALV subgroups. Subgroups A, B and D clustered

together while ALV-E and ALV-C clustered closer to AF 115 isolates.

3. Comparison of the 3’ UTR and LT R

Since ALV-J and sarcoma viruses contain the 3’ UTR, sequence alignment of 3’

UTR of HPRS-103 and all AP 115 isolates and ALV-B/J was performed. The 3’ UTRs of

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all AF 115 isolates and ALV-B/J were similar to that of HPRS-103 with some
substitutions. They also shared a common DR1 region and a complete E element except
AF 115-10, which had a 5-nucleotide deletion and a 6-base insertion (Figure 3.12).
Similarly, all AP 115 isolates and ALV-B/J shared an identical CCAAT enhancer
(ACAAT) with HPRS-103 at the junction of DR1 and the E element. In addition, AF 115
isolates and ALV-B/J had the same core regions of PU1 and PU2 exhibiting one mutation
as that of subgroup J ALV. Sequence analysis of the 3’ UTRs of all AF 115 isolates
indicated the highest identity (95.6%-97.6%) with the same region of the ALV-J isolate
HPRS-103 (Table 3.8). All AF 115 isolates and ALV-B/J were in the same clade as
HPRS-103 except AF 115-10 (Figure 3.13).

Most of the proviral DNA sequences of 3’ LTR of all AF 115 isolates were most
similar to HPRS-103 except for 11—base deletion in the U3 region (Figure 3.14). When
compared with HPRS:103, this deletion removed one of the adjacent 11-base enhancer
repeat motifs, ATGGTATGATC. On the other hand, HPRS-103 exhibited two direct
repeat sequences (GTGGTATGAT and ATGGTATGATC). The R regions of all AF 115
isolates and ALV-B/J were identical to that of subgroup J ALV (HPRS-103), which
exhibited variations fi'om other ALV subgroups A, B, C and D (Figure not shown).

The putative transcriptional regulatory sites of all AF 115 isolates and ALV-B/J
were similar to those of ALV-J and shared by other exogenous ALVS (Figure not shown).
The U3 regions of all AF 115 isolates, ALV-B/J and all exogenous ALV subgroups
included two Y boxes, two PRE motifs, CCAAT LTR enhancer and two CArG boxes. On
the other hand, a CCAAT-like box was found in all AF 115 isolates, ALV-B/J and ALV

subgroups C and J. Moreover, AF 115-10 had one mutation in the CCAAT LTR

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enhancer, CCAAT-like box, and 3’ PRE motif in addition to an extra CCAAT box in the
13-base insertion. On the other hand, AF 115-5 had a mutated 3’ PRE box while AP 115-
7 contained two mutated PRE boxes. Some differences in the transcription regulatory
elements were observed between exogenous and endogenous ALVS (Figure not shown).
Subgroup E ALV (ev-l) had a CCAAT LTR enhancer box downstream of the CCAAT
LTR enhancer box of exogenous viruses. Only one CArG, Y and PRE boxes were present
in the endogenous ALV. The TATA box and PAS were well conserved in all AF 115
isolates, exogenous and endogenous ALVS.

The DNA sequences of the 3’ LTR of the four AP 115 isolates and ALV-B/J
showed high identity with HPRS-103 (subgroup J) ranging from 94.8% to 95.6% (Table
3.9). On the other hand, AF 115-5, AF 115-7 and AF 115-10 showed lower sequence
identities at 89.2%, 88.8% and 84% respectively. The AP 115 isolates were grouped into
two separate clusters (Figure 3.15). Four AF 115 isolates and ALV-B/J (AF 115-4) were
clustered close to subgroup J. On the other hand, a separate clade contained AF 115-5,

AF 115—7 and AF 115-10, being the most divergent.

D. Sequence comparisons of AF 115 isolates and blasted ALVstrains
1. Comparison of the envelope SU glycoprotein gp85

' Blast search of the amino acid sequences of the SU domains of all AP 115 isolates
revealed high similarity to those of viruses belonging to ALV subgroups A and B. The
sequence alignment suggested that the isolates fell into two classes and were very similar
to viruses related either to MAV-l or RAV-2. Six AF 115 isolates were RAV-2-like and

AF 115-5 was MAV-l -like based on sequence similarities in the five clusters of variable

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(vrl, vr2 and W3) and hypervariable (hrl and hr2) regions (Figure 3.16). More
specifically, six AP 115 isolates showed sequence similarities mostly to ALV-26,
followed by RSV-SR-B and RAV-2 and less to AMAV-2, all of which are subgroup B.
On the other hand, AF 115-5 showed most sequence identities to ALV-18, ALV
TymS_90, ALV-25 and ALV-48, followed by ALV-1, ALV-10, ALV-20 and less to
MAV-l, ALV LR-9 and RSA, all of which are subgroup A.

These results were backed up by the sequence distances data (Table 3.10) with
sequence identities between six AF 115 isolates with ALV-26 (94.3% to 97.7%), RSV-
SR-B (93.4%-96.8%), RAV-2 (92.8%-96.2%) and AMAV-2 (94.2%-95.1%). On the
other hand, AF 115-5 sequence identity with these NCBI published sequences ranged
between 78% and 79.9%. Yet, AF 115-5 exhibited sequence identity of 100% with ALV-
18, 99.7% with ALV TymS_9O and ALV-25, 99.4% with ALV-48, 99.1% with ALV-1
and ALV-20 and 98.8% with ALV-10.

Phylogenetic tree of these isolates and reference strains from the GenBank was
established. Figure 3.17 shows the similarities among AF 115 isolates, MAV-l-like and
RAV-2-like isolates already described in graphic format. The phylogenetic analysis
showed that AF 115-5 formed a tight cluster with the published sequence for MAV-l-like
viruses. The remaining six AF 115 isolates were clustered in the same branch with the

published RAV-2-like sequences.

2. Comparison of the envelope TM glycoprotein gp37
NCBI blast search of the amino acid sequences of the TM domains of all AF 1 15

isolates revealed high similarity to those of viruses belonging to ALV subgroup E. The

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sequence alignment suggested that all the isolates were very similar to endogenous
viruses such as subgroup E ALVS (ALV-E SD0501 and RAV-O), ev loci (ev-l, ev-3, ev-6
and ev-21) and recombinant ALVs (ADOL 6803A, ADOL 5701A, ALV TymS_9O and
ALV PDRC-3249) with subgroup E gp37 (Figure 3.18). Moreover, the firsion peptide
sequence was mostly conserved in TM domains of all viruses studied. Based on sequence
comparison in the gp37 of these isolates and published sequences, all AF 115 isolates
were 97.6%-98.5 with ALV-E SD0501 and ev-6, 97.1%-98% with ev-l, ev-3, ALV
TymS_90 and ALV PDRC-3249, 96.6%-97.6% with ADOL 6803A and ev-21, 96.1%-
97.1% with ADOL 5701A and 95.6%-96.6% with RAV-0 (Table 3.11). Phylogenetic tree
of these isolates and matched viral strains from the GenBank showed that all AF 115

isolates are mostly similar to different viruses containing subgroup E gp37 (Figure 3.19).

3. Comparison of the 3’ UTR and LTR

Blast search of proviral DNA sequences of the 3’ UTR of all AF 115 isolates
revealed high association with 3’ UTR of subgroup J ALVS (HPRS-103, UD2, UD-J 1,
ADOL-7501) and recombinant ALVS with subgroup J 3’ UTR (ALV TymS_90 and
ADOL 5701A). Six AF 115 isolates had DR1 and E element sequences similar to those
of ALV-J isolates and ADOL-5701A, a recombinant ALV-A/J. However, AF 115-10 had
similar sequences to ALV TymS_90 based in the rearranged regions (insertion) and
putative transcription elements (Figure 3.20). Specifically, the mutated potential
transcription factors binding sites (PU1 and PU2) in the 3’ UTR were similar in all

viruses studied. Interestingly, the 6-base insertion in AF 115-10 3’ UTR was identical to

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TymS_90 and similar to RSV-NTRE-7 td (recombinant virus between RAV-O and td
RSV-Pr-B) and RSV-td PR2257 (RSV-Pr-C).

The 3.’ LTR of six AF 115 isolates showed sequence similarities mostly to ALV-J
isolates HPRS-103, UD2, UD-J l , ADOL-7501 and recombinant ALV-A/J isolate ADOL-
5701A (Figure 3.21). On the other hand, AF 115-10 mostly showed similar sequences in
the 3’ LTR to ALV TymS_90, a recombinant virus with AEV-like 3’ U3. In the U3
regions, the mutations in the enhancer boxes and the 13-base insertion observed in AF
115-10 were identical to those of TymS_90. The ll-base deletion in U3 of all AF 115
isolates was shared with TymS_90, AEV E26, RSV-NTRE-7 td and RSV-td PR2257.
The transcription regulatory elements were present in all viruses studied.

The 3’ UTR and LTR of six AF 115 isolates exhibited higher sequence identities
with those of ALV-J isolates while that of AF 115-10 was more homologous to that of
ALV TymS_90 (Table 3.12). Most AF 115 isolates showed sequence identities ranging
92.7%- 95.7% with HPRS-103, 90.4%-93.4% with UD2, 89.7%-92.3% with ADOL-
5701A, 89.5%-91.5% with ADOL-7501 and 89.3%-91.4% with UD-J 1. On the other
hand, AF 115-10 sequence identity with these NCBI published sequences ranged between
87.5% and 90.3%. Moreover, AF 115-10 exhibited sequence identity of 97.2% with
TymS_90 while AF 115-5 and AF 115-7 showed higher sequence identity with this
isolate (94.3%) than ALV-J isolates (89.3%-93.6%).

Cladogram constructed with the sequences of the 3’ UTR and LTR of the seven
AF 115 isolates and other NCBI published matching sequences showed that four AF 115
isolates were associated with subgroup J ALVs while AF 115-10 was related to ALV

TymS_90 and AF 115-5 and AF 115-7 were in between the two (Figure 3.22).

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DISCUSSION

The data presented here document partial molecular structure of provrial DNA of
each of the seven AF 115 isolates including the env gene and 3’ UTR and LTR. The
proviral DNA of ALV is usually composed of gag, pol and env genes flanked by LTRs
(Bai et al., 1995a). Among different subgroups, the gag and pol genes are well conserved
while the env gene is highly diverged and the LTR sequences are relatively variable
(Payne, 2001). The major genetic diversity noted among AF 115 isolates were localized
in gp85 of env of isolate AF 115-5 and 3’ UTR and LTR of AF 115—10. This agrees with
previous observation that different regions of the genomic proviral DNA can evolve
independently (Cui et al., 2003a). The genetic diversity in different regions of the seven
AF 115 isolates is most likely to result from mutations in the viral RNA during
replication, selection by immune pressure and recombination events among multiple
ALV subgroups circulating in chicken flocks. This leads to the generation of novel
recombinant ALVS with different genome locations representing different subgroups.

The env gene of ALV encodes two functional regions, the surface (SU) and
transmembrane (TM) domains, which facilitate entry into the target cell. Moreover, it
influences the types of cell targets, and oncogenic spectrum of the virus (Brown and
Robinson, 1988; Chesters et al., 2002). For example,'subgroups A and B ALVs mostly
cause lymphoid leukosis while ALV-J induces myeloid leukosis in chickens (Bai et al.,
1995a, Payne et al., 1992b). Recombinant viruses are believed to be more pathogenic

than their parental counterparts (Aurigemma et al., 1991; Kogekar et al., 1987).

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The SU domain (gp85) of envelope glycoprotein recognizes and binds with
specific host cell receptor and determines subgroup specificity (Bai et al., 1995a; Bova et
al., 1986 and 1988; Bova-Hill et al., 1991; Domer and Coffin, 1986; Hunter, 1997; Rong
et al., 1997). On the other hand, the TM subunit (gp3 7) anchors the SU glycoprotein to
the viral membrane and mediates fusion of the viral and target cell membranes (Fadly and
Payne, 2003; Hernandez et al., 1996). Alignment of the deduced amino acid sequences
among AF 115 isolates revealed several substitutions distributed throughout the gp85
subunit and four in the gp37 subunit. However, the importance of these amino acid
substitutions is not known.

The SU (gp85) of env is known for its considerable genetic variations leading to
antigenic diversity and altered phenotype. Compared to ALV-B/J and other AF 115
isolates, AF 115-5 showed the highest diversity in amino acid substitutions in these five
variable clusters while AF 115-16 exhibited similar variation only in WI region This
genetic divergence displayed by AF 115-5 may reflect changes in host range, subgroup
specificity and receptor interaction. Similarly, the env gene of NTRE-4 was shown to be a
composite of subgroup B hrl region and subgroup E hr2, which infected both C/E and
T/BD cells (Domer et al., 1985; Tsichlis et al., 1980). Moreover, subgroup J ALV
showed high degree of variability in the gp85 region of env among different strains,
isolates and subtypes (F adly and Smith, 1999; Hunt et al., 1999; Silva et al., 2000;
Venugopal et al., 1998).

Despite high levels of sequence variability in gpSS SU of AF 115-5, all isolates
in addition to ALV-B/J contained 14 and 9 conserved cysteine residues in the SU gp85

and the TM gp37 glycoproteins respectively. Some of the amino acid sequences of ALV

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subgroups A-E are very similar containing conserved cysteine residues. These residues
are involved in intrachain disulfide bonds and believed to maintain the overall secondary
structure of retroviral envelope glycoproteins (Domer and Coffin, 1986; Linder et al.,
1992; Valsesia-Wittmann et al., 1994). These results may reflect the structural stability of
envelope glycoproteins of AF 115 isolates.

Like gpSS SU glycoprotein subunit, the gp37 TM glycoprotein subunit exists in
the virus particle as a trimer linked to SU through a disulfide bond (Delos and White,
2000; Einfeld and Hunter, 1988; Hernandez and White, 1998; Hunter et al., 1983). The
TM glycoprotein of ALSV generally contains a stretch of hydrophobic amino acids at an
internal site to the N terminus. This region represents the internal fusion peptide, which is
highly conserved among different subgroups and has been implicated in viral fusion
activity. Generally, a fusion peptide is a helix of 15 to 25 of relatively hydrophobic amino
acids in length, rich in alanine and glycine (White, 1992). A central helix-breaking
proline residue usually exists inside the internal fusion peptide. The predicted fusion
peptide in the TM domain was conserved in all AF 115 isolates and ALV-B/J. This
internal region contained a conserved central proline and was flanked by two conserved
cysteines. These cysteines are believed to be involved in forming a disulfide bond at the
base of the looped firsion peptide. In addition, these cysteine residues are hypothesized to
be essential for proper TM glycoprotein folding, processing, assembly into trimers and
fusion activity (Delos and White, 2000).

The envelope glycoproteins are heavily glycosylated especially the SU region
(Rong et al., 1997). For example, the SU of the ASLV-A enve10pe glycoprotein contains

11 potential N-linked glycosylation sites (NXS/T). There were 12 and 3 conserved

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potential N-linked glycosylation sites (NXS/T) in SU and TM of all AF 115 isolates as
well as ALV-B/J respectively. N-linked glycosylation is important for glycoprotein
function through proper folding (Ellgaard et al., 1999; Helenius and Aebi, 2001) and
stability (Fenouillet et al., 1990; Imperiali and O’Connor, 1999). Moreover, it has been
exploited by viral glycoproteins for immune evasion and receptor usage through direct
and indirect protein—protein interactions (Bolmstedt et al., 1996; Kinsey et al., 1996;
Reed et al.. 1997; Reitter et al., 1998; Schonning et al., 1996; Sjolander et al., 1996;
Willey et al., 1996). Two of the gp85 conserved glycosylation sites in these AF 115
isolates were consecutive, but they are not likely to be both utilized at the same time by
glycosylation enzymes due to steric interference (Wu et al., 1995). Some of the
glycosylation sites may be required for presentation of the adjacent cysteine for proper
disulfide bond formation (Delos et al., 2002). Some amino acid substitutions in SU
glycoprotein resulted in extra or less glycosylation sites in some of the AF 115 isolates.
Whether any of these potential sites are actually glycosylated is not known.

Sequence alignment and distance as well as the phylogenic tree revealed that the
SU glycoprotein of AF 115-5 is highly divergent from the rest of AF 115 isolates and the
naturally occuning recombinant ALV-B/J. Nonetheless, they showed thatthe SU amino
acid sequences of the rest of the AF 115 isolates were similar to each other and to the
ALV-B/J. In addition, sequences encoding the TM (gp3 7) domain of all AF 115 isolates
were highly homologous to each other and ALV-B/J from % identity data and cladograrn.

Since 3’ UTR and LTR were known to contain sequences important in regulating
transcription, translation, splicing, RNA stability, transport and processing; these regions

in AF 115 isolates were sequenced. PCR amplification patterns using primers targeted to

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different regions of 3’ UTR and LTR had suggested that mutations may have occurred in
many of these AF 115 isolates. The 3’ UTR and LTR sequences of all AF 115 isolates
except AF 1 15-10 were similar to the naturally occurring recombinant ALV-B/J.

The 3’ noncoding or untranslated region (3’ UTR) between the 3’ end of gp37 of
env and the LTR of ASLV is conserved and essential for efficient replication and viral
pathogenesis (Ogert and Beemon, 1998; Ogert et al., 1996; Simpson et al., 1997; Sorge et
al., 1983). It has an impact on the development of tumors particularly when it is subjected
to mutations and genetic rearrangements (Hue et al., 2006). Moreover, it contains
powerful regulatory elements, which influence viral gene expression (Bai et al., 1998a;
Robinson et al., 1982; Ruddell, 1995; Zachow and Conklin, 1992). This region is found
in some members of ASLV such as RSV and subgroup J ALV (Bai et al., 1995b and
1998a; Hue et al., 2006; Laimins et al., 1984; Tsichlis et al., 1982). The 3’ UTR
nucleotide sequences can provide information on the phylogenies of the ancestral ALSVS
(Onuki et al., 1987). The 3’ UTR of ALV J consists of a rTM insert, a direct repeat
(DR1) region and an E element (XSR).

Within 3’ UTR, the DR 1 was present and almost identical in all AP 115 isolates
and ALV-B/J. In fact, the DR1 (or F1) element is found at the 5’ end of 3’ UTR, as a
single copy in ALVS (Bai et al., 1998b; Lupiani et al., 2006; Silva et al., 2000). However,
two copies of DR1 are usually flanking the src gene in avian sarcoma viruses (Laimins et
al., 1984). This element has been associated with efficient accumulation and stability of
unspliced RNA in the cytoplasm (Ogert and Beemon, 1998; Ogert et al., 1996), genomic
RNA packaging (Aschoff et al., 1999; Sorge et al., 1983), virion production and

increased spliced src mRNAs in avian sarcoma viruses (ASVS).

162

Most AP 115 isolates shared similar E element with ALV-B/J. In general, the E
(XSR or F2) element is found as a single copy of a hairpin loop sequence downstream of
DR1 in ASVS and ALVS (Lupiani et al., 2006; Payne et al., 1991; Schwartz et al., 1983;
Silva et al., 2000; Skalka et al., 1983). The E element may influence the oncogenic
spectrum and incidence of some ALSVS through activation of cellular or viral oncogenes
(Bai et al., 1995b; Bizub et al., 1984; Laimins et al., 1984; Tsichlis et al., 1982). For
example, some ALVs containing the E element caused more fiequent tumor expression in
certain genetic lines of chickens including recombinant ALV-A/J (Chesters et al., 2006;
Lupiani et al., 2003). Similarly, NTRE-7, a recombinant virus between transformation-
defective Pr-B and RAV-0, was reported to have inherited E element from the RSV
parent (Tsichlis et al., 1982). On the other hand, the E element of AF 115-10 exhibited
few rearrangements (5-nucleotide deletion and 6-base insertion). Whether these
rearrangements in the E element have profound effects on AF 115-10 replication or
oncogenicity in vivo is not known. A putative CCAAT enhancer was observed in all AF
115 isolates and ALV-B/J at the junction of DR1 and the E element. A similar enhancer
element (ACAAT) was reported in the U3 region of ALV-J field isolates from different
countries (Zavala et al., 2007).

The secondary hairpin stern— loop structure of the E element provides a potential
nucleation site for RNA folding and multiple interactions with some host factors. For
example, the E element may contain potential enhancer elements (CCAAT) or responsive
elements (PU1 and PU2) with the consensus sequence A/C/GTT/GNNGC/A/TAAT/G
(Ryden et al., 1993). These elements bind CCAAT/enhancer-binding protein (C/EBP)

transcription factors, which regulate viral gene transcription and protein expression

163

(Baglia et al., 1997; Bowers et al., 1996; Smith et al., 1994). Potential enhancer elements
(C/EBP binding sites) were highly conserved in the E element of all AF 115 isolates and
ALV-B/J except AF 115-10. The two overlapping core regions of PUl sites are not likely
to be both utilized simultaneously by C/EBP transcription factors due to steric
interference. Although the core regions of PU1 and PU2 in all AF 115 isolates contained
one nucleotide mismatch, they can still be potentially functional. Similar results were
observed in different ALV-J field isolates from France (Hue et al., 2006). In addition,
PU1 and PU2 sites from Pr-C RSV exhibited one mismatch compared with the consensus
C/EBP binding site and were still functional and able to bind small amounts of C/EBP
transcription factors (Ryden et al., 1993).

The 3’ LTR of ALV contains a unique 3 (U3) region, a repeat (R) region and a
unique 5 (U5) region. Overall, the 3’ LTR of AF 115-10 was the least conserved among
the isolates and ALV-B/J, followed by AF 115-5 and AF 115-7. The R region was
identical in all AF 115 isolates and ALV-B/J. This was expected since this region is very
vital for reverse transcription when the reverse transcriptase enzyme recognizes a strong
stop signal in R at 5’ end of the virus and then jumps to the homologous R at the 3’ end.
The 5’ end of US was not sequenced.

The U3 region contains several conserved motifs or transcriptional regulatory
elements including promoters and enhancers. Thus, it is believed to play a role in ASLV
replication, infection and oncogenesis (Bowers et al., 1994; Ruddell, 1995; Ruddell et al.,
1989). Some of the conserved protein binding sequence elements in U3 of ASVS and
ALVS include TATA (Laimins et al., 1984), CCAAT (Baglia et al., 1997; Bowers and

Ruddell, 1992; Golemis et al., 1990), CArG (Zachow and Conklin, 1992), Y (Ruddell,

164

1995) boxes and Pentanucleotide repeat elements (PRES). There was a 13-base insertion
in the U3 region of AF 1 15-10 just upstream of the CCAAT enhancer motif. However,
the transcriptional regulatory elements typical of avian retroviruses (TATA, PAS,
CCAAT, CCAAT LTR enhancer, CArG, PRE and Y boxes) were found in all AF 115
isolates and ALV-B/J.

Toward the 3’ end of U3 of all AF 115 isolates and ALV-B/J, there were a highly
conserved TATA box and a key polyadenylation signal (PAS). TATA boxes and TATA-
related motifs are promoters located at the 3’ end of U3 and are involved in initiation of
transcription of ASLVs (Cullen et al., 1985). On the other hand, the key polyadenylation
signal (AATAAA) corresponds to the AAUAAA polyadenylation signal in the RNA
genome of ALSV (Golemis et al., 1990; Guntaka, 1993). CCAAT boxes and CCAAT-
related motifs are enhancer elements, to which transcription factors bind leading to
enhanced gene transcription (Bowers et al., 1996; Cunistin et al., 1997; Ruddell, 1995;
Smith et al., 1994). An additional CCAAT LTR enhancer element with the consensus
sequence 5’T(T/G)NNG(C/T)AA(T/G)3’ is found in the 3’ U3 of RSV and a target for
the Al/EBP and VBP (bZip) DNA binding factors (Smith et al., 1994; Zachow and
Conklin, 1992).

CArG boxes (5’CC(A/T)6GG3’) are enhancers and binding sites for enhancer
factor III (EF III) implicated in mediating transactivation of the RSV LTR enhancer
(Boulden and Sealy, 1990). In the host, CArG boxes are involved in tissue-specific
cellular gene expression (Ruddell, 1995). Y boxes (5’ATTGG3’) are inverted
5’CCAAT3’ enhancer boxes in ASVs and some ALVS (Laimins et al., 1984). They bind

enhancer factor I (EFI) associated with the activation of viral transcription (Greuel et al.,

165

1990; Zachow and Conklin, 1992). Conserved inverse Y boxes (5’ATTGG3’) of ALV-J
are targets for YB-l/EF I DNA binding factors in chickens (Grant and Deeley, 1993).
PRE motifs (5’GGTGG3’) may bind early proteins such as the infected cell protein 4
during Marek’s disease virus (MDV) infection and lead to transactivation of ALSV LTR
(Banders and Coussens, 1994).
All AF 115 isolates and ALV-B/J contained a CCAAT box and a conserved
CCAAT LTR enhancer motif, two PRE motifs, and two conserved CArG and Y boxes.
The presence of duplicate number of these elements in the LTR of AF 1 15 isolates is
consistent with exogenous viruses such as ALV-J (Zavala et al., 2007). LTRs of
endogenous viruses carry weaker or a lower number of enhancers and are thus
tanscriptionally silent (Ruddell, 1995). However, AF 115-10 contained two mutated
CCAAT boxes and 3’ PRE motif while both PRE boxes of AF 115-7 had one mutation
and AF 115-5 contained a mutated 3’ PRE motif. Mutations in PRE boxes have been
reported from different ALV-J isolates fiom France (Hue et al., 2006).
These elements may be targeted by various DNA binding proteins (transcription
factors) to enhance transcription, gene expression and c-myc activation. Some of the
enhancer motifs in the U3 of the LTR of these isolates may act in synergy with the core
regions of PU1 and PU2 binding sites of the E element similar to what has been
suggested for ALV-J isolates (Hue et al., 2006). Several studies have reported that
mutations in some of these enhancers such as CCAAT boxes and CCAAT-related LTR
enhancers negatively affected LTR-driven transcription in transient infections in CEF or
abolished binding of several transcription factors (Bowers and Ruddell, 1992; Ryden et

al., 1993; Smith et al., 1994). Further studies are required to confirm the firnctional

166

potential of these sites (wild type and mutated) as enhancers and targets for DNA-binding
proteins. The conserved sequences in some of these putative sites can be targeted by
primers to improve molecular diagnosis of ALV. The rate of replication and the
frequency of tumors (pathogenicity) of different isolates may be variable since these
elements were conserved in some but not all the isolates and ALV-B/J.

Since 3’ UTR and LTR do not code for structural proteins like the env, it can be
used to determine virus lineages based on genetic sequences not subject to immune
pressure (Zavala et al., 2007). The sequence distances and phylogenies revealed that the
3’ UTR and LTR of all AP 115 isolates except AF 115-10 were similar to ALV-B/J. The

diversity exhibited by the 3’ UTR and LTR of AF 115-10 may have contributed to the
moderately distinct genetic lineage of this variant virus.

Sequence variability in SU (gp85) of the envelope is usually clustered in five
regions, the variable regions vrI, vr2, vr3 as well as the highly variable larger clusters hr]
and hr2 (Bova et al., 1986). The hr] and hr2 regions determine receptor binding
Specificity while the W3 region is responsible for receptor recognition (Bova et al., 198 8).
Sequence comparison based on the five clusters of the SU region (gp85), sequence
distances and phylogenetic trees of all AF 115 isolates with known ALV subgroups
indicated that they belonged to subgroup B except AF 115-15, which belonged to ALV-

A. All AP 115 isolates shared 14 conserved cysteine residues with all ALV subgroups in
the gpSS. There were 14 conserved N-linked glycosylation sites in the SU proteins of six

AF 115 isolates, ALV-B/J and subgroup B ALV. However, the SU of AF 115-5 shared 9
conserved sites with ALV-A.

167

Similar analyses for the TM portion (ng 7) of these isolates suggested they had
similar sequences to ALV subgroups A-E. The ng7 sequences of ALV subgroups A-E
show high sequence identity with few amino acid substitutions. On the other hand, ALV—
J is more diverse and exhibit variable gp37 sequences when compared with the other
subgroups (Bai et al., 1995b and 1998b). Moreover, subgroup J ALV contains a
nonfunctional redundant TM (rTM), which is a partially duplicated copy of the TM
region (Bai et al., 1995b). In fact, all AF 115 isolates shared 6 conserved cysteine
residues with all ALV subgroups in the gp37. There were 2 conserved N-linked
glycosylation sites in the TM proteins of all AF 115 isolates and ALV subgroups.

Since it was established that all AP 115 isolates like ALV-B/J had a 3’ UTR,
these sequences were compared to those of ALV-J (HPRS-103). All AF 115 isolates
showed similar 3’ UTR sequences, % identity, phylogeny and potential enhancer
elements to ALV-J including CCAAT box at the junction of DR1 and E element and core
regions of PU1 and PU2 in the E element. However, AF 115-10 showed sequence
divergence in the E element (5-base deletion and 5-base insertion) compared with ALV-J .

The proviral DNA sequences of 3’ LTR of all AP 115 isolates were similar to
various exogenous ALV subgroups particularly subgroup J. They shared similar
transcription regulatory elements (TATA, PAS, CCAAT and CCAAT related LTR
enhancer motifs, two CArG, two Y and two PRE boxes). However, all AF 115 isolates
had ll-base deletion compared with HPRS-103, which was responsible for deletion of
one of the adjacent repeat elements. These adjacent repeated motifs have been reported to
be variable in numbers in different viral strains of ALSV (Bai et al., 1995b; Patschinsky

et al., 1986). The recombinant avian carcinoma virus MH2E2] has three adjacent repeats,

168

the prototype of subgroup J ALV (HPRS-103) and ASV CTlO has two and ASV Y73 has
one. The variable numbers of these direct repeats in some viral strains have been
hypothesized to be associated with increased virulence or pathogenicity in the host.
Similar sequence alignment was observed among the R and U3 regions (1 l-base deletion)
of all AF 115 isolates and HPRS-103 except for a 13-base addition in AF 115-10.

To confirm the relationships between different AF 115 isolates and different ALV
subgroups in different genomic region, a blast search was performed using sequences of
the envelope and non-coding regions. The sequence comparison of the seven AF 115
isolates with similar NCBI sequences suggested that these viruses are recombinant ALVS.
GenBank blast search for sequences similar to AF115 isolates revealed that the SU
domains of six of these isolates were RAV-2-like and that of AF 115-5 is MAV-l-like.
RAV-2 (subgroup B env) and MAV-l (with an envelope antigenically related to ALV—A)
are laboratory propagated strains isolated from stocks of defective RSV or AMV
associated with LL, ML and other tumors in chickens (Dougherty, 1987). The gp85 of
AF 115-5 showed high association with one (ALV TymS_90) of ten MAV-l-like isolates
from commercial layers associated with fowl glioma in Japan (Hatai et al., 2008).

There were also similarities between our isolates and ALV isolates in the SU
region from another study. The MAV-l-like isolates (ALV-1, ALV-10, ALV-20, ALV-
18, ALV-25, ALV-48) and RAV-2-like isolate (ALV-26) related to our AF 115 isolates
were isolated fiom white leghorn commercial layers in a farm in Canada between 1997
and 1999 (Spencer et al., 2003). It was interesting that our AF 115 isolates came from
white leghorn commercial layers from a farm in Canada between 1997 and 1998.

Sequence analyses of the related viruses revealed that six isolates contained MAV-l-like

169

viruses, three had RAV-2-like viruses and one contained a mixture of the two viruses
(Spencer et al., 2003).

A blast search demonstrated that the TM domains of all AF 115 isolates were
highly homologous to subgroup E ALVS (RAV-0, SD0501, ev-l, ev-3, ev-6 and ev-21).
This was similar to results reported on the first naturally occurring recombinant ALV-B/J
(AF 115-4) fiom the same flocks, which had a gp37 belonging to subgroup E ALV
(Lupiani et al., 2006). Moreover, our isolates showed similar sequences to two
recombinant ALV-A/Js (ADOL 6803A and ADOL 5701A) possessing subgroup E gp37
TM regions, which were isolated fi‘om alv6 CEFS infected with subgroup J ALV (Lupiani
et al., 2000). The TMs of AF 115 isolates showed high identity to one isolate (ALV
TymS_90) associated with fowl glioma in layers in Japan, which also had TM belonging
to endogenous viral loci or subgroup E ALV (Hatai et al., 2008). Another virus that was
related to our isolates in the gp37 region was ALV PDRC-3249, a recombinant ALV with
subgroup A gp85 and subgroup E gp37 and LTR isolated from contaminated commercial
Marek’s disease vaccines (Barbosa etal., 2008)

Comparisons of 3’ UTRs and LTRs of six of the seven AF 115 isolates with
published NCBI sequences showed high association with viruses (HPRS-103, UD2, UD-
J1 and ADOL-7501) belonging to ALV-J and recombinant ALV—A/J (ADOL 5701A). On
the other hand, AF 115-10 showed similar sequences in the 3’ UTR and LTR to ALV
TymS_90, a recombinant ALV isolated fiom a case of fowl glioma in layers in Japan.
ALV TymS_90 had a MAV-l-like SU domain, an ev loci-like TM region, UTR and LTR
associated with different exogenous viruses (A, D and J for UTR and AEV E26 for U3)

and endogenous viruses in the R and U5 regions (Hatai et al., 2008).

170

It is clear fiom sequence comparisons that all seven AP 115 isolates were similar
to several ALV subgroups in different parts of the genome. AP 115 isolates are separated
from the other ALSVS by a number of recombination events although it is not clear how
those events have taken place. This raised the possibility that these isolates might have
formed from recombination events among different ALV subgroups in the field or
hatchery, where infected chicks were in close proximity with ALV-J positive meat-type
chicks. This confirms previous predictions of possible occurrence of natural
recombination among different ALV subgroups circulating in chicken flocks (Gingerich
et al., 2002; Luinani et al., 2003, Nichol, 1996).

The recombination boundary in all AF 1 15 isolates is likely to be between gp85
and gp37 sequences. Another recombination hot spot in these isolates is speculated to be
between gp37 coding region and 3’UTR sequences upstream of the DR1 region. These
recombination hot spots were also predicted in the naturally occuning recombinant ALV
with subgroup B SU domain, subgroup E TM region and subgroup J LTR (Lupiani et al.,
2006). In the case of AF 1 15-10, another recombination boundary may be between 3’
UTR and 3’ LTR sequences among exogenous or endogenous ALVS. For example, ALV
TymS_9O was characterized by sequence analysis to be a recombinant virus belonging to
different exogenous ALVs (subgroup A, B, C, D and J) and endogenous ALV (ev loci,
subgroup E ALV) in different genomic regions (Hatai et al., 2008).

In summary, DNA sequence analysis identified and confirmed that the seven AF
115 isolates were recombinant ALVS with different genomic regions representing
different ALV subgroups. This raised the possibility of the presence of potential

recombination hotspots or boundaries among different ALV subgroups in these

171

recombinant ALVS. Moreover, associations between AF 115 isolates and various ALV
subgroups and strains were predicted based on rearrangements including deletions and
insertions in the 3’ UTR and LTR as well as mutations in different genomic regions such
as gp85 and LTR regions of recombinant ALVS. Furthermore, the stability of the
envelope glycoproteins (SU and TM) were speculated based on the conserved cysteine
residues and predicted N-linked glycosylation sites in these sites. In addition, nucleotide
sequences in the 3 ’ UTR and LTR of AP 115 isolates of recombinant ALVS revealed the
presence of potential transcription regulatory elements, which were mutated in some
isolates and may affect efficiency of transcription of these viruses.

In conclusion, our data suggest that there is a big diversity in the seven AF 115
isolates obtained from a single outbreak of ML in commercial chickens. It is a very
significant finding since it is the first reported outbreak of recombinant ALVS in field
:flocks where various forms of recombinant ALVS were isolated and characterized (ALV-
A/J and ALV-B/J). This confirms that ALV is mutating and evolving very quickly
through recombination, which may have devastating consequences for the poultry
industry. Moreover, this will help in the current understanding of ALV epidemiology and
retroviral evolution. For example, new highly virulent strains of recombinant ALVS may
emerge in the filture, causing new disease problems, higher mortality and serious
production losses for the poultry industry. In addition to the importance of understanding
the molecular structure and relationships among different recombinant strains of ALV in
these AF 115 isolates, it is imperative to explore the virus-host interaction and
transmission of these viruses in biological systems. Future research experiments should

assess the pathogenicity (induction of viremia, shedding, immunity and oncogenicity) of

172

these isolates in the context of virus (dose and strain) and host (age at inoculation and
phenotype) factors.

This huge diversity in ALV isolates may limit the utility of the current diagnostic
procedures used for ALV characterization and negatively affects the prevention and
control of this economically important virus infection in chickens. Therefore, it is
important to incorporate DNA sequence analysis procedures into the current diagnostic
testing of chicken flocks and monitoring of breeders for ALV infection and eradication.
These DNA sequence data can also help in designing molecular diagnostics such as PCR
primers and probes for detection and diagnosis of various ALV isolates in breeders and
field infections. Moreover, the possibility that these recombinants have developed in the
hatchery reiterate the importance of implementing good management practices during
incubation, hatching and rearing of one type of chickens (egg-type or meat-type) from the
same source.

These molecular characterization data are valuable since it can contribute to
improving the design of expression vectors and vaccines. For example, the transcription
regulatory elements in the 3’ UTR and LTR such as promoters and enhancer including
the mutated forms can be incorporated in the design of expression vectors to study their
effect on the efficiency of transcription of cloned foreign genes. Moreover, the efficient
cloning of envelope genes into expression vectors will facilitate understanding their
expression in cells, their protein structure and function as well as their biological
consequences in the host. In addition, the insertion of immunostimulatory genes such as

envelope genes in vaccine vectors (recombinant or subunit vaccines) can allow the study

173

of the induction of cellular and humoral immune responses and protection against ALV

infection and tumor development.

174

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Table 3.2. Relevant accession numbers for viral sequences used in analyses

 

Virus Isolate Designation ALV Subgroup GenBank Accession

 

RAV-1 A AAA67094
RSA or RS-A A NP_040548
RSV-A A AAB50878
ALV LR-9 A AAQ55055
MAV-l A AAA46303
ALV—1 A AF507028
ALV-10 A AF507029
ALV-18 A AF50703O
ALV-20 A AF507031
ALV-25 A AF507032
ALV-48 A AANO6004
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3’ UTR, AEV E26 U3, subgroup E R and US.

176

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PU1 and PU2 are potential enhancer elements. Dark circles are under mutated enhancer residues.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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CHAPTER 4

Conclusions and Future Studies

INTRODUCTION

Avian leukosis virus (ALV) is one of the most field encountered avian
retroviruses associated with different kinds of neoplasms in chickens. ALV is usually
classified into six subgroups A, B, C, D, E and J. Infection with ALV constitutes a
continuous economical threat to the poultry industry due to the emergence of new
mutants and possibly recombinant viral strains. Current efforts for prevention and control
of ALV infection have been compromised by significant genetic and antigenic variations
in different strains and field isolates. This is due to the fact that the continuous emergence
of these new mutants and recombinants hinders current field diagnosis of ALV infection
by standard biological and molecular methods. Therefore, regular monitoring and
improvement of diagnostic methodology to include new ALV isolates, mutants and
recombinants will support the existing efforts for ALV eradication.

Information is currently limited on natural recombination among different ALV
subgroups and its impact on virus isolation, diagnosis and pathogenesis in chickens. The
research studies reported in this dissertation mainly focused on the biological and
molecular characterization of seven new isolates of a naturally occurring recombinant
avian leukosis virus (ALV) associated with myeloid leukosis (ML) in commercial layers.
In addition, these isolates were compared with the first naturally occurring recombinant

ALV-B/J at various levels.

209

CHAPTER 2 CONCLUSIONS

The overall goal of the research studies in chapter 2 was to characterize seven AF
115 isolates of a naturally occurring recombinant ALV (AF 115-1, AF 115-5, AF 115-7,
AF 115-10, AF 115-13, AF 115-14 and AF 115-l6) by biological assays and polymerase
chain reaction. The most significant finding for this research work was that closely and
distinctly related strains of naturally occurring recombinant ALVs were identified using
various diagnostic techniques. In fact, the host range and antigenic relationship of the
seven AF 115 isolates were comparable with those of the naturally occurring recombinant
ALV-BE/J. They indicated that the ALVS present in these isolates were exogenous and
most probably subgroup B and not subgroup J.

PCR analysis results revealed that the seven AF 115 isolates contained ALV-B,
ALV-J and ALV-B/J sequences. Variable PCR amplification patterns of AP 115 isolates
suggested the presence of mutations in different genomic regions of some of these
isolates which were confirmed by DNA sequencing data (chapter 3). These conclusions
were based on the differences in the annealing locations and sequences of PCR primers
used. In addition, similar PCR amplification patterns revealed that AF 115 -1, AF 115-13,
AF 1 15-14 and AF 115- 16 were similar viral strains and closely associated to the
naturally occurring recombinant ALV-B/J. On the other hand, different patterns of PCR
amplification demonstrated that AF 115-5, AF 115-7 and AF 115-10 were distantly

related to the recombinant ALV-B/J (AF 115-4) and the rest of the isolates.

CHAPTER 2 LIMITATIONS

210

There were limitations encountered during conducting the biological assays in
the research studies of chapter 2. Although the host range is considered the most reliable
method for subgroup classification, it was not enough by itself to specifically identify the
nature of ALVS in our isolates. Analysis of the host range data suggested that ALVS from
all isolates belonged either to ALV subgroup A, B or D, but not to J. Moreover, virus
neutralization test revealed specific antigenic relationship of our isolates to ALV-B even
though it is known to be less reliable than host range assay. Previous unpublished studies
used more CEFS and subgroup-specific antisera for the host range and antigenicity assays
on AF 115 isolates. Therefore, only reagents (cells and antisera) giving positive results
were used in our studies, which may have limited our analysis.

There were advantages and disadvantages for using PCR technology in
characterization and establishing relationships among AF 115 isolates. The PCR assay
was rapid and effective in confirming ALV detection in our isolates and predicting
mutations in different genomic regions. The specificity and utility of the used PCR
primers were limited by some of the mutations in our isolates, which may have prevented
the primers from annealing to the desired target sequences. Furthermore, some of the
primers were shown to anneal to sequences from different ALV subgroups to varying
extents through a BLAST search of GenBank. This poses a limitation on the specificity
of some of these primers in detection of certain ALV subgroups. On the other hand, the
sensitivity of PCR detection of ALV in our isolates was not evaluated, but can be

assessed in future studies.

CHAPTER 3 CONCLUSIONS

211

The main objective of the research studies in chapter 3 was to molecularly
characterize seven AF 1 15 isolates of a naturally occurring recombinant ALV (AF 115-1,
AF 115-5, AF 115-7, AF 115-10, AF115-13, AF 115-14 and AF 115-l6) by DNA
sequence analysis. The most significant finding from these research experiments was the
identification and confirmation of the subgroup designation of the different AF 115
isolates by DNA sequence analysis. All AF 115 isolates are recombinant ALVS with
different genomic regions representing different ALV subgroups (Figure 4.1). This was
done by comparing the deduced amino acid sequences of gp85 and gp37 and the proviral
DNA sequences of 3’ UTR and LTR of the seven isolates with the naturally occurring
recombinant ALV-B/J, representative ALV subgroups and related ALV strains through
NCBI blast. Thus, DNA sequence analysis seems to be the golden method to clearly
characterize any ALV isolate in field as well as experimental settings.

Five AF 115 isolates (AF 115-1, AF 115-7, AF 115-13, AF 115-14 & AF115—16)
were closely related to the naturally occurring recombinant ALV-B/J. Similar to ALV-
B/J (AF 115-4), these five isolates had a subgroup B gp85, subgroup E ng7 and
subgroup J 3’ UTR (containing a DR1 and E element) and LTR. On the other hand, AF
115-5 and AF 115-10 displayed sequence variations in the gp85 and 3’ UTR and LTR
regions respectively. AF 115-5 had a similar genomic organization as the other AF 115
isolates except for a subgroup A gpSS. Moreover, the genome of AF 115-10 was similar
to the first five AF 115 isolates except for its 3’ UTR and LTR, which was highly
associated with a recombinant ALV isolate (ALV TymS_90) associated with fowl

glioma. These associations were predicted by the unique sequence rearrangements and

212

mutations in different genomic regions of these recombinant ALVS such as the 3’ UTR
and LTR of AF 1 15-10 affecting putative transcription regulatory elements.

Another significant finding was the identification of conserved cysteine residues
and potential N-linked glycosylation sites in the SU and TM envelope glycoproteins of
all AF 115 isolates. These sequences or sites can affect the envelope glycoproteins
stability and functions. Further studies are needed to elucidate the contributions of these
conserved sequences to the structure and function of these envelope glycoproteins.
Moreover, conserved sequences in the 3’ UTR and LTR of AF 115 isolates included
putative transcription regulatory elements (CCAAT motif, PU1 and PU2 core regions in
the 3’ UTR and TATA box, PAS signal, CCAAT motif and CCAAT-related LTR
enhancer motif and two boxes of Y, CArG and PRE in the 3’ U3 region). In future
experiments, PCR primers can be designed to anneal to these conserved sites in the 3’
UTR and LTR instead of the variable regions in the env and improve molecular diagnosis
of ALV.

On the other hand, AF 115—10 contained extra enhancer elements in the
rearranged sequences of 3’ LTR and had mutations in CCAAT motifs and one of the PRE
boxes. The mutation in PRE box of AF 115-10 was shared by AF 115-5 and AF 115-7.
Some of these elements showed mutations, which may affect their ability to bind
transcription regulatory proteins and thus influence virus transcription. Further studies are
needed to evaluate the functional significance of these enhancer elements (wild type and
mutated) in transient infections through measuring LTR-driven transcription.

In addition, hotspots for recombination in all AF 115 isolates were predicted

based on their sequence analyses and association of their genomic regions with different

213

ALV subgroups and strains. Taken together, the molecular characterization results
suggested that the env of all AF 115 isolates may have risen by recombination between
exogenous and endogenous ALV sequences. Moreover, the gp37 and the 3’ UTR
upstream of the DR1 region of all AF 115 isolates as well as 3’ UTR and LTR of AF

115-10 may be hotspots for recombination among different ALV subgroups (Figure 4.1).

CHAPTER 3 LIMITATIONS

The ClustalW slow accurate method was used for sequence analyses since it is the
most accurate and popular method for typical multiple sequence alignment and their
refinement for phylogenetic analysis. Moreover, phylogenetic trees were constructed
using the neighbor-joining (NJ) distance-based method utilized in MegAlign. However,
the best method for construction of phylogenetic tree depends on the sequence data.
Methods such as maximum parsimony, distance-based, and maximum likelihood are
generally used to find the optimal tree that best accounts for the sequence data. Each of
these methods uses a different type of analysis and has different inherent weaknesses and
strengths. Therefore, it is recommended to reanalyze the sequence data by several
methods and then compare the results. In addition, the reliability or confidence intervals
of the constructed phylogenetic tree can be evaluated by bootstrap analysis procedure. It
is a statistical method that estimates the frequency of the appearance of the same
branches when sequences are resampled. These methods were not addressed in the

analysis, but will be used for filrther analysis of our AF 1 15 isolates.

RESULTS COMPARISON

214

Data from the research experiments in chapter 2 were generally consistent with
those in chapter 3. Biological and molecular assays results of most AF 115 isolates were
similar to those of the naturally occuning recombinant ALV-B/J. Moreover, the
biological (host range and antigenicity) and molecular (PCR and DNA sequencing)
results were consistent in most AF 115 isolates. The only exception was AF 115-5, which
showed discrepancies in the results by different methods. This can be explained by the
possibility of the presence of a mixture of ALVs (NJ and B/J) in this isolate as suggested
by data from the different biological and molecular assays. For example, the host range
data can be interpreted by presence of a mixture of ALV subgroups in isolate AF 115-5.
On the other hand, the virus neutralization and PCR results revealed presence of
subgroup B envelope and ALV-B, ALV-J and ALV-B/J like sequences. However, data
from sequence analysis of AF 115-5 revealed it is a recombinant ALV-A/J.

Similar results to AF 115-5 isolate were reported in an outbreak of subcutaneous
sarcomas in commercial white leghorn layers (Zavala et al., 2006). As with AF 115-5,
virus isolation in either DF-l or alv6 cells were positive, indicating the presence of
exogenous ALVs and probably not antigenically related to ALV-A (Zavala et al., 2006).
On the other hand, the same viruses were partially or completely neutralized by
antibodies against ALV-A and ALV-B in virus neutralization assays. The authors
concluded that there was a possibility of more than one exogenous virus being involved
in this natural infection of commercial layers. Furthermore, a field case of mixed
infection with MAV-l (ALV-A) and RAV-2 (ALV-B) was previously reported also in

commercial white leghoms (Spencer et al., 2003).

215

 

All AF 115 isolates were isolated fi'om commercial layers affected by myeloid
leukosis (ML), but, no subgroup J ALV was found in any of the isolates. Moreover,
inoculation of the naturally occurring recombinant ALV-B/J (AF 115-4) into susceptible
experimental egg-type chickens and different parental lines of commercial white leghorn
layers induced predominately LL (Lupiani et al., 2006; Mays et al., 2006). Inoculation of
commercial layers with MAV-l viruses produced exclusively myelocytomas
indistinguishable histologically from those induced by ALV-J in meat type chickens
(Zavala et al., 2006). It is interesting to speculate that the MAV-l-like virus in AF 115-5
may be responsible for the ML observed in the affected layers.

In short, our data revealed the presence of a huge diversity in the seven AP 115
isolates obtained from a single outbreak of ML in commercial chickens. In fact, this is the
first reported outbreak of recombinant ALVS in field flocks where various forms
(different variant of ALV-B/J) and combination of recombinant ALVS (ALV-A/J and
ALV-B/J) were isolated and characterized (Figure 4.1). In 2000, Lupiani and coworkers
isolated and characterized three laboratory generated recombinant ALV-A/J isolates
where ADOL 5701A represented a different ALV variant from ADOL 5701AA and
ADOL 6803A, which sowed nucleotide deletions in their 3’ UTRs. Coinfection with
genetically divergent strains of HIV -2 and recombination were suspected to occur in
some infected individuals (Gao et al., 1994). Moreover, coinfection of puma by two
different viral strains of feline immunodeficiency virus was reported (Carpenter et al.,
1996). Similarly, coinfection with and recombination by caprine arthritis-encephalitis

virus and maedi-visna virus have been documented in naturally infected goats (Pisoni et

216

al., 2007). Lately, dual infection and recombination were believed to be the source of
new variation in small-ruminant lentiviruses in goats and sheep (Germain et al., 2008).
This wide diversity of ALV isolates in a single outbreak adds to our current
knowledge about ALV evolution and may help in understanding ALV epidemiology.
This fast evolution of ALV may have devastating consequences for the poultry industry
in the future. It is possible that new highly virulent strains of recombinant ALVS may
emerge, causing new disease problems, higher mortality and serious production and
economical losses. In addition, the presence of various forms and combinations of these
recombinant ALVS in our isolates points to the difficulty in diagnosis and prevention of

this economically important pathogen in the field.

RECOMMENDATIONS

The presence of different recombinant ALVs in our AF 115 isolates raises the
possibility that these isolates might have formed from recombination events among
different ALV subgroups in the field or hatchery. The hatchery involvement in mediating
recombination between different ALV subgroups in this outbreak was also suggested
previously (Gingerich et al., 2002). Therefore, it is recommended that hatching,
processing and rearing of chicks from different sources (meat-type or egg—type) or
different breeders be strictly avoided. In the hatchery, stringent biosecurity practices must
be implemented including sanitation (cleaning, disinfection and fumigation) and
monitoring of hatchery building (including egg storage areas and water lines), equipment
(hatchers, incubators and others), eggs and egg transportation equipments, proper

handling and optimal storage of eggs and pest control.

217

In fact, contact transmission of ALV usually occurs among congenitally infected
hatchmates, penmates, fomites and the environment. Thus, general management practices
in the hatchery and farm must be implemented at all levels of production. These
management procedures include using clean eggs and chicks fiom one source, using
disposable chick boxes, rearing one chicken age group, avoiding contact-proximity to
other poultry or animals, mechanical vent sexing, vaccination with separate needles,
rearing on wire-floored cages, regulating movement of personnel, equipment and
vehicles, proper litter and bedding material management and disposal and feed hygiene
and transport. In addition, all farm equipment used for incubation, hatching, and brooding
of fertilized eggs and chicks should be thoroughly cleaned, disinfected and firmigated
afier each use.

Various methods for ALV detection should be fast, economical and suitable for
large-scale use. ALV characterization in addition to ALV isolation and detection are
essential to study ALV epidemiology and evolution and their impact on the poultry
industry. ALV infections are diagnosed by virus isolation and identification in
commercial, pathogen-free and breeder flocks. Virus isolation is carried out in cell
' culture using the appropriate chicken embryo fibroblasts with specific host range,
followed by p27 ELISA. This biological diagnostic assay remains the standard method
for ALV isolation and detection. However, it is recommended that CEFS with different
host ranges are used so that the subgroup is identified. For example, (C/O) CEFS are used
to detect all ALVS while line 0 CEFS (C/E) and DF -1 (C/E) cell line detect exogenous
ALVS. Moreover, different CEFS and cell lines can be utilized to confirm the subgroup of

the isolated ALV such as alv6 (C/AE), 72 (C/ABDE) and DF-l/J (C/EJ) cell line.

218

Molecular diagnostic tests seem to be more specific since it detects, identifies and
confirms the nature of ALV subgroup including mutants and recombinants in various
samples. PCR analysis in conjunction with sequence analysis of ALV isolates can yield
important information about ALV diversity. It is recommended that PCR employing
generic primers (ARK 4836 & REVSP 6683 or RS-7J & REVSP 6683) are initially used
to confirm the presence of ALV. This can be followed by sequence analysis of the
isolated PCR product to characterize the different ALV isolates. However, there are no
PCR primer pairs currently present, which can amplify the region fiom beginning of env
to end of LTR of all ALVs. Therefore, different primers specific for various ALV
subgroups can be used to identify the ALV subgroup.

Detection of ALV mutants and recombinants can be possible by using different
combinations of PCR primers specific for different ALV subgroups as shown by our
current studies. However, since most ALV sequences used for designing these specific
PCR primers are located in the env and LTR regions, which are prone to mutations and
recombination, not all ALV variants will be detected. Therefore, it is recommended that
future PCR primers are designed based on less variable sequences in these regions. For
example, PCR primers should be designed to anneal to conserved sites (transcription
regulatory elements) in the 3’ UTR and LTR and less variable gag and p01 regions. This
can be followed by sequence analysis of different isolates for ALV epidemiological and

evolutionary studies.

FUTURE STUDIES

219

In addition to the importance of understanding the molecular structure and
relationships among different recombinant strains of ALV in these AF 115 isolates, it is
imperative to explore the virus-host interaction and transmission of these viruses in
biological systems. The oncogenicity of different ALV subgroups is based on differences
in env or the LTR (Brown and Robinson, 1988; Chesters et al., 2002). AP 115-5 and AF
115-10 exhibited differences in these two regions when compared with the rest of the
isolates and the naturally occurring recombinant ALV-B/J (AF 115-4). Future research
experiments should assess the pathogenicity (induction of viremia, shedding, immunity
and oncogenicity) of these isolates in the context of virus (dose and strain) and host (age
at inoculation and phenotype) factors.

Although both subgroup A and B are known to induce lymphoid leukosis in
susceptible chickens, AF 115-S may produce different type of tumors since its gp85 is
MAV—l -like. It is expected that AF 115-5 may result in ML when it is inoculated into
susceptible chickens compared with AF 115-10 or AF 115-4 which will cause LL. The
rate of replication and the frequency of tumors of AF 115-10 may be different from the
rest of the isolates due to rearrangements and mutations in the 3’ UTR and LTR.
Chickens inoculated with AF 115-10 may show faster induction of viremia, more
shedding and higher incidence of tumors since AF 115-10 contains an extra CCAAT
element, which may increase its transcription and replication potential.

In future animal studies, ALV-susceptible egg-type chickens should be used since
meat-type chickens susceptible to ALV-B are hard to find. In previous studies, various
parental lines of commercial white leghorn layers (Mays et al., 2006) and experimental

ADOL chickens 1515 x 71 (Lupiani et al., 2006) were shown to be susceptible to the

220

naturally occuning recombinant ALV-B/J (AF 1 15-4). However, there were no major
differences among the different parental lines of commercial chickens and ADOL
experimental chickens. Therefore, it is recommended to use the experimental ADOL
chickens 1515 x 7‘ since they are available and accessible. Later, other lines of chickens
can be used to evaluate their resistance and immune response to AF 115 isolates
representing various forms of naturally occurring recombinant ALVS.

The ALV strains to be used for inoculation of these chickens will include RAV-2
(ALV-B), Hcl (ALV-J), AF 115-4 (ALV-B/J), AF 115-5 (ALV-A/J and ALV-B/J) and
AF 115-10 (ALV-B/J). Each ALV strain will be inoculated into two groups of 30
chickens each either intra-abdominally at hatch or via the yolk sac at 8 days of
embryonation with 103 tissue culture infectious dose (TCIDso/ml). Higher doses (104 and
105 TCIDso/ml) can be used later to evaluate the effect of dose of the virus on
. pathogenicity in susceptible chickens. Another 30 age-matched uninoculated chickens
will be used as controls. All chickens will be bled at hatch, tested for ALV and maternal
antibodies and kept in isolation until termination at 32 weeks of age. At 2, 4, 8, 16 and 32
weeks PI, chickens will be tested for viremia (ALV in serum), shedding (ALV in cloacal
swabs) and immune response (ALV neutralizing antibodies in serum) and observed for
tumors. Dead and live chickens at the end of the experimental period will be necropsied.
Samplesof affected tissues will be prepared, appropriately stained and examined for
microscopic lesions. The type of tumors induced by these ALV strains will be determined

based on characteristic gross and microscopic lesions.

SUMMARY

221

In summary, these studies demonstrated the importance of using different
diagnostic techniques to specifically characterize new emerging ALV isolates such as
naturally occurring recombinant ALVs. The findings fi'om this research work will aid
poultry industry in diagnosis, prevention and control of new emerging mutant and
recombinant ALVs in field flocks and experimental settings. In addition, this research
will be useful to scientists studying mutation, recombination, epidemiology and evolution

of retroviruses.

222

B E

J
gagl poIISUITM_
* i i t 1

AF 115-1,AF 115-7,AF 115-l3,AF 115-14&AF 115-16(ALV-B/J)

 

 

 

 

 

 

 

 

A E J
gag I vol I sv Im—
t 1
AF 115-5 (mixture of ALV-B/J & ALV-A/J)
TymS_90
B E J AEV E

 

 

gag] pol | su ITM-IEI
l t 111

AF 115-10 LTR

Figure 4.1. Schematic representation of DNA proviruses of AF 115 isolates and
homology to known ALV subgroups and strains. Arrows represent potential
recombination boundaries or hotspots. LTR: long terminal repeat, UTR: untranslated
region; gag: group-specific antigen gene; pol: polymerase gene; SU: surface region; TM:
transmembrane region; U3: unique 3’ end sequence; R: repeat region; U5: unique 5’ end
sequence; B: ALV-B, E: ALV-E, J: ALV-J; A: ALV-A; AEV: avian erythroblastosis
virus; TymS_90: recombinant ALV strain with MAV-l (subgroup A) gpSS, subgroup E
gp37, subgroup J 3’ UTR, AEV E26 U3, subgroup E R and US.

223

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