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IMMUNOPATHOGENESIS OF AVIAN LEUKOSIS VIRUS SUBGROUP J IN
WHITE LEGHORN CHICKENS
By

Susan Michelle Williams

AN ABSTRACT OF A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Pathobiology and Diagnostic Investigation
2001

Professor Willie M. Reed

ABSTRACT

IMMUNOPATHOGENESIS OF AVIAN LEUKOSIS VIRUS SUBGROUP J IN
WHITE LEGHORN CHICKENS

By

Susan Michelle Williams

Avian leukosis virus subgroup J (ALV-J) was first described in the early 19905 in
England as the cause of myeloid leukosis. Since its discovery, the virus is now spread
worldwide and broiler breeder companies are trying to rid their breeder flocks of the
infection. Accidental infection has also occurred in commercial laying chickens. The
overall goal of this research was to determine the pathogenesis of ALV-J infection in
white leghom chickens. The objectives were: 1) Compare the pathogenicity of ALV-J in
various lines of white leghorn chickens; 2) Determine the effects of endogenous virus 21
on the pathogenesis of ALV-J in white leghoms; and 3) Determine the tissue tropism of
ALV-J and the susceptibility of the B cell to ALV-J induced transformation in white
leghoms.

For objective 1, chickens from four genetic lines of white leghorn were inoculated
with the U. S. prototype of ALV-J, ADOL Hcl, at either the day of hatch or as 7-day-old
embryos. At 4, 10, 20 and 30 weeks of age, chickens were tested for ALV-J- induced
viremia and antibody, packed cell volumes, and differential white blood cell counts. At 4
and 10 weeks of age, 5 chickens from each treatment group and of each line were
humanely euthanized and bursa] tissues were examined for follicle transformation. All
chickens that died and those that survived the experiment were examined for tumor

formation. Microscopic examination of the grossly affected tissues was also performed

to confirm the diagnosis of tumors. Of all the lines of chickens inoculated with ALV-J at
day of hatch, only Line 0 developed antibodies and cleared the virus. Chickens of all
other lines had various degrees of success in developing antibody and clearing the virus.
The primary tumors observed were lymphoid leukosis (LL) and hemangiomas, regardless
of treatment or genetic line of chicken.

For objective 2, the effect of endogenous leukosis virus 21 (EV21) on the
pathogenesis of ALV-J induced infection and disease was examined. F1 progeny from a
cross between ADOL line 0.44-EV21+ males and ADOL 15B. females were hatched and
characterized as late feathering or early feathering by the length of the primary feathers.
Chicks were inoculated with stain ADOL Hcl of ALV-J at day of hatch. At 4, 10, 18 and
31 weeks of age, chickens were tested for ALV-J viremia and antibody. All birds that
died and those that survived the experiment were necropsied. Chickens harboring EV21
mounted a weak antibody response as compared with those lacking EV21. Although the
incidence of tumors was surprisingly low for birds harboring EV21, overall LL was
diagnosed in five birds and hemangioma in one bird.

Tissue tropism and B cell transformation assays were studied in chickens of line
1515 X 7}, a highly susceptible line to ALV-induced infection and tumors. Chicks were
inoculated as 7-day-old embryos with strain ADOL Hcl of ALV-J. At 2 and 6 weeks of
age, various tissues were tested by immunohistochemistry utilizing a monoclonal
antibody specific for gp85. Viral antigen was found in all tissues examined except
skeletal muscle. At 4 and 10 weeks of age, bursa] tissues were examined for the presence
of transformed follicles using the methyl green pyronine stain. Results indicated that

ALV-J could transform bursa] follicles. Infection with ALV-J may be manifested as LL.

I want to dedicate this body of work to my family.

iv

ACKNOWLEDGMENTS

I would like to thank everyone involved in this project. To my husband, Orlando,
for all your love and support (all those extra miles you drove so I could do this). To my
son, Jonathan, for making me realize that there is life besides grad school. To the
members of the Tuskegee Michigan State family, thanks for all the fun times and support
for my time spent here and especially Dorothy Reed, Claudette Buckingham, and Ruby
Perry for taking care of Jonathan when I needed your help. To Pat Lowrie for the
motherly advise when I first came to MSU as a Vetward Bound Student and when I
retumed as a grad student. To the scientists and technicians at the USDA ADOL lab,
thank you for all your help, input and insight in tumor virus biology and life in general.
To Melanie Flesberg for all your help in bleeding what seemed like thousands of birds,
your patience with all my “dumb” questions and all the “chick” parties in necropsy,
thanks a million and I’ll remember “the only good chicken is a dead chicken”. To the
former graduate students of ADOL, Carol Cardona, Ping Wu and Hsiao-Ching Lui,
thanks for all your help, after hours grad student pity parties, inspiration and friendship.
To Sharon Thon, for your photographic and slide making skills, thanks for all your help.
To my church family at Friendship Baptist Church, thanks for all your prayers and
support, especially Mamise Roberts, Dr. Charles Lowrie, and the extraordinary women’s
group. And finally to my committee, Dr. Willie Reed, Dr. Aly Fadly, Dr. Mick Fulton,
Dr. Margo Holland and Dr. Scott Fitzgerald for all the support, mentoring, and guidance
you offered me during this process and Dr. Jon Patterson for joining my committee at the
end. Once again, thank you very much for this opportunity to grow professionally and

Personally.

TABLE OF CONTENTS

Page
LIST OF TABLES ................................................................................. viii
LIST OF FIGURES ................................................................................. ix
LIST OF ABBREVIATIONS ................................................................................. xii
CHAPTER 1
Introduction and Literature Review ................................................................ 1
Retroviridae .................................................................................. 1
Avian Leukosis-Sarcoma Viruses and Diseases ......................................... 7
Introduction .......................................................................... 7
Disease Incidence and Distribution .............................................. 8
Etiology ............................................................................. 10
Virus Replication .................................................................. 12
Endogenous Viruses ............................................................... 21
Pathogenesis and Pathogenicity ................................................ 23
Viral Induced Neoplasms of Chickens ......................................... 30
Immunity ........................................................................... 36
Genetic Resistance ................................................................ 37
Diagnosis ........................................................................... 39
Prevention and Control ........................................................... 44
Subgroup J ......................................................................... 46
Research Objectives ............................................................... 54
References ................................................................................... 55
CHAPTER 2
Response of Various Lines of White Leghorn Chickens to Infection with Subgroup J
Avian Leukosis Virus .............................................................................. 80
Abstract ...................................................................................... 81
Introduction ................................................................................. 82
Methods and Materials ..................................................................... 83
Results ....................................................................................... 86
Discussion .................................................................................. 133
References ................................................................................. 1 3 8
CHAPTER 3
Role of Endogenous Virus 21 (EV21) in the Response of White Leghorn Chickens
10 Infection with Subgroup J Avian Leukosis Virus .......................................... 140
Abstract .................................................................................... 141
Introduction ................................................................................ 14]

vi

Page

Methods and Materials ................................................................... 143
Results ...................................................................................... 145
Discussion ................................................................................. 148
References ................................................................................. 15 1

CHAPTER 4

Tissue Tropism of Subgroup J Avian Leukosis Virus in White Leghorn Chickens ...... 154
Abstract .................................................................................... 155
Introduction ................................................................................ 1 5 5
Methods and Materials ................................................................... 157
Results ...................................................................................... 161
Discussion ................................................................................. 178
References ................................................................................. l 83

CHAPTER 5

Conclusions and Future Directions .............................................................. 186
References ................................................................................. 193

vii

LIST OF TABLES
Page

Table 2.1 Development of ALV-J or ALV-A induced viremia and antibody in five lines
of white leghorn chickens inoculated with virus as 7-day-old embryos or at day of age..89

Table 2.2 Bursa] transformation induced by ALV in five lines of white leghorn chickens
inoculated as 7-day-old embryos or at day of hatch ............................................ 90

Table 2.3: Packed cell volume values in five lines of white leghorn chickens inoculated
with ALV-J or ALV-A as 7 day old embryos or at day of hatch ............................. 95

Table 2.4: Diagnosis of ALV induced tumors in five lines of white leghorn chickens
inoculated with ALV-J or ALV-A as 7-day-old embryo or at day of age .................. 96

Table 3.1: Development of ALV-J induced viremia and antibody in white
leghorn chickens harboring or lacking EV21 .................................................. 146

Table 3.2: Characterization of ALV-J positive chickens in regards to antibody status. 147

Table 4.1 Distribution of gp85 expression in 1515 X 7. Chickens Inoculated as Embryos
with ALV-J ......................................................................................... 163

Table 4.2 Development of ALV-J or ALV-A induced viremia and antibody in chickens of
a highly susceptible cross to ALV induced tumor formation ................................ 163

Table 4.3 Bursa] follicle transformation induced by ALV-A or ALV-J in a highly
susceptible cross to ALV induced tumor formation .......................................... 164

viii

LIST OF FIGURES

Figure 1.1. A schematic figure of a retrovirus virion illustrating various Page
locations of structure and proteins ............................................................... 12
Figure 1.2. Summary of reverse transcriptase activity ......................................... 17
Figure 2.]. Line 0 heterophil comparison ........................................................ 97
Figure 2.2. Line 0 lymphocyte comparison ..................................................... 97
Figure 2.3. Line 0 eosinophil comparison ....................................................... 98
Figure 2.4. Line 0 monocyte comparison ........................................................ 98
Figure 2.5. Line 1515 heterophil comparison ................................................... 99
Figure 2.6. Line 1515 lymphocyte comparison .................................................. 99
Figure 2.7. Line 1515 eosinophil comparison ................................................... 100
Figure 2.8. Line 1515 monocyte comparison ................................................... 100
Figure 2.9. Line 71 heterophil comparison ...................................................... 101
Figure 2.10. Line 71 lymphocyte comparison .................................................. 101
Figure 2.11. Line 71 eosinophil comparison ................................................... 102
Figure 2.12. Line 71 monocyte comparison .................................................... 102
Figure 2.13. Line 63 heterophil comparison .................................................... 103
Figure 2.14. Line 63 lymphocyte comparison .................................................. 103
Figure 2.15. Line 63 eosinophil comparison ................................................... 104
Figure 2.16. Line 63 monocyte comparison .................................................... 104
Figure 2.17. Line 1515 X 71 heterophil comparison ........................................... 105
Figure 2.18. Line 1515 X 7. lymphocyte comparison ......................................... 105
Figure 2.19. Line 1515 X 71 eosinophil comparison ........................................... 106
Figure 2.20. Line 1515 X 71 monocyte comparison ............................................ 106

ix

Page
Figure 2.21. HeterOphil count comparison of all ALV-J infected lines combined over
time ........................................................................................ 107

Figure 2.22. Lymphocyte count comparison of all ALV-J infected lines combined over
time ......................................................................................... 107

Figure 2.23. Eosinophil count comparison for all ALV-J infected lines combined over
time ......................................................................................... 108

Figure 2.24. Monocyte count comparison for all ALV-J lines combined over

time ......................................................................................... 108
Figure 2.25. Control group average percent for heterophils ................................. 110
Figure 2.26. Control group average percent for lymphocytes ............................... 112
Figure 2.27. Control group average percent for eosinophils ................................. 114
Figure 2.28. Control group average percent for monocytes ................................. 116
Figure 2.29. Day of Hatch inoculated group average percent for heterophils ............. 118
Figure 2.30. Day of Hatch inoculated group average percent for lymphocytes ........... 120
Figure 2.31. Day of Hatch inoculated group average percent for eosinophils ............ 122
Figure 2.32. Day of Hatch inoculated group average percent for monocytes ............. 124
Figure 2.33. Embryo inoculated group average percent for heterophils ................... 126
Figure 2.34. Embryo inoculated group average percent for lymphocytes .................. 128
Figure 2.35. Embryo inoculated group average percent for eosinophils ................... 130
Figure 2.36. Embryo inoculated group average percent for monocytes .................... 132
Figure 4.1. Immunohistochemistry on bursa of Fabricius sections ........................ 165
Figure 4.2. Irnmunohistochemisty on spleen sections ....................................... 166
Figure 4.3. Immunohistochemistry on kidney sections ...................................... 167
Figure 4.4. Immunohistochemistry on nerve sections ....................................... 168

Page

Figure 4.5. Immunohistochemistry on proventriculus sections ............................. 169
Figure 4.6. Immunohistochemistry on liver sections ......................................... 170
Figure 4.7. Immunohistochemistry on testis sections ........................................ 171
Figure 4.8. Immunohistochemistry on pancreas sections .................................... 172
Figure 4.9: Immunohistochemistry on adrenal gland sections .............................. 173
Figure 4.10: Immunohistochemistry on bone marrow sections .............................. 174
Figure 4.11: Immunohistochemistry on ovary sections ....................................... 175
Figure 4.12: Immunohistochemistry on heart sections ....................................... 176
Figure 4.13: Immunohistochemistry on thymus sections .................................... 177

xi

30g

Ab
ADOL
AEV
ALSV
ALV
ALV-A
ALV-J

ART-CH
att

CA
CEF
COFAL
c-myc
DNA
EAV
env

EV21
FeLV

gag
gp37
gp85
gs
HEM
HPRS

Kb

LDLR
LL
LPD
LTR
MA
MGP
ml
ML
NC

p27

LIST OF ABBREVIATIONS

30 gauge

anfibody

Avian Disease and Oncology Laboratory
avian erythroblastosis virus
avian leukosis sarcoma virus
avian leukosis virus

avian leukosis virus subgroup A
avian leukosis virus subgroup J
avian myeloblastosis virus
avian retrotransposons-chicken
attachment site

centigrade

capsid

chicken embryo fibroblasts
complement fixation for avian leukosis
cellular myc gene
deoxyribonucleic acid

ancient endogenous virus
envelope gene

endogenous virus locus
endogenous virus 21

feline leukemia virus

gram

group specific antigens gene
glycoprotein 37

glycoprotein 85

group specific antigen
hemangiomas

Houghton Poultry Research Station
integrase

infectious units

kilobases

kilodalton

low density lipoprotein receptor
lymphoid leukosis
lymphoproliferative disease
long terminal repeats

matrix

methyl green pyronine

milliliter

myelocytomatosis or myeloid leukosis
nucleocapsid

nanometer

protein 27

xii

SEAR
src
SU

TNFR
tRNA
tv-a
tv-b
tv-c

U3

U5

V+, A-
V+, A+
VI

WBC

primer binding

polymerase chain reaction

packed cell volume

polymerase

polypurine

triidothyronine

L-thyroxine

repeat

reticuloendotheliosis virus
ribonucleic acid

ribonuclease H

regional poultry research laboratory
reverse transcriptase

reverse transcriptase polymerase chain reaction
simian immunodeficiency syndrome
subgroup E receptor

sarcoma gene

surface protein

transmembrane protein

tumor necrosis factor receptor
transfer ribonucleic acid

tumor virus-a

tumor virus-b

tumor virus-c

unique sequences at the 3' end of genome
unique sequences at the 5' end of genome
virus positive, antibody negative
virus positive, antibody positive
virus isolation

virus neutralization

white blood cell

xiii

CHAPTER 1

Introduction and Literature Review

I. Retroviridae

A. Introduction

The Retroviridae is a large family of viruses that primarily infect mammals.
Retroviruses are characterized as an enveloped virion with a dimer ribonucleic acid
(RNA) genome, composed of two identical single stranded RNA molecules. The RNA
molecule has a plus sense orientation meaning that it can serve as a template for
translation of proteins. However, retroviruses use a deoxyribonucleic acid (DNA)
intermediate stage before transcribing RNA for protein translation. The genome ranges
from 7 to 13 kilobases (Kb) in length. The genomic structure that is conserved in all
retroviruses includes the 5' R and U5 regions, group specific antigens (gag), polymerase
(pol), envelope (env) genes and the 3' U3 and R regions. Some viruses have additional
genes while others have the basic three. These additional genes account for the larger
genomes (Coffin 1992).
B. Morphology

Retroviruses have a distinct morphology when viewed ultrastructurally. Four
types of particles can be found. The A Type particles are only found inside cells. They
have a clear center that is surrounded by a shell. They can be intracytoplasmic or
intracisterna]. The B Type particles have doughnut shaped cores when they bud and

eccentrically located cores within a budded particle. The C Type particle has a crescent

shaped core at budding and centrally located cores in virions. The D Type particles have
more elongated electron dense cores in the virions (Weiss et al. 1982).
C. Major Viral Genes and Encoded Proteins

There are three major genes identified in all retroviruses, encoding a number of
essential proteins for the life cycle of the virus. Italics are usually used to name a gene
and two to three capital letters for proteins. The gag gene encodes proteins that are the
major structural elements of the capsid. There are three GAG proteins common to all
retroviruses. Some make a fourth GAG protein. First are the matrix (MA) proteins that
line the inner face of the virion envelope. MA proteins are involved in budding of the
virus. The capsid (CA) protein forms the core shell of the virion (Dickson et al. 1982;
Dickson et al. 1985). In most retroviruses, the CA protein is the detectable antigen and is
the basis for identification assays. The nucleocapsid (NC) is the protein closely
associated with the genomic RNA. It has been reported to promote RNA-RNA duplex
formation (Prats et al. 1988; Prats et al. 1990) which is presumed necessary for genome-
primer association and genome dirnerization. At the end of the gag gene, the protease
enzyme is encoded. It is responsible for all the proteolytic cleavages that generate the
mature GAG and POL proteins during viral maturation.

The pol gene is 3' downstream of the gag gene and encodes for two major
proteins, reverse transcriptase (RT) and integrase (IN). The main function of the POL
proteins is viral synthesis and integration into the host DNA. The RT product includes
enzymes needed for DNA synthesis—RNA directed DNA polymerase, DNA directed
DNA polymerase, and ribonuclease H (RNase H). Each of these enzymes is necessary to

create double stranded DNA from the single stranded RNA viral genome. However, the

error rate in RNA directed DNA polymerase is high when compared to DNA-DNA
polymerase (Battula and Loch 1976). This error rate is the cause of the variability found
in retrovirus genomes. The RT is highly conserved among retroviruses. The 1N protein
has two enzymatic functions, DNA cleavage and strand transfer (Grandgenett and Mumm
1990). It accomplishes these functions by cleaving the host DNA target and the viral
DNA 3’ ends then ligating the viral and cellular DNA together (Katzman et al. 1989;
Rice et al. 1996; Katz et al. 1998).

The envelope or env gene encodes the surface proteins that are needed for
recognition of specific cell receptors. The polypeptide precursor is created by RNA
splicing and then further processed into two proteins, the surface protein (SU) and the
transmembrane protein (TM). The SU protein is a larger protein and is glycosylated.
The SU protein also includes the site for host-cell receptor interaction with the virion
(Coffin 1992) and host ranges that help classify closely related viruses into subgroups in
certain species (Weiss et al. 1982). The TM protein is smaller and is the C-terminal
cleavage product. It serves as an anchor for the SU protein complex to the viral envelope
and mediates fusion of the envelope to the host cell membrane (Coffin 1992).

D. Long Terminal Repeats

The process of reverse transcription creates repeated sequences at the ends of the
proviral genome, referred to as long terminal repeats or LTRs. These are terminal
noncoding regions that are essential in cis for viral replication. They are composed of
three regions, the U5, 3 unique sequence near the 5' end of the genome, R, a short
sequence directly repeated at each end of the genome that is used during reverse

hanscription, and U3, 3 unique sequence that contains signals used by the provirus to

regulate transcription and processing of transcripts at the 3' end of the genome. After
reverse transcription has occurred the regions are fused together in the following order
U3-R-U5 and are found at each end of the resulting proviral genome (Coffin 1992).
E. Groups of Retroviridae
1. Avian Leukosis-Sarcoma Virus (ALSV) Group

This group includes of both exogenous and endogenous viruses of domestic fowl
(Payne 1992). These viruses fall into the C-Type virion and their genomes encode the
basic three genes, gag, pol, and env. Sometimes the exogenous viruses carry oncogenes
like src (sarcoma) found in Rous sarcoma virus (Parker et al. 1981). The viruses are
further divided into ten subgroups (A—J) according to host range, viral envelope
glycoprotein antigens, and viral interference patterns (Duff and Vogt 1969; Okazaki et a1.
1975; Domer et al. 1985; Bova et al. 1988; Payne et al. 1992). However, viruses of
ALSV that affect chickens belong to six subgroups (A, B, C, D, E and J) (Payne et al.
1991; Payne 1992).
2. Mammalian C-Type Virus Group

Viruses of this group also include exogenous and endogenous viruses from many
different mammals, including rodents (Kozak and Ruscetti 1992), carnivores (Hardy Jr.
1993), and other avian viruses not included in ALSV group. (Barbacid et al. 1979; Gazit
et al. 1979). These viruses are similar to the ALSV group with simple genomes and there
are many oncogene-containing strains that have been described (Coffin 1996). Examples
of viruses in this family are murine leukemia viruses, feline leukemia virus (F eLV),
reticuloendotheliosis virus (REV) and lymphoproliferative disease of turkeys (LPD).

Mouse viruses are classified by species distribution of their receptors: xenotropic viruses

use receptors found in most species except mice, ecotropic viruses replicate only in mice
cells, and polytropic and amphotropic viruses use different receptors found on mouse and
non-murine species (Kozak and Ruscetti 1992; Coffin 1996). F eLVs are classified into
subgroups A, B, and C. Subgroup B contains the endogenous viruses (Hardy Jr. 1993;
Coffin 1996).
3. B—Type Virus Group

The only infectious agents in this group are the endogenous and exogenous mouse
mammary tumor viruses (Kozak and Ruscetti 1992; Coffin 1996). These viruses also
have simple genomes with the addition of the sag gene for superantigen activity (Coffin
1996) and no oncogene has been described with the viruses. Mammary carcinomas are
the sequela to infection with these viruses.
4. D-Type Virus Group

This group includes both endogenous and exogenous viruses from primates and
sheep (Coffin 1996). These also are simple genomic viruses with no oncogene-
containing members. Examples include Mason-Pfizer monkey virus, simian
immunodeficiency syndrome (SAIDS) virus, and Jaagsiekte disease virus in sheep (York
et a1. 1992). B and D-Type viruses are closely related and both form intracytoplasmic A
particles (Coffin 1996).
5. HTLV-BLV Group

This group only includes exogenous viruses of humans and cattle. This group of
viruses contains at least two genes that are for gene expression, tax and rex, besides the

basic three genes and no oncogenes. Examples of this group include bovine leukemia

virus (a B cell lymphoma) and human T-cell leukemia virus —1 and -2 (a T-cell
lymphoma) (Coffin 1996).
6. Lentivirus Group

Viruses in this group are complex exogenous viruses that infect humans, feline,
bovine, primates, sheep and goats. The prototypes of these viruses were called “slow
viruses” because of the slow progression of the disease and very long incubation periods.
Various other genes are found in the genome besides the basic three. tat and rev are
most common with nef and vzf also occurring in some of the viruses as well as VP)“ and
vpu. Examples from this group include human immunodeficiency virus-1 and —-2, simian
immunodeficiency virus, feline immunodeficiency virus, bovine immunodeficiency virus,
Visna/maedi virus, caprine arthritis-encephalitis virus, and equine infectious anemia
virus. These viruses mainly induce neurological and immunological diseases versus
neoplastic disease that can manifest with the previously mentioned groups.
7. Spumavirus Group

This group is referred to as the “foamy” viruses because of the vacuolation in cell
culture that occurs following inoculation (Coffin 1996). Infection with these viruses is
not associated with any known disease (Loh 1993; Coffin 1996). They are complex
viruses with additional proteins encoded at genes bell, be12, and bel3. Examples of this
group are simian foamy virus, human foamy virus, and feline syncytium-forming virus.
They are among the largest of the retroviruses with approximately 11,000 nucleotides

(Maurer and Flugel 1988; Loh 1993).

8. Unclassified fish retroviruses

Retroviruses of fish have recently been described (Martineau et al. 1992; Petry et
al. 1992; Eaton et al. 1993; Bernard and Bremont 1995; Hart et al. 1996; Zhang et al.
1996; LaPierre et al. 1999). Examples are walleyed dermal sarcoma virus, walleyed
epidermal hyperplasia virus and snakehead fish virus. Walleyed dermal sarcoma virus is
now the largest retrovirus with approximately 13,000 nucleotides (Martineau et al. 1992).
The lengths of the genomes sequenced so far indicate that other proteins are present but

have not been fully characterized (LaPierre et al. 1999).

II. Avian Leukosis-Sarcoma Viruses and Diseases

A. Introduction

The term, lymphoid leukosis (LL), was first coined by Biggs (1961) and
Campbell (1961). This name superseded visceral lymphomatosis that Jungherr (1941)
had termed the disease in 1941. The earliest case of a leukotic disease was first report by
Roloff (1868) and Capan'ni (1896) who described fowl leukemia. Confusion with
Marek’s Disease, a neoplastic disease involving T cells, was cleared away when the
etiological agent for Marek’s Disease was finally discovered in 1967 (Churchill and
Biggs 1967; Nazerian et al. 1968). Burmester (1947) provided proof of a viral etiology
for LL.

In 1908 the first reported experimental transmission of erythroblastosis was
documented (Ellerman and Bang 1908). Later, both Burmester and coworkers (1959)
and Beard (1963) describe the viral etiology and pathogenesis of various strains of

leukosis viruses causing erythroblastosis. Strains are called avian erythroblastosis viruses

or AEVs. Experimental transmission of myeloblastosis was first demonstrated by
Schmeisser (1915). The most investigated leukosis virus strain is the BAI-A AMV
(Payne and Fadly 1997). Pentirnalli reported the first description of myelocytomatosis in
1915. Most strains that cause myelocytomatosis such as MC29 and HPRS-103 also cause
other types of neoplasms (Mladenov et al. 1967; Beard 1980; Payne et al. 1992).

Nephromas and nephroblastomas as well as other types of tumors are caused by
ALV. Nephromas are usually categorized as either adenomatous or carcinomatous while
nephroblastomas are of the complex Wilm’s tumor type (Payne and Fadly 1997). The
only viruses that can experimentally induce nephroblastomas are BAI-A AMV
(Burmester et al. 1959; Beard 1980), MAV-2 (N) (a myeloblastosis associated
virus)(Watts and Smith 1980) and virus strain 1911(Payne et al. 1993). Nephromas can
be induced by a variety of well characterized laboratory viruses as well as field strains.

Osteopetrosis was first described in the 1920’s (Pugh 1927) as sporadic diffuse
osteoperiostitis. Other names for the disease include Marble bone and thick leg disease
(Payne and Fadly 1997). In 193 8, Jungherr and Landauer suggested the term
osteopetrosis gallinarium. They were also the first to reproduce the disease and noted
that it was associated with LL with some frequency (Jungherr and Landauer 1938).
Several other researchers were able to reproduce the disease without LL tumors
(Campbell 1963; Franklin and Martin 1980; Hirota et al. 1980).
B. Disease Incidence and Distribution

Leukosis-sarcoma viruses are found worldwide but actual clinical disease
incidence is quite low. Subgroup A, B and, most recently, I of ALV are found in

commercial flocks in the United States in the field with subgroup A ALV more common

than subgroup B (Payne and Fadly 1997). All subgroups of ALV can be detected either
by virus isolation or antibody assays (Spatar et al. 1975; Payne and F adly 1997). Egg-
and meat-type chickens as well as other species such as pheasants, partridges and quail,
are susceptible to infection with ALV. Natural infection of Japanese quail, pigeons,
geese, and Pekin and Muscovy ducks has not been reported (Chen and Vogt 1977; Vogt
1977). However, the susceptibility of experimental infection with ALV has been
documented in these species as alternative models for infection (Nehyba et al. 1990;
Geryk et a1. 1996; Salter et al. 1999; Trejbalova et al. 1999). Endogenous retroviral
genes can be found in cells from almost all chicken lines and some other species of
galliform birds. These genes can be complete or defective viruses of subgroup E
(Robinson 1978; Crittenden 1981; Smith 1987; Crittenden 1991; Dirncheff et al. 2000).
They are inherited in a Mendelian fashion at discrete loci on the chromosome and termed
endogenous viral loci or ev loci.

The incidence of actual clinical LL in commercial chickens has decreased over
the years due to eradication programs. There are occasional losses that can be up to 30%
(Jordan 1990). Most losses are noted in egg laying chickens and breeder stocks.
Subgroup J viruses cause myelocytomatosis and has resulted in significant economic
losses for broiler breeder flocks in the United States and abroad (Payne er a1. 1992; Fadly
and Smith 1997; Fadly 1998; Fadly and Smith 1999). Field cases of erythroblastosis and
myeloblastosis are very uncommon (Payne and Fadly 1997). Other tumors that occur
frequently with ALV infection include hemangiomas (25%) and renal tumors (19%)
(Campbell and Appleby 1966) in broilers. Osteopetrosis occurs sporadically and males

tend to be more affected than females (Payne and Fadly 1997).

C. Etiology
1. Classification

The avian leukosis-sarcoma viruses as well as all other retroviruses are
characterized by the presence of the enzyme reverse transcriptase. However, not all of
those viruses have complete genomes and various terms have been used to describe the
viruses. The terms “defective” and “complete” describe retroviruses that either lack
some aspect of the viral genome and cannot replicate without a helper virus or a complete
virus that can replicate without help. The term “acutely transforming” describes viruses
that cause neoplasms in a short period of time, usually in a few days to weeks. These
viruses carry a viral oncogene that allows for transformation and tumor formation
quickly. (Weiss et al. 1982). An example of an acutely transforming complete virus is
Rous sarcoma virus. It contains all the genes necessary for replication and v-src, a viral
oncogene (Duesberg et al. 1977). The opposite term, “slowly transforming”, describes
viruses that take many weeks to months for tumor development. These viruses do not
carry a viral oncogene and have to insert their genomes near a host proto-oncogene.
(Weiss et al. 1982)
2. Subgroups

Six of the ten different subgroups of ALV are found in chickens: A, B, C, D, E
and J. Classification of ALV into various subgroups is based on assays that determine
host range, interference patterns, and viral envelope antigens. Host range is determined
by growth of virus in embryo fibroblasts from various avian species. Interference
patterns look at interactions between members of different and same groups. Viral

envelope antigens are detected by viral and serum neutralization tests (Weiss et al. 1982).

10

Subgroups F and G viruses are endogenous and are found in three species of pheasants,
ring-necked (Hanafusa and Hanafusa 1973; F ujita et a1. 1974), golden (Fujita et al.
1974), and Lady Amherst (Hanafusa et al. 1976). Subgroup G viruses, isolated by
Hanafusa et al (1976), had different gs (group specific) antigen from the rest of ALSVs
and the viral proteins were also different from ALV and REV. On this basis they were
placed in a new class termed pheasant virus (Hanafusa et al. 1976) but retained the
Subgroup G designation. Subgroup H is an endogenous virus group isolated from
Hungarian partridge (Hanafusa et al. 1976). Subgroup I is also an endogenous virus
group isolated from Garnbel’s quail (Troesch and Vogt 1985). Various defective strains
of ALSVs lack an envelope gene and thereby have no subgroup. They can replicate with
help of helper viruses and they take on the designation of the helper virus’ envelope
(Payne and Fadly 1997).
3. Morphology

The diameter of the ALV particle is 80-120 nm. It is spheroidal in shape with
knobbed spikes projecting from the envelope. The spikes are about 8nm in diameter.
The freshly budded virion is not mature until the cleavage of the GAG precursor protein
which results in an electron dense central core (Coffin 1996). Figure 1.] illustrates a
schematic drawing of retroviruses. The genome consists of two single-stranded
molecules of RNA with capping at the 5 ' end and polyadenylation at the 3 ' end. The
buoyant density in sucrose is characteristic for C-type retroviruses at values of 1.15-

1.17 g/ml (Robinson and Duesberg 1968; Bauer 1974).

11

RNA Genome

 

Envelope
Matrix MA Capsid CA
Protease PR

Nucleoprotem NC Transmembrane

TM
Integrase IN

Receptor

Reverse Transcriptase binding SU

RT

 

Figure 1.1. A schematic drawing of a retrovirus virion illustrating various locations of
structure and proteins. Adapted from Fields Virology 3rd ed.
D. Virus Replication

Retrovirus replication involves the enzyme reverse transcriptase that makes the
group unique. The first interaction between the virus and the host cell is attachment of
the virion to the cell via a specific cell surface receptor. Interactions between the receptor
and the envelope glycoproteins allow or prevent viral entry. The virion glycoproteins are
arranged such that the TM protein or glycoprotein 37 (gp37) serves as an anchor for the
SU protein or glycoprotein 85 (gp85) via disulfide linkages (Sommerfelt 1999).

The receptor for ALV-A has been defined and recently mapped on the chicken
genome. ALV-A receptor is genetically related to the human low-density lipoprotein
receptor (LDLR) (Bates et al. 1993; Young et al. 1993). The receptor mapped to the tv-a
(tumor virus A subgroup) locus (Bates et al. 1998) which also confers susceptibility to

infection for subgroup A. Subgroups B, D and E susceptibilities are linked to the tv-b

12

locus. The receptor for subgroups B and D is CAR] and appears to be a member of the
tumor necrosis factor receptor (TNFR) family (Brojatch et al. 1996). The gene for
CAR] was recently mapped to the tv-b locus (Smith et al. 1998). Subgroup E ALV’s
receptor (SEAR) was discovered and cloned from turkey cells (Adkins et al. 1997) and is
almost identical to CARI (Adkins et al. 2000). Both receptors are located in the tv-b
locus and only differ in animo acids at residue 62 (Adkins et al. 2000). Subgroup C ALV
receptor is located in the tv-c locus (Crittenden 1991) but no gene has been identified yet.
The receptors for the remaining subgroups have not been identified.

The envelope protein exists as a homotrimer (three SU and TM subunits
covalently bonded). After the SU protein binds with the appropriate receptor, the current
model of entry is a conformational change of the SU protein and fusion to the cell
membrane. The following proposed model for fusion involves the trimeric envelope
subunits binding to the host cell receptor. More receptors migrate to the area and are
bound to the SU subunits, resulting in a cooperative conformational change in both SU
and TM subunits. The activated fusion peptide is inserted into the host membrane and the
receptor is released by the SU subunits. TM subunits can laterally diffuse to form a
fusion pore (Damico et al. 1998). This all takes place independently of pH. This model
is similar for the influenza virus hemagglutinin glycoprotein except that a low pH is
needed (White 1990). After the firsion pore is complete, the virion core is released into
the cell cytoplasm (Hunter and Swanstrom 1990). What happens to the various capsid
proteins after fusion is not well defined (Coffin 1996).

The next step is to begin creating DNA from the RNA viral genome. In 1964,

Ternin (1964,1976) suggested the idea that a provirus was necessary for RNA tumor

13

viruses such as ASLV to reside in infected cells as DNA. In 1970 Temin isolated the
polymerase that created DNA from RNA templates using Rous sarcoma virus (Temin and
Mizutani 197 0) thereby proving his hypothesis of a provirus existence. To begin
synthesis of the DNA, the RT (p68) enzyme needs a primer to attach the nucleotides to
when transcribing the RNA. In all retrovirus genomes, a specific tRNA molecule is base
paired, an 18 nucleotide sequence, to the primer binding (PB) region on the viral genome
(Fu et al. 1997). For all ASLVs, it is tRNA tryptophan (tRNAT'l’) (Faras et al. 1974;

F aras et al. 1974; Waters et al. 1975; Waters and Mullin 1977).

Once synthesis begins, the RT enzyme goes towards the 5' end of the viral
genome, transcribing the U5 and R regions into minus strand DNA using the RNA
dependent DNA polymerase activity. The newly created RNAzDNA hybrid is acted upon
by the RT’s RNase H activity which degrades RNA in RNAzDNA hybrids (Moelling et
al. 1971). This allows for base pairing to occur with the 3' R region and the newly
synthesized R in the minus strand DNA. This strand transfer or “jump” allows continued
synthesis of the minus strand DNA. The capsid structure and the presence of
nucleocapsid, NC, protein help facilitate the alignment afier the “jump” (Allain et al.
1994). The remainder of the RNA template is transcribed into minus-strand DNA and
degraded by the RNase H activity except for a polypurine (PP) tract located just 5' to the
U3 region. This tract resists the RNase H activity and leaves the RNA bound to the DNA
to serve as a primer for synthesis of the plus-strand DNA (Resnick et al. 1984; Smith et
al. 1984; Coffin 1996). The resultant minus-strand of DNA looks like this: 3'——-PB-gag-

pol-env-PP-U3RU5-tRNAT'l’u5'.

l4

The plus-strand synthesis begins using the RNA PP region not digested by the
RNase H activity as a primer. The RT, now using the DNA dependent DNA polymerase
activity to transcribe the plus-strand DNA molecule, begins to elongate from the primer
to the 5' end of the template including the tRNA that encodes for the original PB site.
Elongation stops at the correct site due to a modified base in the tRNA, a mlA residue,
that cannot be copied (Coffin 1996). This allows for perfect alignment after the second
“jump” to the PB region on the 3' end of the minus-strand DNA. Once the double
stranded DNA provirus is complete; the remaining RNA primers are removed by the
RNase H activity of the RT (Coffin 1996). Figure 1.2 summarizes the viral RNA to
proviral DNA conversion. The error rate in incorporating incorrect nucleotides with
reverse transcriptase is quite high when compared to DNA polymerase because it lacks a
3' to 5' exonuclease proofreading mechanism. RT has an error rate of about one
nucleotide per 9,000-17,000 nucleotides for ASLV (Battula and Loeb 1976) as compared
to DNA polymerase from Escherichia coli, which has an error rate of one in 100,000
nucleotides (Battula and Loeb 1976). This error may have contributed to the highly
variable strains of ALV-J subgroup that have recently been described (Venugopal et al.
1998; Venugopal 1999; Silva et al. 2000).

The next step is integration of the proviral genome into the host cell. How the
DNA gets into the nucleus has not been fully described for ASLVs. Murine leukemia
virus (MLV) can be found in the cytoplasm for many hours before they appear in the
nucleus. It seems that mitosis is important for MLV to get into the nucleus and
integration occurs in post replication DNA (Haihosseini et al. 1993). The same is not

true for lentiviruses. Controversial evidence suggests that nuclear localization signals in

15

MA (Bukrinshy et al. 1993) or Vpr (Heinzinger et al. 1994) direct the preintegration
DNA through the intact nuclear membrane (Bukrinsky et al. 1992). Two other forms of
provirus exist in the nucleus. They are covalently closed circles with one or both LTR
(Coffin 1996). Once believed to be intermediates in integration (Panganiban and Temin
1984), now it is known that they are functionally dead ends of aberrant transcripts that
cannot be integrated (Randolph and Champoux 1993).

The terminal ends of the LTRs are highly conserved in all retroviruses (Varmus
and Brown 1989). The LTR’s outside edges have inverted repeats that serve as the
retroviral attachment (att) site which function in integration (V armus and Brown 1989).
The repeats are about 20 nucleotides long and the terminal four nucleotides are 5'-
AATG. . .CATT-3' (V armus and Brown 1989). The site of integration in the host DNA
has been determined to be random for retroviruses as a whole group (Coffin 1996).
However, when examining B cell lymphomas in chickens, the integration site seems to be
in the first intron of c-myc (Robinson and Gagnon 1986). The integrase enzyme, IN
(p32), cleaves the two terminal nucleotides off leaving a —OH on the 3' ends of the
proviral DNA. IN also cleaves the host DNA in a straggered manner to allow strand
transfer of the proviral DNA into the host DNA. Once inserted, the host cell machinery
fills the the gaps and ligates the ends together. Overall, this process results in duplication
of host DNA at the ends of the proviral DNA (Luciw and Leung 1992).

Expression of the provirus is now controlled by the host cell systems. The LTRs have the
signals that are recognized by the cellular transcription machinary. The 5' LTR acts as
the promoter for viral RNA synthesis by RNA polymerase II, the same enzyme that

creates mRNA. All retroviruses have a TATA box in the U3 region, 20-30 base pairs

16

 

 

 

1 PB gag pol env 1 U3 I R]
ll ¢ 1 l 1
PB gag pol env U3 R
[1 I l I
ll 1 T7 1
¢ R. L;
PB
gag p01 pp
I I _
l l
4

 

env' U3' R' U55?

4
PB' gag' pol' env' U3' R' [15th
$ w R us

 

 

 

PB' gag' pol' env' U3' R' U5'
U3 R U5 PB
U3 R US PB gag pol env U3 R U5
{—> ‘——>
LTR LTR

Figure 1.2. Summary of reverse transcriptase activity. Adapted from
Fields Virology 3rd ed.

17

upstream from the initiation site (Lewin 1990). The TATA-binding protein (TBP)
recognizes the TATA box and binds. Upstream from the TATA box is a region called
the CCAAT box, which is important in determining the efficiency of the protomer
(Lewin 1994). There are also enhancer elements still further upstream in the U3 region.
The role for enhancers is thought to be to increase the numbers of transcription factors in
the area of the promotor (Lewin 1994).

Once all the transcription factors have been recruited to the area of the promoter
and put into place, the polymerase can create RNA transcripts (viral genome). The newly
synthesized RN As are capped on the 5' ends with a 7-methylguanosine designated as
7mG and methylated by host enzymes (Stoltzfus 1988). A polyadenylation (poly(A)) site
in 3' U3 adds a poly(A) tail to the end of the transcripts. The 5' U3 poly(A) site is not
found in the transcripts of ALSVs (Coffin 1996).

The newly synthesized RNA transcript can now travel down three different
pathways. First is to be genomic RNA that gets packaged into the new virions. Second
is to be messager RNA (mRNA) for translation of gag encoded polyproteins. Third is to

be precursors for subgenomic viral transcripts (V arrnus and Swanstrom 1982; Varmus
and Swanstrom 1985; Stoltzfus 1988). The subgenomic messages are created by splicing
the leader sequence from the 5' end to an acceptor site within the genome. This results
With all spliced transcripts with the same 5' and 3' ends as the viral genome (Luciw and
Leung 1992). The env gene is expressed from a spliced transcript for all retroviruses
(Luciw and Leung 1992). The 5' donor site is usually located in the leader sequence
“PStfearn from gag in most retroviruses; for ALSV it is found six codons into gag (Coffin

1996). What determines which pathway the RNA goes is unknown.

18

The gag and pol genes are translated into proteins via the typical fashion of the
ribosomal subunits binding to the capping group and then scan the RNA for the GAG
AUG start codon. At the end of gag is a translational terminator. pol is in a different
reading flame (Coffin 1996) from gag and requires a frame shift in the —1direction in
order for the ribosome submits to continue downstream and translate POL proteins
(Coffin 1996). This slippage occurs about 5% of the time (Jacks et a1. 1988) when the
tRNA recognizes a specific sequence in the ribosomal acceptor site. For ALSV these
sequences are A AAU UUA plus a psuedoknot structure after the seven nucleotides
(Charnorro et al. 1992).

The ENV protein is synthesized on polyribosomes associated with the rough
endoplasmic reticulum. All retroviral ENV precursor proteins stay anchored to the
membrane by the hydrophobic membrane spanning domain near the carboxy end (Perez
and Hunter 1987). Modifications take place in the Golgi body. Then the ENV precursor
protein is cleaved into SU(gpSS) and TM (gp37) (Hunter and Swanstrom 1990). The
cleavage always takes place at a characteristic amino acid sequence. For example, in
RSV, the sequence is Arg/Lys-x-Lys-Arg (Dong et al. 1992).

Virion assembly is becoming more understood for retroviruses because of the
research on HIV. There are two mechanisms of assemby depending on the retroviral
8T 011p. With B-, D-type, and spurnavirus groups, capsid assembly occurs in the
cytoplasm while the others assemble at the cell membrane (Coffin 1996; Sakalian and
Hunter 1998). How the capsid precursor protein gets to the assembly site is still
unknown, but is mediated by the MA domain of the GAG (Krausslich and Welker 1996).

Once the GAG and GAG-POL precursors are at the plasma membrane, self assembly into

19

capsids occurs (Sakalian and Hunter 1998). The genomic RNA also needs to get to the
plasma membrane area in order to be included with in the virion. How this occurs is also
unknown but is believed to migrate with the viral proteins(Krausslich and Welker 1996).
Two copies of the viral RNA genome are packaged (Berkowitz et al. 1996). The
packaging signal lies within the 5' untranslated region of the genome and is termed ‘1’
(Mann et al. 1983). And a specific host cell tRNA is incorporated in the virion before
budding occurs (F aras et al. 1974; Waters and Mullin 1977). For all ASLVs, it is tRNATrp
(F aras et al. 1974; Faras et al. 1974; Waters et al. 1975; Waters and Mullin 1977).

Three regions in the GAG protein have essential functions for budding of the
virus to occur (Craven and Parent 1996; Sakalian and Hunter 1998). They are designated
M, I, and L. M, the membrane binding domain, is located at the amino terminus of the
GAG protein (Nelle and Wills 1996; Verderarne et al. 1996). Any mutations in this area
prevent assembly from occuring (Wills et al. 1991). I domain or the interaction domain
functions in holding GAG proteins in place near one another for assembly to occur
(Craven and Parent 1996). I appears to be located in the NC domain of GAG and confers
density to the virion by interaction of NC and RNA, directing the alignment of proteins
(Craven and Parent 1996). The L or late domain is essential for budding or releasing of
the particle from the cell membrane (Wills et al. 1994). It is located near the MA
cleavage site, in the p2b region of GAG (Craven and Parent 1996). How the L domain
functions is unknown in the budding mechanism (Craven and Parent 1996).

After the particle buds from the cell membrane, it undergoes maturation to

become infectious. The viral protease (PR) enzyme is responsible for cleavage of the

20

GAG polyprotein into its three major proteins, CA (p27), MA (p19) and NC (p12). Once
the cleavage has taken place, the virion is mature and infectious (Vogt 1996).

E. Endogenous Viruses

A unique feature of retroviruses is their ability to be inherited in the gerrnline in a

relatively stable manner (Coffin 1982). There are three well-established retrovirus-like
families in the normal chicken genome plus a newly described family. These families
include the endogenous viral or ev loci, the EAV or ancient endogenous viral elements
(Dunwiddie et al. 1986; Boyce-Jacino et al. 1992), and avian retrotransposons of
chickens or ART-CH (Gudkov et al. 1992; Nikiforov and Gudkov 1994). There are about
fifty copies of EAV and ART-CH in a normal chicken genome. The newly described
family is the ev/J family (Benson et al. 1998; Ruis et al. 1999). Just recently ev/J family
was determined to be a member of EAV by sequence analysis (Sacco et al. 2000). There
is also a highly repetitive element known as CR1 (Stumph et al. 1984).

There have been at least 29 different ev loci identified (Payne and F adly 1997)
and there is, on average, 5 ev loci per chicken (Rovigatti and Astrin 1983). Endogenous
viruses are assigned the subgroup E designation. They can be complete infectious virions
or defective viruses that partially express proteins from the gag gene and/or env gene.
Sometimes they are transcriptionally silent like ev-l (Coffin 1982). Protein expression
from these ev loci can interfere with interpreting diagnostic tests like the enzyme-linked
immunosorbent assay (ELISA) and complement-fixation test for ALV (COFAL) as well
as the chick helper factor (chf) test, because they will give positive reactions. The
PYOtOtype virus for subgroup E is RAV-O. It was first described as originating from

Spontaneous production in line 7 chicken embryo cells (V ogt and Friis 1971).

21

The phenotypic character of endogenous viruses has been determined by what
viral products are expressed in uninfected cells. The first example is gs, which refers to
group specific antigens encoded by the gag gene. Dougherty and Di Stefano (1966)
described the first endogenous virus in a COF AL assay on uninfected cells. To be
classified as gs+, the cells must have the ev3 locus (Astrin and Robinson 1979; Coffin
1982). Another example is the chf. To be chf+, cells must produce endogenous
subgroup E ENV proteins that complement env gene defective RSV(—) to allow
production of infectious particles without a helper virus (V ogt 1967). Several loci encode
for the chf+ phenotype, ev3, ev6, and ev9 (Payne and Fadly 1997).

One ev locus, ev21, has become very important for the poultry industry. It
encodes for a complete virus known as EV21. EV21 has been mapped to the Z
chromosome and is linked to the dominant sex linked gene K which regulates slow
feathering (Warren 1925). Late feathering can be differentiated from early feathering and
is commonly used for sex determination of newly hatched chicks by chicken breeders and
growers (Warren 1930). EV21 can be transmitted congenitally to the progeny and
therefore increases the susceptibility of chicks to exogenous ALV (Harris et al. 1984;
Bacon et al. 1988; Smith and Fadly 1988; Smith et al. 1991).

Endogenous viruses rarely cause tumors because of the weak promoter activity of
the LTR (Motta et al. 1975; Payne and F adly 1997). The endogenous viral loci can be
beneficial or detrimental to the host depending on whether they are expressed or not by
creating resistance or tolerance (Payne and Fadly 1997). Also, endogenous viral genes
have been shown to be nonessential for chicken survival (Astrin et al. 1979). Chickens

that lack ev genes are designated line 0 (Crittenden and Fadly 1985). Line 0 chickens are

22

useful research tools for chickens experiments as well as providing cells lacking ev loci
for biological assays. Line 0 chicken embryo fibroblasts (CEF) are resistant to infection
with subgroup E viruses and are designated as C/E.

F. Pathogenesis and Pathogenicity

1. Natural and Experimental Hosts

Chickens are the natural hosts for all subgroups of ALV (Payne 1987). Other
species like pheasants, partridges and quail are also natural hosts but only for certain
subgroups. Turkey cells are susceptible to ALV A through E and J (Payne et a1. 1992) but
rarely do turkeys develop tumors after infection. Experiments with subgroup A ALV and
turkeys inoculated as embryos or at hatch show that turkeys develop non-neoplastic
lesions like inflammation and lymphoid proliferation in various visceral organs (El-

Mubarak et al. 1983). Osteopetrosis had been reported in turkeys experimentally
(Holmes 1964). Just recently, turkeys were experimentally infected and developed
tumors with strain 966, an acutely transforming ALV-J (Venugopal et al. 2000). Rous
sarcoma virus has the largest host range and can result in tumor formation in chickens,
turkeys, pigeons, ducks, guinea fowl, pheasants, rock partridges, and Japanese quail
(Payne and Fadly 1997).

2. Transmission

Exogenous ALVs can be transmitted in two ways: horizontally by bird to bird
contact, either direct or indirect, and vertically from hen to progeny via the egg (Cottral et
al. 1954; Rubin et al. 1961; Rubin et al. 1962). Vertical transmission is very important in
terms of epidemiology for virus spread. Vertical transmission results in chicks that are

Viremic tolerant that can spread the infection to their hatch mates. Viremic chicks

23

maintain the infection and transmit virus to the next generation. Horizontal infection is
thought to be important also in maintaining a vertical transmission rate because
sometimes a small percentage of highly susceptible chickens become Viremic tolerant
when exposed to virus soon after hatch. Together, these two methods of transmission
allow the virus to maintain itself in the chicken population (Payne and Bumstead 1982).
Endogenous ALVs are transmitted genetically in the germ cells of both male and
females and by contact among susceptible chickens. For subgroup E viruses that are
defective and cannot give rise to infectious particles, they can still express some proteins
that influence how the chicken responds to infection with an exogenous ALV (Crittenden
et al. 1982). If the endogenous virus can produce a complete infectious virus, for
example EV21, then it can be transmitted like an exogenous ALV. Many chickens are
resistant to such infections because of the endogenous viral genes within their genome
(Crittenden 1981; Crittenden and Astrin 1981).

There are four serological categories in mature chickens with regards to ALV
infection: l-virus negative, antibody negative (V-, A-); 2-virus negative, antibody
positive (V -, A+); 3-virus positive, antibody positive (V+, A+); 4-virus positive, antibody
negative (V+, A-) (Rubin et al. 1961; Rubin et a1. 1962). Category 1 contains birds that
are in an infection free flock and genetically resistant birds in a susceptible flock.
Chickens that are genetically susceptible and in an infected flock fall into the other three
categories. Most will be V-, A+ and a small percentage will be V+, A-. The remaining
category consists of birds in the midst of seroconversion when tested. The V+, A-
chickens transmit virus to the majority of their progeny (Rubin et al. 1962; Payne et al.

1982). These congenitally infected embryos fall into the V+, A- category once they

24

hatch. A small percentage of the V-, A+ hens can transmit the virus to their progeny on
an intermittent basis, which may be due to a low antibody titer in those hens (Tsukamoto
et al. 1992).

Viral shedding in the oviduct comes from the albumen secreting glands (Payne
and F adly 1997). This results with virus in the albumen but does not necessitate that the
chick will be virus positive when hatched. Several studies have shown that 1/8th to ‘/2 of
the embryos were infected from eggs with virus in the albumen (Spencer et al. 1977;
Payne et al. 1982; Tsukamoto et al. 1992). This may be due to maternal antibodies in the
yolk that were sufficient enough to neutralize the virus. Another explanation for this
phenomenon, is inactivation of the virus during storage before incubation. Storing the
fertile eggs at various temperatures resulted in decreased viral titers in the albumen, thus

lowering the prospect of the developing embryo to be infected (Fadly and Okazaki 1979).
3 . Host and Environmental factors

The age of the host at exposure determines the rate of development of tumors.
Usually as the host ages, resistance to tumor formation increases (Burmester et al. 1960).
However, route of inoculation also is important. Administering the virus via the oral or
nasal cavities in young birds can lead to tumors but usually not after three weeks of age.
Using an intravenous route, birds are susceptible for longer periods of time (Burmester et
al. 1960). Females are more susceptible to LL than males. Testosterone seems to
increase resistance (Burmester and Nelson 1945) to tumor formation. The genotype of a

bird is also important and will be discussed later.

25

4. Viral/Cellular Oncogenes

Viruses that carry oncogenic or one sequences have been isolated from many
animal species. The sequences were traced back to cellular genes that fall into one of
several categories: growth factors, grth factor receptors, signal transduction,
intracellular tyrosine kinases, serine/threonine kinases, and transcription factors (Gordon
1985; Lewin 1994). Viral and cellular sequences are termed as v- and c- oncogene,
respectively. The differences between a v-onc and c-onc usually is that c—onc contains
introns and is expressed at low levels while a v-onc does not have introns and is
expressed in high levels (Lewin 1994).

Examples of v—oncs in avian retroviruses are src, myc, myb, erb, and rel. The first
four are found in ASLVs and rel is found in strain T of reticuloendotheliosis virus. Rous
sarcoma virus was the first retrovirus isolated and characterized along with its oncogene,
src (Bishop and Varmus 1982). The protein was named pp60""". It is characterized as
an intracellular tyrosine kinase (Bishop and Varmus 1982; Lewin 1994). There are at
least eight subfamilies of tyrosine kinase. The c-src family of proteins is activated by
many different signals. Some include mitosis, growth factors, cytokines, and G proteins
(Erpel and Courtneidge 1995). The proteins that SRC interacts with demonstrate its
multifunctionality. They include proteins involved in cytoskeleton organization, cell to
cell adhesion, cell to cell communication, cell to substrate adhesion, and RNA processing
(Brown and Cooper 1996).

What makes v-src different than c-src is the truncation and replacement of
sequences encoding the COOH terminus so that Y527 (Tyrosine 527) is deleted (Parson

and Weber 1989). This conserved sequence and constant position serves as the major site

26

uh".

of phosphorylation because without it, the v-src protein has increased kinase activity with
no inhibitory regulation that phosphorylation at Y527 provided (Cooper et al. 1986). By
examining what pathways c-src protein interacts with in the cell, the over production of
the protein v-src could easily cause uncontrolled cell growth and transformation.

The oncogene v-myc was determined to be responsible for transformation by the
virus MC29 that causes myelocytomatosis (Sheiness et al. 1978; Reddy et a1. 1983;
Enrietto and Hayman 1987). A fusion protein of GAG-MYC which has 452 N terminal
residues encodes the transforming protein and 416 of these residues come from exons 2
and 3 of c-myc (Reddy et al. 1983). MC29 that has v-myc, is a transforming defective
virus because part of gag, all of pol and most of env are missing (Reddy et a1. 1983).

The cellular component c-myc was first described in 1980 (Sheiness et al. 1980).
c-myc belongs to the myc family of genes which include B-myc, L-myc, N-myc, and s-
myc. It encodes the transcription factor c-MYC, which interacts with another protein
MAX to control gene expression (Dang et al. 1999). There are over forty target genes
that c-MYC can act upon as an up regulator or down regulator for cell grth (Dang et
al. 1999). Because of the vast number of target genes that regulate cell grth affected
by c—myc, the potential for cell transformation is high if the highly regulated c-myc
becomes unregulated for some reason. While most of the research on c-myc as a proto-
oncogene has been in human carcinogenesis, viral induction of c-myc was described in
the early 1980’s (Crittenden and Kung 1984). In chickens infected with avian leukosis
Virus, the integration of the proviral DNA upstream of c-myc can cause transformation of

B cells due to the strong promoter properties in the LTR (Crittenden and Kung 1984).

27

Avian myeloblastosis virus (AMV) contains the oncogene v-myb along with avian
E26 leukemia virus. Both are defective viruses. In AMV, most of the env gene has been
replaced by v-myb (Duesberg et a1. 1980). There are ten substitutions in v-myb compared
to c-myb in the shared region (Gerondakis and Bishop 1986). These substitutions affect
the phenotype of the transformed myeloid cells (Introna et al. 1990). In the DNA
binding region, there are four amino acid substitutions in v-myb that affect the ability of
the MYB protein to transform different cell types, to control specific genes, and to be
controlled by other host proteins (Introna et al. 1990). Truncation of c—myb can cause
transformation. Truncation of the N—tenninus only is highly transforming while
truncation of C-temrinus only is weakly transforming (Lipsick and Wang 1999). As the
immature cells of the hematopoietic and lymphoid systems go through their
differentiation process, c—myb is expressed on the cells. As they become mature, this
expression decreases rapidly (Chen 1980; Westin et al. 1982). It is also expressed in
other tissues that can become cancerous. Therefore, the narrow spectrum of tumors seen
with AMV may be due to the mutations specific to v-myb versus c-myb having a limited
potential as an proto-oncogene (Lipsick and Wang 1999).

The frnal v-oncs are found in avian erythroblastosis virus (AEV). It is v-erb that
is two separate oncogenes, v-erbA and v-erbB. Both genes are necessary for full
transformation of erythroblasts because v-erbA does not cause transformation itself, but
increases the transformation activity of v-erbB (Lewin 1991). Recent research indicated
that v-erbA expression has two different effects on target cells: it prevents the

erythroblasts that have been transformed by v-erbB from spontaneous maturation into

28

erythrocytes and expands the range of conditions that allow the erythroblasts to propagate
(Lewin 1991).

The cellular homolog is c-erbA which encodes the nuclear receptor for thyroid
hormone (Sap et a1. 1986; Weinberger et al. 1986; Sawyers et al. 1991). The oncogenic
gene v-erbA is a 75 kDa fusion product of gag and v-erbA (Thonneyer and Barriahmad
1999). Functionally, the two proteins are different: c-erbA protein binds to
triiodothyronine (T3) but v-erbA protein has little affinity for T3 ligand. It can no longer
be stimulated to activate transcription of the avian erythrocyte anion transporter gene
(Zenke et al. 1990). The truncation at the 3' end of v-erbA is responsible for this loss
because that region had been mapped to the very end of the 3' end of c-erbA (Zenke et al.
1990).

The gene c—erbB encodes the epidermal growth factor receptor (Sawyers et al.
1991). The v-erbB product has lost the regulation of expression by lacking the ligand
binding domain and therefore tyrosine protein kinase function of v-erbB may be
constitutively activated (Kris et al. 1985).

5. Non-neoplastic Conditions

Sometimes when chickens are exposed at a young age to certain strains of ALV,
other disease manifestations besides neoplastic conditions can occur. Examples include
hepatitis, anemia, immunodepression and wasting with infection of RAV-l, RAV-60,
MAV-2 and subgroups B and D (Crittenden et al. 1982; Smith 1986). The
immunodepression was described as decreased lymphoid cells in the bursa and spleen,

decreased antibody response, and hypergammaglobulinemia (Smith 1986).

29

In addition to tumor disease conditions, ALV infection has been shown to
decrease egg production in layer breeder hens (Gavora et al. 1980; Payne and Fadly
1997 ). Various researchers have determined that ALV infection can also affect grth
and performance of the chickens (Gavora et al. 1980; Crittenden et al. 1983). In males,
ALV in the semen does not affect sperm production, but quality may be affected slightly
(Segura et al. 1988).

G. Viral Induced Neoplasms of Chickens-Gross and Microscopic Lesions
1. Marek’s Disease

Marek’s disease (MD) is caused by an alpha herpesvirus. It induces lymphoid
tumors in visceral organs. It also causes paralysis and in the case of infection with very
virulent viruses, bursa] and thymic atrophy are the only lesions seen after death. There are
vaccines available to control MD tumors.

Gross lesions can consist of a single lesion or a combination of the following
lesions. There is unilateral enlargement (2-3X normal size) of peripheral nerves, which
are edematous and have grey to yellow discoloration. Lymphoid tumors can occur in one
or more organs resulting in enlargement due to diffuse cellular infiltrate or due to nodular
formation. The tumors are grey to white in color. (Riddell 1987; Calnek and Witter
1 997).

Microscopically, MD tumors are composed of pleomorphic lymphoid cells. The
neoplastic cells have been determined to be mostly T cells, with a few B cells admixed
With a few plasma cells. There is also lymphocytic infiltration in nerves, lymphoid cell
Cufling in the cerebellum, infiltration in the feather follicles by lymphoid cells, and

soII‘letirnes, lymphoid infiltration in the iris resulting in a lesion known as “Marek eye”.

30

MD can also affect the bursa, causing interfollicular tumors with a pleomorphic lymphoid

cell population (Riddell 1987; Calnek and Witter 1997).

2. Reticuloendotheliosis

Reticuloendotheliosis is a group of diseases caused by a retrovirus in the
Mammalian Type C group called reticuloendotheliosis virus (REV). REV causes three
syndromes: An acute reticulum cell neoplasm, a chronic lymphoid neoplasm, and a
runting syndrome. The chronic lymphoid neoplasm and runting syndrome is caused by
nondefective REVS in nature.

With REV infection in chickens there can be two forms of the lymphoid
neoplastic disease, a bursa] form and a nonbursal form. Grossly, the lymphoid tumors
can be diffuse or nodular and grayish white in appearance. The bursa] form mainly
affects the bursa of Fabricius and liver. The neoplastic cells are B cells, which makes this
form of REV indistinguishable from LL. The nonbursal form resembles Marek’s disease
with thymus, heart, liver and spleen affected with lymphomas. The neoplastic cells have
been determined to be T cells (Riddell 1987; Witter 1997).

3. Lymphoproliferative Disease

Lymphoproliferative disease of turkeys (LPD) is a disease cause by a retrovirus in
the Mammalian Type C group of viruses. Grossly, the predominant lesion is
Splenomegaly. The spleen can be pale pink and mottled or contain miliary white to gray
foci. Sometimes these foci occur in pancreas, kidneys, thymus, lungs, heart, gonads, and

intestinal wall. There can be peripheral nerve enlargement (Riddell 1987; Biggs 1997).

31

Microscopically, the characteristic lesion is lymphoproliferation of pleomorphic
cells like lymphocytes and lymphoblasts, plasma cells and macrophages scattered
throughout the lesion. Nerve lesions are more focal in distribution than diffuse and
consist of the same pleomorphic cells (Riddell 1987; Biggs 1997).

4. Lymphoid Leukosis

Grossly visible tumors can be seen in various visceral organs but most commonly
affected organs are liver, spleen and bursa of Fabricius. Tumors are smooth, soft and
glistening. Cut surface appears to be slightly gray to off white. Neoplasms can be
nodular, miliary or diffuse in distribution or a combination of these. The nodules can
range fiom 0.5 mm to 5 cm in diameter and occur by themselves or in clusters. The
miliary form can be seen best in the liver as numerous, small nodules no larger than 2mm
in diameter evenly distributed throughout the organ parenchyma. The diffuse form
causes the organ to be uniformly enlarged with a slight gray color and a very friable
texture (Whiteman and Bickford 1989; Jordan 1990; Payne and Fadly 1997).

Microscopically, all lymphoid leukosis tumors are focal and multicentric in
origin. Organs are affected diffusely with coalescing foci. The cells displace and
compress the normal parenchyma rather than infiltrate between normal cells. The cell
type consists of large, lymphoblastic cells that are all in the same stage of development,
i.e., a monomorphic population. The cytoplasm is very basophilic and the nucleus has
prominent nucleoli and margination and clumping of chromatin (Riddell 1987; Payne and
Fadly 1997). There have been two reports of viral inclusion bodies seen in the

myocardium (Gilka and Spencer 1985; Nakamura et al. 1988).

32

5. Erythroblastosis

Diffuse enlargement of the liver and spleen, and sometimes the kidneys, are the
most characteristic gross lesions of erythroblastosis. These organs are usually cherry red
to deep mahogany in color and are soft and fiiable. The liver may be finely mottled due
to degeneration surrounding the central veins of the lobules. The bone marrow is
hyperplastic, very soft or watery, cherry red to deep mahogany and often has
hemorrhages. The affected birds appear anemic and have petechia] hemorrhages in
various organs (Jordan 1990; Payne and F adly 1997).

Microscopically, the bone marrow can have rapidly proliferating erythroblasts
that fail to mature in the blood sinusoids. In advanced cases, there is little to no adipose
tissue and small islands of myelopoiesis with sheets of homogeneous erythroblasts filling
the marrow. When organs are involved, changes are usually due to hemostasis with
accumulation of erythroblasts in the sinusoids and capillaries. As the sinusoid distends, it
results in pressure atrophy of the parenchyma. The cells always remain intravascular
unlike LL or myeloblastosis. The erythroblast has a large round nucleus with fine
chromatin and one to two nucleoli and abundant amounts of basophilic cytoplasm. The
cell itself is irregular shaped and can have pseudopodia (Riddell 1987; Payne and Fadly
1 997).

6. Myeloblastosis

Gross lesions of myeloblastosis include enlarged and friable parenchyrnal organs.
There can be gray diffuse tumor nodules in the liver and occasionally in other visceral
organs. The bone marrow is firm and reddish gray to gray. With advanced cases, grayish

infiltrations in the liver, spleen and kidneys are diffuse and give the organ 3 mottled

33

appearance or sometimes a granular appearance. There is usually an anemia that may
cause paleness to be noted in the comb as well as hemorrhage from feather follicles due
to clotting deficiencies (Jordan 1990; Payne and Fadly 1997).

Microscopic lesions include massive intravascular and extravascular
accumulations of myeloblasts and some promyelocytes in the parenchyma of various
organs. There is infiltration and proliferation outside the sinusoids and around portal
tracts in the liver. The bone marrow has extensive myeloblastic activity in the
extrasinusoidal areas. The myeloblast is a large cell with slightly basophilic cytoplasm
and a large nucleus with 1-4 acidophilic nucleoli which may not stain well (Riddell 1987;
Payne and Fadly 1997).

7. Myelocytomatosis

Gross lesions of myelocytomatosis consist of tumors that are characteristic and
usually easily recognizable. The tumors occur on the surface of bones and near cartilage,
although any organ can be affected. They usually develop on the costochondral junctions
of the ribs and the inner sternum. Sometimes the cartilaginous bones of the mandible and
nares can be affected. Flat bones of the skull also can be affected. Myelocytomas are
dull, yellow-white, soft and fiiable or caseous, and diffuse or nodular. They sometimes
have a thin layer of bone covering them that can be broken easily (Jordan 1990; Payne
and Fadly 1997).

Microscopically, the tumors consist of uniform myelocytes with little stroma.
Their nuclei are large, vesicular and eccentrically located. A distinct nucleolus is

Common also. The cytoplasm is filled with eosinophilic spherical granules. The cells are

34

similar to myelocytes found in normal bone marrow (Riddell 1987; Payne and F adly

1997)
8. Nephromas and Nephroblastomas

There are two types of ALV-induced tumors of the kidney: 1) nephroblastomas of
the complex Wilrn’s, and 2) adenomas or carcinomas. Grossly, the tumors can range
from small grayish nodules embedded in the parenchyma to large, yellowish gray
lobulated masses that replace most of the normal kidney tissue. The tumors can be
pedunculated and connected by a fibrous vascular stalk. The larger tumors are often
cystic and can occur in both kidneys (Payne and Fadly 1997).

Nephromas microscopically can vary greatly. When the tubules are affected,
there can be primitive abnormal glomeruli among the abnormal tubules. Frequently
cystic adenomas are seen. Hemangiomas and endotheliomas sometimes occur within the
nephroma (Riddell 1987; Payne and Fadly 1997).

Nephroblastomas have great variation histologically. Both epithelial and
mesenchyma] elements are affected to varying degrees. The epithelial structures can vary
from enlarged tubules with invaginated epithelium with malformed glomeruli to irregular
masses of distorted tubules to large, cuboidal undifferentiated cells with little to no
tubular formation (Riddell 1987; Payne and Fadly 1997). There may be islands of
keratinizing stratified squamous epithelial structures (pearls), bone, or cartilage (Ishiguro
et al. 1962). Metastases of these tumors are rare.

9. Osteopetrosis
Gross lesions include changes in the diaphysis of the tibia and/or tarsometatarsus.

Other bones that can be affected are other long bones, pelvic bones, shoulder girdle and

35

ribs. The lesions are typically bilaterally symmetrical and begin as pale yellow foci
against the normal bone. The periosteum is thickened and the abnormal bone is spongy.
The lesion is circumferential and advances to the metaphysis, resulting in a fusiform
looking bone. The lesion can vary in severity from a slight exostosis to massive
asymmetric enlargement with almost complete occlusion of the bone marrow cavity
(Jordan 1990; Payne and Fadly 1997).

Microscopically, the periosteum is thickened over the lesion due to increased
number of basophilic osteoblasts. There is a size increase and irregularity of the
haversian canals. There is also an increase in size and number of lacunae and their
positioning is altered. Osteocytes are numerous, large and eosinophilic and the new bone
is basophilic and fibrous (Riddell 1987; Payne and Fadly 1997).

10. Hemangioma

Grossly, hemangiomas can appear as dark red, circumscribed and raised nodules
on the skin or surface of visceral organs. When the tumor ruptures in an organ, blood
clots are found in the peritoneal cavity. Also the chicken will appear pale and anemic.
Exsanguination can occur.

Microscopically, the neoplasms are composed of blood distending channels lined
by endothelial cells. The endothelium may proliferate as dense solid masses, or may
form a latticework with small capillary-sized spaces or large vessels supported by
collagenous cords. (Riddell 1987; Payne and Fadly 1997).

H. Immunity
Both humoral and cell-mediated immunity are involved with ALV infection.

After becoming infected from hatch-mates or their surroundings, most chicks have a

36

transient viremia then develop virus-neutralizing antibodies that rise to a high titer and
persist throughout the life of the bird (Rubin et al. 1962; Solomon et al. 1966).
Antibodies to ALV are passed from the dams to progeny, and passive immunity lasts for
3-4 weeks after hatch (Payne and Fadly 1997). The cytotoxic T lymphocyte activity has
been shown to influence immtmity to ALV (Bauer et al. 1976).

Reports on Rous sarcomas illustrate that a tumor-bearing host will also respond to
tumor-associated cell surface antigens or TASAs. This response serves to retard grth
of the tumor or cause regression. How this process works is not understood well.
Irnmunodepression has been associated with ALV infection in some instances. However,
it has also been documented that B and T cell functions are normal with a subgroup A
infection (F adly et al. 1982) and, most recently, that heterophil, macrophage and
lymphocyte functions in chickens with a subgroup J infection are the same as uninfected
controls (Stedman et al. 2000).

1. Genetic Resistance
1. Cellular Resistance to Infection

Genetic cellular resistance to ALV infection means that the virus cannot infect the
cell. Various reports suggested that a single autosomal dominant gene was responsible
for controlling the susceptibility to RSV (Prince 1958; Waters and Burmester 1961).
This was later proven with subgroup A RSV infection using various assays (Crittenden et
al. 1964; Crittenden et al. 1971). It is now lmown that independent autosomal loci
control the responses to-infection by subgroups A, B, and C viruses. They are called tva,
Wb, and tvc, respectively. These are the same loci that have genes that encode the various

receptor proteins for those subgroups mentioned in a previous section. There is some

37

evidence of a linkage between tva and tvc loci (Payne and Pani 1971). Subgroup D
viruses are controlled by the tvb locus (Pani 1975). Each locus has alleles for
susceptibility and resistance with resistance being recessive (Crittenden 1968). These
loci are inherited in a simple Mendelian fashion.

Inheritance of subgroup E ALV resistance is much more complicated. There are
two autosomal loci involved, tve and 1" (Payne et al. 1971). The I” gene is dominant for
resistance and can block susceptibility when the tve susceptible allele is present.
However, it has been reported that tvb alleles are required for susceptibility to subgroup B
infection and there is a controversy over the existence of the tve locus (Crittenden et al.
1973; Pani 1974; Pani 1976). One study suggests that the 1‘ locus is actually an ev locus
that expresses ENV glycoproteins which block the receptor for subgroup B infection
(Robinson et al. 1981).

Genetic resistance to subgroup J ALV has not been documented in chickens.
However, host range assays demonstrated that several avian species were resistant to
infection (Payne et al. 1991; Payne et al. 1992). These avian species include Common
pheasant, Japanese Green pheasant, Golden pheasant, Japanese quail, guinea fowl, Pekin
and Muscovy ducks and geese (Payne et al. 1992).

2. Resistance to Tumor Formation

Genetic resistance to tumor formation has mainly been examined using Rous
sarcoma virus. The major histocompatibility complex (MHC) or B complex in chickens
was determined to influence the outcome of RSV inoculation in the wing web beginning

in the 1970’s. The B locus was involved with regression of tumors induced by RSV

38

(Collins et al. 197 7). A MHC-linked gene allowed regression of RSV-induced tumors, R-
Rs-l. Its allele, r—Rs-l, allowed progression of the tumor (Schiennan et al. 1977).

As more knowledge was discovered about the B locus, three regions were
determined to make up the locus, the B-F, B-G and B-L regions (Pink et al. 1977). Three
different laboratories reported results that the gene that controlled regression lies within
the B-F region (Collins and Briles 1980; Plachy and Benda 1981; Birkmeyer and
Nordskog 1982). It also seems that the MHC gene associated with regression also
restricted metastasis of the tumors (Collins et al. 1977). Recent research confirms that a
gene or genes in the B-L/B-F regions or closely linked regions are responsible for the
regression of Rous sarcomas (Auclair et al. 1995).

Genetic resistance to LL tumor development is less understood that genetic
resistance to infection (Crittenden 1975; Schiennan and Collins 1987). However,
resistance documented in RPL line 6 chickens has been attributed to the bursa] cells and
their intrinsic ability to become infected or not (Purchase et al. 1977).

J. Diagnosis

1. Virus Isolation

Virus isolation used to be carried out by chick inoculation, either subcutaneously,
intramuscular, intraperitonea], or intracerebral (Payne and Fadly 1997). These are slow
and expensive ways to isolate virus. Currently, virus isolation is carried out in cell
culture, which is faster and more economical. Suitable sample materials for virus
isolation include plasma, serum, tumors, whole blood, peripheral blood mononuclear
cells (F adly and Witter 1998), infected organs, feces (Burmester 1956), albumen or 10-

day-old embryos (Spencer et al. 1977), and feather pulp (Spencer et al. 1983). To

39

perform virus isolation in cell culture the correct chick embryo fibroblasts (CEF) are
necessary. To detect all endogenous and exogenous ALVs, CEFs of the genotype C/O
(susceptible to all subgroups) are necessary. To detect only exogenous ALVs, CEFs of
the genotype C/E (cells resistant to subgroup B only) are necessary. There are various
CEFs that have resistance to various subgroups. An example is ALV6 CEFs which are
C/AE (resistance to subgroups A and E) developed by Crittenden and Salter (Salter and

i Crittenden 1989; Crittenden and Salter 1990; Salter and Crittenden 1991) via transgenics.
Recently, a genetically engineered cell line that is resistant to subgroup J ALV (C/I) has
been developed (Hunt et al. 1999).

Because most ALVs do not produce cytopathic effects in cell culture, a method
for indirect detection of the virus is essential. Most diagnostic tests for ALV use
detection of gs antigen p27, the viral capsid protein, as an indicator for the presence of
the virus. The most common tests used for detection of p27 are COFAL (Complement
Fixation for Avian Leukosis) (Sarma et al. 1964) and ELISA.

The most commonly used test for detecting p27 is the enzyme-linked
irnmunosorbent assay or ELISA (Smith et al. 1979; Tsukamoto et al. 1991). ELISA kits
for detection of p27 are now commercially available. Commercially available rabbit
anti-p27 IgG and rabbit anti-p27 conjugated to horse-radish peroxidase can be used for
in-house preparation of ELISA plates. Using ELISA, ALV or gs antigen can be detected
directly on samples or indirectly on cell culture inoculated with samples. Interpretation
of the results of the direct ELISA for p27 must be done with care due to the possibility of
detecting endogenous p27 from ev loci. Serum has been shown to be unsuitable for use

in direct assay for p27 (Payne et al. 1993).

40

2. Serology

Samples of plasma, serum or egg yolk can be used for antibody determination
(Payne and Fadly 1997). The virus neutralization test is the most sensitive test for
detecting antibodies to ALV. This test can be conducted in microtiter plates. The correct
indicator virus for the desired subgroup and known positive antibody should be used.
Equal volumes of sample and virus are mixed together and incubated for 45minutes. The
entire mixture is then placed on CEFs. After culture for 7-9 days, the p27 ELISA is used
on the lysates to determine the presence of p27. If the ELISA test is negative, there are
neutralizing antibodies in that sample. If the ELISA test is positive, there are no
neutralizing antibodies in the sample (F adly and Witter 1998). Direct method for
detecting ALV antibody has been developed using the ELISA (Smith et al. 1986). Kits
for detection of antibodies of subgroups A, B and J ALV are commercially available.
False positives can occur with microtiter virus neutralization for various technical reasons
and cross-reactions can occur if endogenous virus antibody is present (F adly and Witter
1998).
3. Phenotypic Mixing Assays

Samples of plasma or serum are suitable test materials for phenotypic mixing
(PM) assays. The test requires C/O and C/E CEFs and virus stocks of RSV(Rous-
associated virus —0) [RSV (RAV-0)]. RAV-O is the prototype subgroup E ALV. C/O
CEFs are co-cultured with RSV (RAV-O) for 1 day. The test sample is added and cultured
for 5-7 more days. When discrete foci form from RSV (RAV-O) transformation,
supernatant fluid is collected and centrifuged. The supernatant is then placed on C/E

CEFs to detect RSV (with sample ALV envelope), not the original RSV (RAV-O), and

41

cultured for 5-7 days. Production of foci is considered positive (Okazaki et al. 1975;
Fadly and Witter 1998). This assay forms the basis for the non-producer (NP) cell
activation test (Rispens and Long 1970; Rispens et al. 1970). The NP activation test
requires NP cells that have been transformed by defective RSV strain (RSV Bryan high-
titer strain) and do not produce virus that is detected in tests using C/E CEFs. A Japanese
quail cell line transformed by an envelope defective BH RSV, (R(-)Q), is also needed.
Test samples are inoculated onto susceptible CEFs and co-cultivated with NP cells. The
test is positive for ALV by testing for the presence of RSV. The R(-)Q cell test uses non
producing R(-)Q cells and C/E cells infected with the test sample. Cocultivation of the
two cell lines activates the R(-)Q cells to produce infectious RSV having the envelope of
the sample exogenous ALV (Crittenden et al. 1979).
4. Detection of Viral Nucleic Acids

Two main methods for detecting viral nucleic acids include Southern blotting and
hybridization and polymerase chain reaction (PCR). There are reported PCR methods for
detection of proviral DNA for subgroups A and J (Van Woensel et al. 1992; Smith et al.
1998; Smith et al. 1998; Silva et al. 2000). However, these tests require that no mutations
have occurred where the primers anneal in order to amplify the desired sequences. If
there is a mutation that interferes with the primer, the test will permit false negatives.
Southern hybridization uses specific probes labeled radioactively to detect proviral DNA
in tissues or tumors (Weiss et al. 1982).
5. Immunohistochemical Tests

Direct and indirect fluorescent antibody tests can be used to detect antigen in

infected cell cultures (Kelloff and Vogt 1966; Payne et al. 1966). Other

42

immunohistochemical staining methods have been used to detect group-specific antigen
in a variety of tissues (Di Stefano et al. 1973; Gilka and Spencer 1984; Schnegg et a1.
1994; Arshad et al. 1997; Arshad et al. 1999). Various staining methods include avidin-
biotin complex and soluble enzyme immune complex. Each can use various enzymes to
convert the chromo gens into a colored product like horseradish peroxidase, alkaline
phosphatase, glucose oxidase or beta-galactosidase. Using antibodies against gs antigen
(p27) may also detect endogenous viral p27.

6. Differential Diagnosis

Differential diagnosis of tumors of poultry must include three diseases, avian
leukosis, Marek’s disease, and reticuloendotheliosis. In the 1970s, lymphoproliferative
disease of turkeys was also included in the differential diagnosis of tumors in Europe and
Israel.

MD causes paralysis. In the case of infection with very virulent viruses, bursa]
and thymic atrophy are the only lesions seen after death. MD can cause mortality in birds
as young as four weeks of age and as old as 20 weeks, sometimes older. To reach a
diagnosis of either MD or LL, one must evaluate the history of the flock, age of the flock,
clinical signs, gross and microscopic lesions. There are various sources of information
that can help a clinician or pathologist make that decision (Page et al. 1969; Siccardi and
Burmester 1970; Yamamoto et al. 1972; Fadly and Witter 1998).

Historically, there have been few reports of RE infection causing neoplastic
disease in the United States (Crittenden et al. 1979; Witter and Crittenden 1979; Hayes et
al. 1992; Drew et al. 1998; Miller et al. 1998). In other parts of the world, it occurs more

frequently (Sasakj et al. 1993; Witter 1997; Payne 1998). There was a recent report of

43

vaccine contamination with REV that resulted in lymphomas in commercial chickens
(Fadly et al. 1996).

To determine if the blusa] tumors seen with REV are caused by REV or ALV, one
must complete virologic assays or conduct PCR on the tumors to determine which
etiologic agent is the cause (Aly et al. 1993; Fadly and Witter 1998).

K. Prevention and Control

Because there is no effective or practical treatment or vaccination for prevention
of ALV infection, eradication is the preferred method of control. The key to eradication
is to break the vertical transmission fi'om dam to progeny. To establish an ALV-free
flock, chicks must come from hens that are not infected or do not shed the virus, then are
reared and maintained in a clean environment (Payne and Fadly 1997). Many methods to
produce ALV-free flocks have been recommended. The use of immune, non-virus
shedder hens to repopulate a flock is one method. These antibody-positive hens are
selected on the assumption that they would be less likely to shed virus than hens without
antibody. Three chicks per hen are tested for the presence of virus and hens that are
negative are used to repopulated the flock (Hughes et a1. 1963). Another method to
produce an ALV negative flock is to choose hens that are non-immune, non-virus
shedders. Here the assumption is that hens without antibody have not been exposed or
infected by the virus, therefore, they are less likely to be intermittent shedders than
antibody positive hens (Levine and Nelsen 1964). A third method to produce an ALV
negative flock is to select nonviremic hens regardless of antibody status. This method
could take up to four generations before becoming leukosis free and infection was not

ruled out (Zander et al. 1975).

The application of eradication programs to commercial flocks has been based on
information on relationships between hens, eggs, embryos and chicks (Spencer et al.
1977). Three associations came out of this research: 1) egg albumen may include gs
antigen and exogenous ALV, and both are usually present together; 2) there is a strong
association between ALV or gs antigen in egg albtunen and ALV in vaginal swabs; and
3) there is an association between ALV in vaginal swabs or albumen and ALV in newly
hatched chicks and embryos (Spencer et al. 1977). Several tests can be used to detect
virus in swabs but it is well known that not all birds will be identified with one test
(Payne and F adly 1997). Therefore, depending on the company’s philosophy about
eradication or reduction of shedding, their eradication programs would be different.

An example of a complete eradication program involves four steps: 1) negative
dams (negative vaginal swabs or egg albumen tests) to select fertile eggs (Okazaki et al.
1979; F adly et al. 1981; Payne et al. 1982; Crittenden et al. 1984); 2) small group rearing
and avoid contact transmission between groups (F adly et al. 1981); 3) test chicks for
virus by biologic assay or by PCR and discard all positive chicks and their pen mates
(Okazaki et al. 1979; Fadly et al. 1981; Fadly et al. 1981; Payne et al. 1982; Payne and
Howes 1991); 4) rear ALV-free groups in isolation.

Because chicks are most susceptible to infection, various good management
practices can help reduce the chance of infection. Thorough disinfection of all
incubators, hatchers, brooding houses, and equipment with a detergent will disrupt the
viral envelope and render the virus inactive (Payne and Fadly 1997).

Another method for prevention and control is to select breeders that are

genetically resistant to ALV. Birds that lack the receptor for a certain subgroup cannot

45

become infected with that subgroup. See previous section on Genetic Resistance. At
least one primary broiler breeder company has selected chickens resistance to subgroup A
through genetic selection (McKay 2000).

The use of vaccines to control ALV has not been successful at all. Burmester
tried to inactivate ALV by various methods and could not produce an antibody response
to the inactivated product. Attempts to produce a modified live or attenuated ALVs also
have failed in that all strains tested produced tumors (Okazaki et al. 1982). Recently,
recombinant ALVs expression various envelope proteins have been created as vaccines
(McBride and Shuman 1988; Chebloune et al. 1991; Wright and Bennett 1992).

L. Subgroup J Avian Leukosis Virus
1. Introduction

In 1991 the first description of a novel subgroup (J) of ALV (ALV-J) affecting
chickens was documented in the United Kingdom (Payne et al. 1991). The isolates came
from commercial meat-type chickens. The acronym HPRS, which stands for Houghton
Poultry Research Station, was assigned to each isolate plus a number. Isolate HPRS-100
was isolated fiom tissues of a line 20 chicken with ascites syndrome. Isolates HPRS-101
and 102 were isolated from vaginal swabs of normal hens from line 20. Isolate HPRS-

103 was isolated fi'om a vaginal swab from a normal hen of line 23. Isolated HPRS-104
was isolated from a myeloid leukosis tumor from a bird in line 20. Payne’s group
conducted various tests such as interference assays, virus neutralization assays and host
range assays, on these novel subgroup viruses to determine what subgroup they belonged
(Payne et al. 1991). The results concluded that these isolates, I-IPRS-100-104, were

members of the first new subgroup described since 1970 (V ogt 1977). HPRS-103 was

46

identified as the prototype. Since then, various reports of subgroup J infection indicated
that it has spread worldwide (Fadly and Smith 1997; Fadly 1998; F adly and Smith 1999;
Nakamura et al. 2000; Payne 2000).

2. Subgroup Designation

The host range of HPRS-103 strain of ALV-J has been defined (Payne et al.
1992). Cell cultures from various avian species (V ogt 1977; Troesch and Vogt 1985)
including two jungle fowl species were used to define host range of the new isolate. Cell
cultures from line 0 chickens, Japanese quail, ring-necked pheasant, domestic guinea
fowl, turkey, Pekin duck, Muscovy duck, domestic goose, red jungle fowl and Sonnerat’s
jungle fowl were used in this host range study. The host range focus assay (Spencer
1987) was conducted by creating RSV pseudotypes with the prototype virus for
subgroups A, B, C, D, E, and HPRS-103, the prototype for ALV-J. Of all the species
tested only chicken, red jungle fowl, Sonnerat’s jungle fowl, and turkeys were susceptible
to RSV (HPRS-103) infection. Because the new isolate RSV (ALV-J) failed to grow in
remaining cell cultures, the presence of subgroup F, G, and I were ruled out.

The new subgroup was confirmed when sequence analysis of the env gene
revealed that is was different from all other ALV subgroups that affect chickens (Bai et
al. 1995). Genetic analysis of the gp85 protein (SU) revealed many deletions, amino acid
substitutions, and amino acid insertions when compared to subgroups A-E. The gp85 of
HPRS-103 had only a 40% overall average identity with subgroups A-E ngSs while A-E
have 80-85% identity to each other (Bai et al. 1995). These differences led the

researchers to confirm the novel subgroup is different and should be identified as J.

47

3. Disease manifestations

a. Neoplastic conditions

 

To determine the oncogenic properties of subgroup J ALV, Payne and
collaborators used the HPRS-103 prototype and various commercial lines of meat-type
chickens and experimental strains of Leghom chickens (Payne et al. 1991; Payne et al.
1992). They reported a variety of tumors with myeloid leukosis (ML) most common,
followed by renal adenomas, hemangiomas, histocytic sarcomas, granulosa cell trunor,
mesothelioma, pancreatic adenocarcinoma, and unclassified leukemias. The myeloid
leukosis seen was similar to myelocytomatosis caused by MC29, an acutely transforming
virus (Mladenov et al. 1967). Most of the tumors developed in meat-type chickens with
few tumors in the Leghorn strains (Payne et al. 1991; Payne et al. 1992).

In the field, various ages of meat-type chickens have been affected. Fadly and
Smith (1997, 1999) reported that flocks from great grandparents, grandparents, parents,
and broilers haven been affected and as young as four weeks of age with myeloid
leukosis. Other field observations include histocytic sarcomatosis in the spleen and
sometimes liver with ML (Arshad et al. 1997). A case report from Japan, thoroughly
described the lesions seen histologically in the bone and ossifying cartilage of the trachea
and larynx (N akamura et al. 2000). They described the marked proliferation of
myelocytes in the marrow of various bones and periosteum. Also noted was proliferation
of myelocytes in the dura mater of the spinal cord or periosteum of the vertebrae that
caused pressure atrophy of the cord.

Just recently, a report on the experimental induction of erythroblastosis with

subgroup J infection was published (V enugopal et al. 2000). Acutely transforming virus

48

strains were isolated from field cases of erythroblastosis and given to day-old chicks or
ll-day-old embryos. The earliest cases of myelocytomatosis and erythroblastosis
occurred at 22 and 23 days post inoculation in chickens infected as embryos. Similar
findings regarding in vitro transformation with ALV strain 966 (Payne et al. 1993) were
noted with these strains even though in vivo they transformed erythroblasts. Moscovici
and others (Moscovici et al. 1981) reported that leukemic viruses often select one target
cell lineage to transform in vitro while in viva multiple lineages can be affected. The
report by Venogopal et al did not determine whether erbA or erbB oncogenes were
included in the genomes of these acutely transfonning virus strains of ALV-J.

A report of experimental infection of turkeys with strains HPRS-103 and 966
ALV-J was recently published (V enugopal et al. 2000). The results indicate that day old
turkey poults can become infected with subgroup J and seroconvert at about 10 weeks
post-infection. No tumors were observed in this group most likely due to the short time
period. However, turkey poults inoculated with the acutely transforming strain 966 did
develop tumors within 4 weeks of age, the most common time period. Nine of the 12
poults that received the virus intravenously developed tumors while zero out of eight
poults inoculated intraperitoneally with virus developed tumors. The tumors were
described as multiple discrete nodules in the liver. Other gross lesions included
enlargement of the kidneys and white foci on the surface of the spleen. Microscopically,
the lesions in the liver consisted of myeloblasts in the parenchyma and blood vessels.
These tumors were similar to tumors produced by the MC29 virus in turkeys (Schaff et

al. 1978).

49

b. Non-neoplastic Conditions

 

Although there have been anecdotal remarks that infection with ALV-J is
responsible for a variety of conditions such as immune depression, increased flock
variability and poor feed conversion, no reports have been published to document these
remarks. The only report of body weight suppression is from an experimental flock of
broiler breeders and their congenitally infected progeny (Stedman and Brown 1999).
Results fiom this report demonstrated that congenital ALV-J infection affected weight
gain as compared to uninfected, age-matched controls as early as one week of age. They
also noted that the infected chicks had delayed maturation and feather development. An
explanation for this difference in weight gain offered by Brown et al (Brown et a1. 2000)
suggested that the differences in L-thyroxine (T4) levels between the two groups was
related to the development of hypothyroidism in ALV-J infected birds. These birds had
less T4 measured in their sera when compared to uninfected birds which, then led to
differences in weight gain between the groups.

4. Theories of Evolutionary Beginnings

Genetic analysis revealed that ALV-J has interesting sequences (Bai et al. 1995).
Bai and others detected an E element that had only been detected in sarcoma viruses
(Schwartz et al. 1983) and had an env gene similar to sequences found in the EAV family
of endogenous viruses (Boyce-Jacino et al. 1989; Boyce-Jacino et al. 1992). EAV E5] is
an env gene that is defective in the SU region due to stop codons and frame shifts
(Boyce-Jacino et al. 1992) and are very closely related to the env gene of HPRS-103 as
determined by high stringency Southern blotting and sequence analysis (Bai et al. 1995).

This would explain the 40% identity with other ALV gp855. The E element is found just

50

upstream to the 3' LTR. It has only been detected in replication competent RSVs before
and never in ALVs. The function of the E element is unknown. They also discovered an
insertion of a redundant TM segment that is truncated just downstream from the whole
TM (gp3 7). Bai and coworkers (1995) believe that a recombination event occurred with
either EAV E51 or similar sequences to create HPRS-103. Also, their analysis provided
possible origins for other parts of the HPRS-103 genome fi'om other exogenous ASLVs.

Work by two different groups have identified the sequences similar to E5] that
are present in subgroup J ALV. Benson et al (1998) named these sequences ev/J. From
their work, they identified between 6-10 copies of the ev/J proviruses per genome and
that some of the elements segregate in the chicken population. Further work by the same
group (Ruis et al. 1999) determined that ev/J isolates are proviruses that have very little
variation between them, demonstrate a weak similarity to ASLVs, retain enough
sequences that encode for assembly, budding and/or infectivity. RNA can be expressed
by some members which encoded GAG and ENV related polypeptides in chick embryo
cells (Ruis et al. 1999). They also noted that the LTRs of ev/J and ART-CH only had
four different base pairs in the R and U5 regions when compared.

Work by Smith et al (1999) determined that the EAV-HP sequence is different
from E51 and most likely was recombined with in producing HPRS-103. They also
determined that EAV-HP was in the genome in relatively few copies. They mapped
EAV-HP loci to 4 chromosomes on the chicken genome map, 1, 3, 4, and W. Further
work with sequence analysis compared ev/J and EAV-HP and found them to be virtually
identical (Sacco et a1. 2000). They also determined that EAV-HP has 96% identity with

ART-CH on the 5' end of the genome and 99% identity with ART-CH on the 3' LTR R

51

and U5. Another interesting point they found was the EAV-HP U3 is a weak promoter of
transcription.

This subgroup is the first one for ASLV to arise by recombination events between
endogenous and exogenous viruses (Smith et al. 1999). However, this is not the first
documented case for retroviruses in general. Feline leukemia viruses have been reported
to evolve in this manner (Roy-Burman 1995). This report demonstrated that endogenous
feline retroviruses were able to recombine with exogenous viruses in the envelope region
and result in new viruses, among other traits, that are antigenically different from existing
viruses.

5. Antigenic Variants

Antigenic variants have been documented not only in avian retroviruses but also
in all retroviruses as well with other animal and human viruses. Chubb and Biggs (1968)
reported that viruses within the same subgroup could be antigenically different based on
antisera. Fadly and Smith (1997, 1999) documented this phenomenon with subgroup J
isolates HPRS-103 and ADOL Hcl, the U. S. prototype. Work by Benson et al (1998)
determined that U. S. isolates ADOL Hcl, ADOL R5-4 and HPRS-103, the European
prototype arose from a common ancestor and not as separate recombination events.

Two reports have been published about the variability of subgroup J env gene and
how it can result in antigenic variants (V enugopal et al. 1998; Silva ct al. 2000).
Venugopal and others (1998) examined 12 ALV-J viruses in order to determine that they
were variants from HPRS-103. Only two viruses reacted with the neutralization assays
while nine were PCR positive (Smith et al. 1998). All reacted with a specific monoclonal

antibody to gp85 made from HPRS-103. They suggest that the variants arose due to

52

selection pressure most likely caused by immune responses. The United States group
looked at envelope genes from various ALV-J isolated from different farms (Silva et al.
2000). They noted that the U. S. strains were more related to themselves than to the
European strains. Also, the U. S. strains seem to continue mutating at a fairly high rate.
These changes cause difficulty in diagnosis using traditional methods such as virus
neutralization assays (which virus to choose as indicator virus), or whether ELISA kits
for antibody based on the gp85 region will it detect all variants of ALV-J.

Diagnosis of subgroup J ALV can be difficult because of the many variants of the
virus. Virus isolation will detect all subgroups in samples containing infectious virus
particles. Direct ELISA for antibody detection (V enugopal et al. 1997) has limited value
due to the antigenic variation (V enugopal et al. 1998; Venugopal 1999; Silva et al. 2000).
PCR tests that have been published detect proviral DNA in the samples (Smith et al.
1998; Smith et al. 1998; Silva et al. 2000). PCR tests run the risk of false negatives due
to mutations in the proviral DNA where the primers are designed to anneal. RT—PCR
(reverse transcriptase-polymerase chain reaction) has been documented to detect ALV-J
(Smith et al. 1998) along with nested RT-PCR which increases the sensitivity of the test
(Garcia et al. 2000). However, all these PCR based tests may not detect the virus if there
are mutations in sites of the genome where primers must anneal.

A recently published report examined infection rates in congenitally infected
commercial broiler breeders raised in experimental conditions (Witter et al. 2000). They
wanted to determine when was the optimal time for testing and detecting dams that would
transmit virus to their progeny and what types of conventional tests would best detect

shedder dams. From their results, 95% of the hens that would transmit virus to progeny

53

were detected using routine assays for virus and GSA in the albumen. However, the
remaining 5% were difficult to detect, if at all, using the assays in the report (Witter et al.
2000). This remaining percentage is important for the breeding companies due to the
high rate of horizontal transmission of ALV-J after hatch.
M. Research Objectives

Most of the research on ALV-J has been conducted with meat-type chickens
because ALV-J has been associated with major problems in meat-type chickens. Fadly
(1998) reported one case of ALV-J ~1ike induced disease and tumors in commercial laying
chickens in North America. The reason for this outbreak in egg-laying chickens was co—
mingling of infected meat-type chickens with commercial layer chickens in the hatchery.
Furthermore, in the Netherlands, experiments were conducted to determine susceptibility
of two lines of commercial layers, a brown Leghorn line and a white Leghorn line, to
ALV-J infection (Albers and Derkx 2000). Studies conducted by Payne et al (1991,
1992) demonstrated that laying chickens are susceptible to ALV-J infection but less
likely to develop tumors from the infection.

The overall research goal for the current work was to determine the pathogenesis
of ALV-J infection in white Leghorn chickens. The objectives were to: 1) compare
pathogenicity of subgroup J ALV in various lines of white Leghorn chickens; 2)
determine the effects of host factors on the pathogenesis of subgroup J ALV in white
leghoms; and 3) determine the tissue tropism of ALV-J in white leghoms and the

susceptibility of the B cell to transformation by ALV-J.

54

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79

CHAPTER 2
Response of Four Genetic Lines of White Leghorn Chickens to Infection with

Subgroup J Avian Leukosis Virus

80

1. Abstract

The response of four lines of white leghorn chickens to infection with ALV-J was
studied. The lines of chickens evaluated were lines 0, 151,, 7,, and 6,. Chickens of each
line were inoculated as either 7-day-old embryos or at the day of hatch. Uninoculated
chickens fi‘om each line were also included as controls. The two treatment groups
involving each line received the ADOL-Hcl strain of ALV-J at a dose of 10" infectious
units per chicken. At 4, 10, and 30 weeks of age, samples for virus isolation and virus
neutralization were collected. At 4 and 10 weeks of age, bursa] tissue was collected for
methyl green pyronine (MGP) staining and examined for ALV induced pre-neoplastic
bursa] lesions, also known as transformed follicles. Tumor incidence and type were
recorded for each line and treatment group. At 4, 10, 20, and 30 weeks of age,
hematological assays of packed cell volume (PCV) and white blood cell (WBC)
differentials were performed. Subgroup A ALV served as positive controls for the
experiment using the highly susceptible 1515 X 71 cross of chickens with the exact same
protocol as ALV-J inoculated groups. The embryo inoculated groups remained Viremic
tolerant throughout the entire study. Groups inoculated at day of hatch had various
serological responses. Line 0 developed neutralizing antibodies and cleared the virus by
10 weeks of age. The other three lines had varying degrees of success in developing
neutralizing antibodies and viral clearance, with some birds at 30 weeks of age still
harboring the virus in the presence of neutralizing antibodies. Results from the bursa]
transformation assays indicated that ADOL-Hcl could induce transformed follicles in
white leghorn chickens. PCVs did not differ between lines or treatment groups.
Lymphocytes were the most common cell type detected for all lines, regardless of virus
treatment used. Heterophils followed lymphocytes as the second most fiequently
detected cell type while eosinophils and monocytes comprised a very small portion of the
cell counts. Differential WBC counts were difficult to interpret for all groups due to the

variability of cell counts among the control birds. Tumor type and incidence varied. LL

8]

and hemangiomas were the main manifestations with a total of 14 birds diagnosed with

each. There was only one bird diagnosed with myelocytomatosis.

11. Introduction

Avian leukosis virus is the most common retrovirus that causes neoplasms in
poultry. ALVs have been classified into subgroups (A-I) based on host range in embryo
fibroblasts of different avian species, interactions between virus-specific cell receptors
and viral envelope glycoproteins and serum neutralization assays (F adly and Witter
1998). ALV causes a variety of neoplasms and other production problems. A novel
subgroup J was first isolated in the United Kingdom in the early 19905 (Payne et al.
1991) and subsequently isolated in the United States Giadly and Smith 1997). ALV-J
causes myeloid leukosis as its main manifestation while other subgroups cause LL.
ALV-J seems to manifest tumor formation more readily in broiler breeder chickens,
whereas infection of white leghorn chickens results in a lower incidence of tumor
formation (Payne et al. 1991; Fadly and Witter 1998). White leghorn chickens naturally
infected with subgroup J -like ALV also develop tumors (Fadly 1998).

Much is known about subgroups A and B ALVs in leghorn chickens. Disease is
usually typified by bursa] transformation and metastasis of transformed B lymphocytes to
the liver, spleen and other visceral organs after along period of time (Payne and F adly
1997). Early lesions include hemangiomas fhemangiosarcoma of the liver, lung and
kidney (Payne and F adly 1997). Problems such as testicular atrophy or gonad non-
maturation can lead to production losses. Broiler breeders infected with ALV-J tend to
have tumor formation on the keel or sternum, ribs, liver and kidneys (Payne et al. 1991;
Payne and Fadly 1997). These tumors are characterized as myelocytomatosis and are
histologically distinct from LL. Renal tumors of adenomas or carcinomas are also
common. Ages of birds affected by tumor formation and increased mortality usually

begins at 15 weeks. ALV-J induced tumors have been noted in broiler parent flocks as

82

young as four weeks of age (Fadly and Smith 1997). Payne et al (1992) experimentally
infected various genetic lines of meat-type and leghorn chickens. They determined that
line 0 chickens (leghoms in general) had a lower incidence of tumor formation than the
meat type lines of chickens. They also noted the lack of LL in the leghoms
experimentally inoculated with HPRS-103. Other lines involved in Payne’s study were
Brown leghorn, line 151, line 6, and line N plus three lines of meat-type chickens. The
objective of this study was to determine the response of four genetic lines of white
leghorn chickens to infection with strain ADOL-Hcl (U .S. prototype of ALV-J). The
response of chickens to infection with ALV-J was compared to that of line 1515 X 7,

infected with ALV-A.

III. METHODS AND MATERIALS

A. Chickens: Five lines of white leghorn chickens from the USDA, Avian Disease
and Oncology Laboratory, East Lansing, M1 were used. They included line 0, 151,,
7,, 63 and F, of line 1515 X 7,. Line 0 is not an inbred line, it lacks endogenous viral
(ev) loci, is resistant to ALV-E infection, and susceptible to infection and tumor
development by ALV-A, -B, and -C. Line 151, is an inbred line that contains ev loci
and is susceptible to ALV infection and tumor development. Line 7, is an inbred line
that contains ev loci and undefined for ALV-A infection and tumor formation. Line 63
is an inbred line that contains ev loci and is susceptible to ALV-A infection but
resistant to tumor development (Bacon et al. 2000). The breeder flocks for all lines are
free of many avian pathogens, including ALV, as determined by routine serological
surveys. Chickens were housed by line and treatment and kept in plastic isolators with

positive airflow for 30 weeks and given food and water ad libitum.

B. Viruses: Strain ADOL-Hclof ALV-J (F adly and Smith 1999) was used to infect

chickens of lines 0, 1515, 7,, and 6,. The titer of the virus stock was 10’ infectious units

83

(IU)/ml. Rous-associated virus-1 (RAV-1), a subgroup A ALV, with a titer of 10‘5 IU/ml
was used to infect the F, progeny of 1515 X 7, chickens. Each treatment group was

given 10" IU/chicken either via the yolk sac or intraperitoneally of the assigned virus.

C. Experimental Design: Five lines of chickens were divided into three treatment
groups per line comprising a total of 15 groups. Group 1 in each line (75 embryos/line)
was inoculated as 7-day-old embryos via the yolk sac using a 30g 1/2-inch needle. Group
2 in each line (50 chicks/line) was inoculated intraperitoneally at day of hatch using a 30g
1/2 inch needle and group 3 in each line (20 chicks/ line) served as uninoculated controls.
RAV-l infected chickens (1515 X 7,) served as the positive control for Groups 1 and 2. At
hatch (embryo inoculated only-10 chicks/line), 4, 10 and 30 weeks of age, whole blood
samples were collected and plasma was used for virologic assays. The experiment was
terminated at 30 weeks of age. All birds were necropsied either at time of death or at
termination of the experiment.

Bursa] transformation was determined by methyl green pyronine (MGP) staining.
At 4 and 10 weeks of age, five chickens from each of the fifteen groups were randomly
chosen and euthanized. Bursa] tissue was collected and plica separated and placed flat in
cassettes, usually 2 to 3 cassettes per bird. Bursa] tissues were fixed in 4%
paraforrnaldehyde in phosphate buffered saline solution. Tissues were embedded in
paraffin, sectioned, and stained with MGP stain (Siccardi and Burmester 1970; Carson

1990) and examined histologically.

D. Virological and serological assays: Virus isolation (VI) and virus neutralization
(VN) assays were conducted on all plasma samples at each sampling period using line 0
(C/E) CEF. Briefly, VI was performed using 100 microliters of sample plasma added to
CEF grown in 4% calf serum Leib McCoy media containing penicillin, streptomycin and

amphotericin B plus 0.04IU of heparin per plate. Media was changed the following day

84

to 1% calf serum Leib McCoy media containing above antibiotics and fungicide.
Cultures were allowed to grow for 7- 9 days. Plates were frozen and thawed twice (Fadly
and Witter 1998). Cell lysates from CEF cultures were tested by the gs antigen p27
ELISA (Smith et al. 1979). VN assays were performed according to procedures by F adly
et al (1998). Briefly, test samples were diluted 1:5 with Leib McCoy media without
serum and heat inactivated for 30 minutes at 56° C. Dilute virus stocks of RAV-l or
ADOL-Hcl were added such that each plate of a 96 well microtiter plate received 500-
1000 viral particles/ml. Equal volumes of test samples were added to the microtiter plates
and incubated for 45 minutes. Appropriate positive and negative control samples were
included. Cells were added (5X10" cells/well) in 4% calf serum Leib McCoy containing
antibiotics and fungicide, and incubated 7-9 days at 37° C. Cultures were frozen and
thawed twice and the cell lysates were tested for the presence of p27 by ELISA. VN
assays were performed separately with both RAV-l and ADOL-Hcl for indication of

accidental cross contamination.

E. Hematological assays: At 4, 10, 20 and 30 weeks of age, differential WBC counts
and PCVs were performed. Blood smears were stained with 3 Wright stain using an
automated stainer (Wescor 7100 Aerospray TM Slide Stainer). Differential WBC counts
were done manually and based on counts of 100 WBC using a light microscopic at 40X.
PCVs were performed using microhematocrit tubes (Oxford Labware by Sigma) and spun
for 3 minutes. Each tube was hand read using the Lancer Critocap Microhematocrit

Capillary Tube Reader card.

F. Pathology: Necropsy was performed on all birds that died during the experiment and
at termination. Tissue samples for histology were taken from birds with gross tumor
formation. Normal tissue samples taken from control birds fi'om each line included liver,

spleen, gonads, intestines, heart, lung, and kidney. All samples were fixed in 10% neutral

85

buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and

eosin.

G. Statistical analysis: Statistical analysis was performed with the SAS 8.0 program.
Viremia and antibody data, bursa] transformation data, and tumor incidence data were
all examined with a chi-square test. Differential WBC counts for each line of chicken
were analyzed by the Wilcoxon Rank-Sum test due to the nonparametric distribution of
the data. Additional analysis was performed using the 2-way analysis of variance
(ANOVA) with the Dunnett’s t Test when comparing control lines to treatment groups
lines for differences in cell means and comparing line 0 to the other three lines within
treatment groups for differences in cell means. Results were considered significant at a
level of P _<_ 0.05 for chi-square, Wilcoxon Rank-Sum and 2-way ANOVA tests.
Results were considered significant when within the 95 % confidence interval for the

Dunnett’s t Test.

IV. RESULTS

A. Viremia and antibody assays: The whole blood sampling of the embryo inoculated
groups consisted of 10 chicks randomly selected from each line at hatch. At hatch, 48 out
of 50 were VI positive. The two negative chicks, one line 0 and one line 7, were
inoculated with ALV-J. VI and VN results for 4, 10, and 30 weeks sampling periods are
presented in Table 2.1. The embryo inoculated group, regardless of line of chicken or
virus, were Viremic tolerant (viremia and lacking antibody) the entire experiment. All ten
ALV-A infected positive control chicks sampled at hatch were positive. Two other ALV-
A embryo inoculated positive control chickens seroconverted at 10 weeks of age
indicating that inoculation of those two chickens as embryos was not successful.
However, they became infected soon after hatch as all birds in that group were virus

positive at four weeks of age. The negative control groups in each line were negative for

86

virus and antibodies throughout the experiment. Day of hatch ALV-J infected chickens
had variation in viremia and antibody production along the different genetic lines. Line 0
responded sooner with antibody production at four weeks, 27% Ah +, followed by line 7,
with 17% Ab +, line 63 with 6% Ab + and none positive in line 151,. Line 0 was also able
to neutralize the virus sooner than other lines. By ten weeks, there were no line 0 VI+
chickens. Interestingly, line 1515 developed neutralizing antibodies but could not
neutralize circulating virus as demonstrated by the fact that all remaining chickens at 30
weeks were VI-l- (30/30) and 13/30 also had neutralizing Ab. Lines 63 and 7, also had
varying degrees of success in producing neutralizing Ah but could not completely
neutralize circulating virus as demonstrated by the presence of virus at 30 weeks in the
presence of neutralizing Ab. ALV-A, day of hatch inoculated chickens began to test
positive for antibodies at 4 weeks of age (3%). By 10 weeks, 96% tested positive for
antibody with 5/27 still virus positive. At the end of the 30 week time period, all
chickens had died.

B. Bursal Transformation: Bursa] follicle transformation is detectable by using a MGP
staining technique (Carson 1990). The transformed follicles appear pyroniphilic (more
eosinophilic) compared to surrounding follicles. Also, the normal architecture is
disrupted such that the cortex and medulla are indistinguishable from each other. Results
are presented in Table 2.2. The bursa] tissue sample was considered positive if one
follicle had evidence of transformation after examining all sides for that chicken.
Negative controls had no transformations in any line. Line 1515 had a 40% transformation
rate in the day of hatch inoculated group at 10 weeks versus none in its embryo
inoculated counterpart. . Line 0 had a 20% rate at 4 weeks in each group that transformed,
while line 7, day of hatch inoculated group and line 63 embryo inoculated group had a
20% transformation rate at 10 weeks. ALV-A inoculated chickens had a 60% follicle

transformation rate at 4 weeks of age regardless of time of virus inoculation. At 10

87

weeks, both embryo and day of hatch inoculated chickens had a 100% bursa] follicle

transformation rate.

C. Hematological assays: The PCV results did not demonstrate any differences between
lines of chickens or treatment group. Results are presented in Table 2.3. There was an
occasional chicken that had a low PCV of 13-17 but no overall anemia could be detected
for any treatment group or genetic line of chicken.

Differential WBC counts for all five lines indicated that lymphocytes were the
most common cell type counted regardless of genetic line, treatment protocol, or virus.
The one exception was for the 30 week old line 0 control chickens where heterophils
were more numerous on a percent average basis. Heterophils were the next most
common cell type. Eosinophils and monocytes had minimal cell counts with usually no
more than an average of 2 cells per 100 examined regardless of line or treatment group.

Results of differential WBC counts are in Figures 2.1-2.20. Line 0 control
chickens mean percent for lymphocytes was increased compared to day of hatch
inoculated birds and embryo inoculated chickens for the 4 week time period (61% versus
60% and 51% respectively) and the10 week time period (62% versus 59% and 53%
respectively). At 20 and 30 weeks, the results were reversed with day of hatch
inoculated chickens (66% and 60%) and embryo inoculated chickens (86% and 79%)
having increased lymphocytic mean percents compared to the control chickens counts
(65% and 44%) (Figure 2.2). With regards to heterophils (Figure 2.1), the mean percent
for control line 0 chickens were decreased compared to day of hatch and embryo
inoculated chickens at the 4 week time period (3 7% versus 38% and 47%) and 10 week
time period (33% versus 39% and 46%). At 20 and 30 weeks, control chickens (34% and

56%) have increased heterophil mean percents

88

Table 2.1: Development of ALV-J or ALV-A induced viremia and antibody in five

lines of white leghorn chickens inoculated as 7 day old embryos or at day of hatch.

VI and Ab Testing-—No.+/No. tested (%)

 

 

 
 
  
  

Age Group Line V1 Ab
46 =4 113,; extinct) 440/40(100)1 , 040(0)?"
, it ,2 " 49 ,g, .. 13/44(30)1 3‘ 1274c(27)2 395
36; a .3._, .3. H 0720(0) sew/20(0):
""* 1““ Iiriel‘515‘““’ 46740(100) " 6740 (0)
2 44744 (100) 0744 (0)
3 0719 (0) 0719 (0)
4.4396143 47% ‘4: A.,,Lmef63g fil/Zl?€100£_ 07,3151» 4.
:34 4 a, , A 1607/2100) 4‘34 226% g,
" cf 2.35.5 ' 3.,7» 4+ I2-Q(). r... 491
1 tine 71' 3733’ (160') 73 (0)
2 18/18(100) 3/18(17)
3 0/18 (0) 0/18 (0)
,s'“ -5: 431 ’ Line1515 x-71- 4.45745 (100 4 . 30745(0)ae _4
- . ..-~. 2' ~ .~ ,. 4 : 34./34(100' mam-c
. 4 3 I", ‘3‘, ,. 0716(0).“ .-.,, «MO/16(0) ‘1
1 Line 0 26726 (100)3 0726 (0)4
2 0737 (0)3 28737 (76)4
3 0715 (0) 0715 (0)
,4 4 .55”. "'1 Lane 1515 334734000)- 0734 (0)54 .
1443;: 3 2‘ 43:, 36736 3.7) 44437371364
.3 4' '_'. v . 4 8 0/1 0/14 0 "14‘
16 532518“ '”1 116363” 157156030) 0713((0))
2 11712 (92) 1712 (8)
3 0718 (0) 0718 (0)
,4 4 :71 . Line-'21 ‘ :463'272000) 072(0)6-: ”7"
,7 ' , 2.1. 3 ’ 3714 1_) .3714‘(96
4"; 3 g, 4314' 07 )3 30711(0
’ ’ 1 ‘ L1ne1515X71 34735 (97)7 2735 (6)8
2 5727(19) 7 26727 (96) 8
3 0711 (0) 0711 (0)

 

 

 

 

 

 

 

89

 

Table 2.1 Continued
Age Group Line VI Ab
«11,3; ;Line30 4 ’ 777 (100)9 3077(03‘10 T";
:4 1 2- . _ = ,0/28 (0)96 26728('93)19‘ :4
413’ 3 6 .54, .‘ 6 _ '0'76 0) ' 076(0)
1‘ Linelsls‘ 17717 00) 0717(0)11 “ "
2 30730 (100) 13730 (43)11
3 079(0) 079 (0)
30 weeks 1 , Line 63 878 (100) 33 4'43 0783(0)
.1 .2 , , 475 (88) , ' (17,5 (20),,
. ~ 3. : 1“ ' 0712 712 0
1 “‘ '1’" “‘ L1n6‘7, - ‘“ 1717100)) 071 (0 '6 “"“
2 576 (83) 676 (100)[2
3 0/5(0) 0/5(0)
4,; 3.4:; =41" 4 31.154141635143547,- «; 131(100) 3.4 4071(014; 434 4
413. 3?} > 2 >3 6: ‘ ‘ £71 . ”In “. ' ?
~ 4' 34 ,7"- 0/5(0) "’ 5 2951973: 11

 

 

 

 

 

 

 

Groupl, inoculated as embryos; group 2, inoculated at one day of age; group 3,
uninoculated controls.

a: all but three chicks accidentally died in first week of life due to management
problems.

b: all chickens in this group died before 30 weeks of age.

Entries with the same number in superscript are statistically different from each other

with a p—value of <0.05 for that assay.

Table 2.2: Bursal transformation induced by ALV in five lines of white leghorn
chickens inoculated as 7 day old embryos or at day of batch.
Age at Testing--No. +/No. tested (%)

of hatch

    

compared to day of hatch (33% and 39%) and embryo inoculated chickens (13% and
21%).

Line 1515 control chickens had increased mean percent heterophil count for all
time periods when compared to day of batch and embryo inoculated chickens (Figure
2.5). Embryo inoculated chickens heterophil counts were increased at 4 (23%) and 10
(18%) weeks compared to day of batch inoculated chickens (5% and 8% respectively). At
20 and 30 weeks the day of hatch inoculated group (13% and 12%) is slightly increased
compared to the embryo inoculated group (11% and 11%). Control chickens had
decreased mean percent lymphocyte counts for all time periods when compared to day of
hatch and embryo inoculated chickens (Figure 2.6). Day of hatch inoculated chickens
mean percent lymphocyte counts were increased at 4 and 10 weeks (95% and 91%)
compared to embryo inoculated birds (74% and 81%). The groups switch at 20 and 30
weeks with the embryo inoculated chickens (88% and 89%) slightly increased compared
to day of hatch inoculated chickens (86% and 87%).

Line 71 control chickens had decreased mean percent heterophils for all time
periods when compared to day of hatch inoculated chickens (Figure 2.9). With regards to
the mean percent lymphocyte counts, the day of batch inoculated chickens were
decreased compared to control chickens(Figure 2.10). Due to the very low number of
chickens in the embryo inoculated group, no comparisons were made at any time period.

Line 63 control chickens had increased mean percent heterophil counts at 4, 10
and 30 weeks of age compared to day of hatch and embryo inoculated chickens (Figure
2.13). At 20 weeks, controls were equal to day of hatch chickens for heterophils (4.8%)
while embryo inoculated chickens were increased (5.7%). For mean percent lymphocyte
counts (Figure 2.14), the control chickens were decreased at 4, 10 and 30 weeks of age
when compared to day of hatch and embryo inoculated chickens. At 20 weeks, the
controls (95%) were equal to the day of hatch inoculated (95%) and slightly increased
compared to the embryo inoculated (94%).

91

The cross 151, X 7l (ALV-A infected) control chickens had decreased mean
percent heterophil counts compared to day of hatch and embryo inoculated chickens at all
time periods (Figure 2.17). However, the control chickens had increased mean percent
counts of lymphocytes at all time periods when compared to day of hatch and embryo
inoculated chickens (Figure 2.18). Comparisons at 30 weeks only consisted of controls
and embryo inoculated chickens because all remaining day of hatch chickens had died.
Analysis of the 30 week data was not performed due to the small number of embryo
inoculated chickens left at this time period.

Each line was analyzed for differences between control group and inoculated
treatment groups. There were significant statistical differences found between line 0
control chickens and embryo inoculated chickens for heterophil and lymphocyte counts at
weeks 4,10, 20 and 30 (Figures 2.1 and 2.2) and for monocytes between control chickens
and day of hatch chickens at week 10. Line 151, had significant statistical differences
between control chickens and both day of hatch inoculated chickens and embryo
inoculated chickens for heterophil counts at weeks 10, 20 and 30, lymphocyte counts at
weeks 4, 10, 20, and 30, and eosinophil counts for weeks 4, 10, and 30 (Figures 2527).
Line 63 had significant statistical differences between the control chickens and both day
of hatch inoculated chickens and embryo inoculated chickens for heterophil and
lymphocyte counts at weeks 4 and 10, and eosinophil counts at week 30 (Figures 2.13-
2.15). In line 7I there were significant statistical differences between control and day of
hatch chickens for heterophil and lymphocyte counts at weeks 4, 10 and 30, eosinophil
counts at week 4 and monocytes at weeks 4 and 20. Due to the low number of line 71
embryo inoculated chickens that survived to 4 weeks of age, comparisons were made
only between control and day of hatch groups (Figures 2.8-2.12).

Statistical analysis of the differential WBC counts revealed that when all 4 lines
of ALV-J infected chickens are combined together for that specific treatment group, there

were significant differences seen for heterophils between the control lines and both day of

92

hatch and embryo inoculated lines at 4 and 30 weeks and there were differences between
the control lines and embryo inoculated lines at 10 and 20 weeks (Figures 2.21). For
lymphocytes, there were significant differences between the control lines and both day of
batch and embryo inoculated lines at 4 and 30 weeks and there were significant
differences between the control lines and embryo inoculated lines at 10 and 20 weeks
(Figures 2.22). For eosinophils, there were significant differences between the control
lines and embryo inoculated lines at 4 weeks (Figure 2.23). For monocytes, there
significant differences between the control lines and both day of hatch and embryo
inoculated lines at 4 and 10 weeks and there were significant differences between control
lines and embryo inoculated lines at 30 weeks (Figure 2.24).

Comparing the four lines, (lines 0, 151,, 63, and 7,) for differences between cell
counts was conducted by comparing each line to line 0 results for each treatment group
and time period. Line 0 was chosen for comparison because it is only non-inbred line
in the study. In the control group, there were significant differences in heterophil and
lymphocyte average percentages noted at 4, 10, and 30 weeks between line 0 and all
three remaining lines and at 20 weeks between line 0 and 1515 and 63 (Figures 2.25-
2.26). For eosinophils, there were significant differences noted at 20 weeks between
line 0 and 63 and at 30 weeks between line 0 and 151, and 63 (Figure 2.27). For
monocytes, there were significant differences noted at 4 and 30 weeks between line 0
and 63 and at 10 weeks between line 0 and 1515 (Figure 2.28). Comparing day of hatch
inoculated line 0 to the three remaining day of hatch inoculated lines revealed
significant differences in the heterophil and lymphocyte average percentages at all time
periods (Figures 2.29-2.30). For eosinophils, there were significant differences noted
at 4 weeks between line 0 and line 1515, at 20 weeks between line 0 and lines 151, and
63, and at 30 weeks between line 0 and 1515 (Figure 2.31). For monocytes, there were
significant differences between line 0 and lines 1515 and 63 at 10 weeks (Figure 2.32).

Due to the low number of embryo inoculated line 7, chickens that survived to 4 weeks,

93

 

no analysis for line 7, compared to line 0 embryo inoculated chickens will be noted on
Figures 2.33-2.36 although line 71 data was included. Comparing embryo inoculated
line 0 to embryo inoculated lines 1515and 63 revealed significant differences in the
heterophil and lymphocyte average percentages at all time periods (Figure 233-234).
For eosinophils, there were no significant differences noted at any time period. For

monocytes, there were significant differences between line 0 and 63 (Figure 2.36).

D. Histopathology: All chickens that died during the experiment and at termination
were subjected to necropsy. All gross tumors were noted and tissue samples taken for
microscopic examination. Results are presented in Table 2.4. In treatment group 1
line 0, there was one chicken with erythroblastosis and one chicken with myeloid
leukosis. The other lines had hemangiomas and renal tumors as the main non-myeloid
or non-leukosis manifestation as well as LL. The single chicken with myeloid leukosis
had involvement of the heart, lung, liver and kidney. In treatment group 2, besides
tumors, line 0 and 1515 had some male chickens with atrophic testes at 30 weeks. Line
63 had no tumor formation. One line 0 chicken developed a chondrosarcoma. Group 1
ALV-A infected chickens developed the greatest number of tumors overall, as
expected. There were 28 chickens with LL and 4 with hemangiomas. Group 2 ALV-
A infected chickens again developed the most tumors with 23 developing LL, one with
myelocytomatosis, three with hemangiomas and one developed renal tumors. Negative

control chickens did not develop tumors.

94

Table 2.3: Packed cell volume values in five lines of white leghorn chickens inoculated
with ALV-J or ALV-A as 7 day old embryos, at day of hatch, or uninoculated controls

Range Average

    

Age Group Line # of samples
V . 7‘" , ‘ . 7 . _, . _ .57 _

   
  

Line] 515 44 36-44 40

4 weeks Line 63 15* 32-40 35
Line 7, 19 28-35 30

Line 1515 x 7, 34 28-35 31

 

LinelSI, 33* 32-38 35
Line 6, 15 27-35 31
Line 7, 2 19-30 25

 

 

Line 1515 x 7, 11 29-34 32

 
 

 

first“: "

. 1“,. »_

me! ‘
LinelSIs 30 13-53 40
30 weeks Line 63 5 32-46 42
Line 7, 6 26-43 33
Line 1515 x 7, 0" nt nt

,t

        

* Indicates fewer samples evaluated due to human error.

95

“ No samples tested because all birds had died by 30 weeks of age.
nt- not tested.

1- Embryo inoculated

2- Day of hatch inoculated

3- Uninoculated controls

Table 2.4: Diagnosis of ALV induced tumors in five lines of white leghorn chickens

inoculated with ALV-J or AVL-A as 7 day old embryo or at day of age.

 

 

 

Group Line # with LLb MLc HEMd RT‘ Other‘
Tumor/#at
risk‘

1 Line 0 I III? 7 1 l 2 I
1 515 7/17 0 0 4 l 2
6, 1/5 1 0 0 0 0
7, 1/1 0 o 1 0 o
1515 x 7, 28/29 28 0 4 o 0

2 L1ne 0 7/29 2 O 4 0 1
1515 6/ 3 l 4 0 3 O 1
6, 0/5 0 o o 0 o
7, 2/6 0 o 1 1 o
1515 X 7, 26/26 23 1 3 1 0

3 Line 0 0/6 0 0 0 O U
151, 0/8 0 0 o o o
6, 0/12 0 o o o 0
7, O/ 8 0 0 O 0 O
1515 x 7, 0/5 0 o o 0 0

 

 

 

Group 1, inoculated as embryos. Group 2, inoculated at day of age, group 3,
uninoculated controls.

a: # at risk=# died with tumors + # survived to end of experiment

b: LL=lymphoid leukosis

c: ML=myelocytomatosis

d: HEM=hemangioma

e: RT=renal tumors

f: Other: chondrosarcoma, erythroblastosis, fibromas

96

 

 

 

Percentage of cells
\-
9

    

 

 

 

 

4 weeks 10 weeks 20 weeks 30weeks
Age oI'CIIIckenI
[-Controls .DIy ofHItch DEmbryol

 

 

Figure 2.1: Line 0 heterophil comparison. Mean percent number of heterophils over
time. Change in letters indicates a statistically significant difference between the control
group and the indicated treatment group at that time period.

100

 

90

 

 

 

 

 

 

Percentnge of Cells

 

 

 

 

 

4 weeks 1

weeks 30weeks
Age of Chlckens
EControls IDay of Hatch ElEmbryo]

Figure 2.2: Line 0 lymphocyte comparison. Mean percent number of lymphocytes over

time. Change in letters indicates a statistically significant difference between the control
group and the indicated treatment group at that time period.

97

Percentage of Cells

 

4 weeks 10 weeks 20 weeks 30weeks

Age of Chlekens

 

lIControlr llDay offllteh DEmhryoJ

 

Figure 2.3: Line 0 eosinophil comparison. Mean percent number of eosinophils over
time. Change in letters indicates a statistically significant difference between the control
group and the indicated treatment group at that time period.

4

3.5

Percentage of Cells
N

 

4 weeks 10 weeks 20 weeks 30weeks

Age of Chickens

 

1IControls IlDay ofHateh DEmbryoi

 

Figure 2.4: Line 0 monocyte comparison. Mean percent number of monocytes over
time. Change in letters indicates a statistically significant difference between the control
group and indicated treatment group at that time period.

98

35

 

30 ,,

 

 

 

Percentage of Cells

 

 

 

 

4 weeks In weeks 20 weeks 30weeks

Age of Chicken

 

1lCoatrols leyotH-teh DEmbryo’

 

Figure 2.5: Line 151, heterophil comparison. Mean percent number of heterophils over
time. Change in letters indicates a statistically significant difference between the control
group and the indicated treatment group at that time period.

   

 

 

Percentage of Cells

 

 

 

 

 

 

 

1
1
1

4 weeks It) weeks 2 weeks JDweeks

Age of Chickens

 

1ICoItrels lDay ot‘HateI Ell-Embryo]

 

Figure 2. 6: Line 1515 lymphocyte comparison. Mean percent average for lymphocytes
over time. Change 1n letters indicates a statistically significant difference between the
control group and the indicated treatment group at that time period.

 

 

2.5

 

 

 

 

 

 

 

 

Percentage of Cells

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4 weeks 10 weeks 20 weeks 30weeks

Age of Chlckeas

 

[IControls IDay of Hatch DEmbryfl

Figure 2.7: Line 1515 eosinophil comparison. Mean percent number of eosinophils
over time. Change in letters indicates a statistically significant difference between the
control group and the indicated treatment group at that time period.

 

 

 

 

 

 

 

 

Percentage of Cells

 

 

 

 

 

 

 

 

 

 

 

4 weeks 10 weeks 20 weeks 30weeks

Age of Chickens

 

[IControls IDay ot‘ Hatch DEmhryo1

Figure 2.8: Line 1515 monocyte comparison. Mean percent number of monocytes
over time. Change in letters indicates a statistically significant difference between the
control group and the indicated treatment group at that time period.

100

Percentage of Cells

 

4 weeks 10 weeks 20 weeks 30weeks

Age of Chickens

 

1. Controls l Day of Hatch El Embryo I

 

Figure 2.9: Line 7, heterophil comparison. Mean percent number of heterophils over
time. Change in letters indicates a statistically significant difference between the control
group and the indicated treatment group at that time period.

 

 

Percentage of Cells
(ll
0

    

 

 

 

 

 

 

4 weeks 10 weeks 20 weeks 30weeks

Age of Chlckens

 

Eontrols lDay of Hatch CIEmbryo}

 

Figure 2.10: Line 7, lymphocyte comparison. Mean percent number of lymphocytes
over time. Change in letters indicates a statistically significant difference between the
control group and the indicated treatment group at that time period.

101

 

2.5

 

 

 

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10 weeks 20 weeks 30weeks

Age of Chickens

 

{IControls BDay of Hatch DEmbryo,

 

Figure 2.11: Line 7, eosinophil comparison. Mean percent number of eosinophils over
time. Change in letters indicates a statistically significant difference between the control
group and the indicated treatment group at that time period.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 2.12: Line 7, monocyte comparison. Mean percent number of monocytes over
time. Change in letters indicates a statistically significant difference between the control
group and the indicated treatment group at that time period.

102

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4 weeks 10 weeks 20 weeks 30weeks

Age of Chickens

 

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Figure 2.13: Line 63 heterophil comparison. Mean percent number of heterophils over
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group and the indicated treatment group at that time period.

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4 weeks 10 weeks 20 weeks 30weeks

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Figure 2.14: Line 63 lymphocyte comparison. Mean percent number of lymphocytes
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103

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4 weeks 10 weeks 20 weeks 30weeks

Age of Chickens

 

llControls IDay of Hatch CIEmbryo1
Figure 2.15: Line 63 eosinophil comparison. Mean percent number of eosinophils over

time. Change in letters indicates a statistically significant difference between the control
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4 weeks 10 weeks 20 weeks 30weeks

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Figure 2.16: Line 63 monocyte comparison. Mean percent number of monocytes over
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104

 

 

 

 

 

 

 

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4 weeks 10 weeks 20 weeks 30weeks

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llControls llDay of Hatch DEmbryo;
Figure 2.17: Line 1515 X 7, heterophil comparison. Mean percent number of heterophils
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4 weeks 10 weeks 20 weeks 30weeks

Age of Chickens

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significant difference between the control group and the indicated treatment group at that
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105

 

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Figure 2.23: Eosinophil percent comparison of ALV-J infected lines combined over
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Figure 2.24: Monocyte percent comparison of ALV-J infected lines combined over time.
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108

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V. DISCUSSION

In this study, we repeated some of the work of Payne et a1 (1992) utilizing some
of the same genetic lines of chickens infected with an antigenically different strain of
ALV-J. Our studies were conducted with the antigenically distinct U. S. strain, ADOL-
Hcl , whereas Payne’s work was conducted with the HPRS-103 strain of ALV-J first
isolated in the U. K. Antibodies made against HPRS-103 do not neutralize ADOL-Hcl
while antibodies against ADOL-Hcl neutralize HPRS-103 (Fadly and Smith 1997; Fadly
and Smith 1999). Venugopal et a1 (1998) also noted antigenic variability of 12 field
isolates compared to HPRS-103. Only two of those field isolates were neutralized by
HPRS-103. The 12 isolates were determined to be ALV-J by PCR (Smith et al. 1998).
Silva et a1 (2000) also described the genetic variability among U. 8. isolates in the
envelope region. Since there is antigenic differences between the two prototypes, this
study was conducted to determine if there are differences in response to infection with a
different strain of virus in various genetic lines of chickens. The highly susceptible F,
progeny of 1515 X 7, infected with ALV-A served as the positive control.

Experimental inoculation of embryos before the immune system has developed is
one way to reproduce natural congenital infection with ALV. Congenitally infected
chicks with ALV usually remain Viremic tolerant throughout their lives, serving as a
source of infection for hatch mates. The results obtained in this experiment demonstrated
the same outcome of Viremic tolerance in four genetic lines of white leghorn using the
U. S. prototype of ALV-J. However, the results were dramatically different in chickens
inoculated at day of hatch compared to those inoculated as embryos. Day of hatch
inoculated line 0 chickens were able to seroconvert and neutralize circulating virus by 10
weeks of age. Line 1515 had all chickens (30/30) harboring virus at 30 weeks of age
while 13/30 had detectable antibody at 30 weeks of age. Line 7, had virus detected in 5/6
chickens and antibody detected in 6/6 chickens at 30 weeks of age. Line 63 had limited
antibody detected while the majority of the chickens harbored virus at 30 weeks of age.

133

Previous experiments done with ALV-A (RAV-1) and different genetic lines of chickens
with various ev genes develop antibodies at a slower pace than line 0 which lacks ev
genes (Crittenden et al. 1987; Crittenden 1991). The ev genes in chickens have been
studied and have been shown to interfere with the immune system when challenged with
exogenous ALVs (Crittenden et al. 1987). The chickens in this experiment had ev genes
(except line 0) and our results supported previous work which demonstrated that the ev
genes interfere with the immunological response to ALV infection.

The four virus negative chickens in Group 1, ALV-J (lines 0 and 7,) and ALV-A
embryo inoculated, illustrated that embryo infection is not always 100%. The inoculum
in these cases may not have had enough viral particles to establish an infection. One line
0 chick and one line 7, chick ALV-J Group 1 were not virus positive and did not survive
the first week of life. The two, ALV-A, Group 1, chickens were exposed to the virus at
hatch fiom their infected hatch mates and were not detected until 10 weeks when
seroconversion occurred. They were not a part of the original 10 chicks that were
randomly selected to test at day of hatch for the presence of virus. Because detection of
the antibody did not occur until 10 weeks of age and only one chicken had cleared the
virus, they were kept in the experiment.

Tumor susceptibility in ALV infection does not correlate to serological status and
the ability to produce antibodies (Payne and Fadly 1997). The loci WA and th (trunor
virus subgroup A and B) are important in determining the cellular resistance or
susceptibly of the bird to infection (Payne and F adly 1997). However, this is not always
the case. For example, line 63 is susceptible to infection by subgroup A but rarely
develop tumors, mainly due to lack of target cells in the bursa of Fabricius, not because of
the WA or th loci (Purchase at al. 1977). This perhaps will hold true with subgroup J
infection. Only one chicken in the Line 63 groups developed a tumor and it was of
lymphoid origin and only one chicken had bursal transformation at ten weeks of age.

Both of these line 63 chickens were in the embryo inoculated treatment group. Line 7, is

134

undefined for ALV-A infection and tumor formation. In this study, line 7, was
susceptible to infection and tumor formation with ALV-J. There was bursal
transformation at 10 weeks (20%) with three chickens (one embryonically challenged and
two day of batch challenged) developing tumors. Inoculation as embryos or at day of
batch can also influence tumor incidence (F adly et al. 1987). Viremic tolerant chickens
are more likely to die from LL than chickens that have antibody (Rubin et al. 1961). This
is due to the lack of antibody response, allowing the circulating virus to continuously
replicate in embryo inoculated chickens and therefore increase the chance of integration
by the proviral DNA next to a proto-oncogene. Line 0 and 15I5 illustrated this
phenomenon when inoculated with ALV-J as embryos or at day of hatch (Table 3). The
other two lines did not have enough chickens survive to the end of the experiment to
make a valid statistical comparison. Line 1515 X 7, is highly susceptible to ALV-induced
tumor formation. This was evident by the fact that 96% (28/29) of the chickens in the
embryo inoculated group developed tumors and 100% (26/26) of chickens inoculated at
day of hatch developed tumors before 30 weeks of age. Both groups had a few chickens
with more than one tumor type present.

Bursal transformation due to infection with subgroup A ALV demonstrates the
ability of the virus to integrate next to a proto-oncogene (usually c-myc) in B cells and
result in LL. The MGP staining technique was used to demonstrate the presence of RNA
within a cell, which indicates that the cell is active in the cell cycle. When retroviruses
integrate next to a proto-oncogene, the strong promoters within the LTRs direct the over
expression of the downstream host genes. The overexpressed proto-oncogene protein
disregards the normal cellular grth regulatory system within that cell (Lewin 1994).
These cells are candidates for transformation and stain brightly eosinophilic with the
MGP stain due to the large amounts of RNA being produced. The results from these
studies indicates that ALV-J can transform bursal follicles and may account for the

lymphoid leukosis manifestation that was observed as a predominate lesion in the white

135

leghoms used in this study (Table 3). The oncogene involved was not determined in this
study. With regards to the ALV-A infected chickens, they responded as expected with
100% of the bursas examined having transformed follicles. The highly susceptible line
1515 x 7, allows the integration of the proviral DNA near c-myc, resulting in a high
frequency of bursal follicle transformation.

Differences in WBC counts among chickens may be difficult to interpret due to
the wide range of normal white blood cell counts (Lucas and J arnroz 1961; Sturkie and
Griminger 1976; Campbell 1988). Most authors agree that in order to identify ill birds,
large differences from the normal leukogram must be noted (Lucas and Jamroz 1961;
Sturkie and Griminger 1976; Campbell 1988). Comparisons were made only on a mean
percent basis because a complete blood count was not performed. Therefore, no analysis
on actual decreases or increases in cell counts was performed. Within control Group 3,
there were statistical differences between all genetic lines of chickens for heterophil and
lymphocyte levels at all sampling periods. These differences could be due to the genetics
of each line (Lucas and J arnroz 1961; Shen et al. 1984) or due to the overall health status
of the chickens. The control chickens may have had incidental physical injuries that could
have influenced their leukograms. Various authors explain the differences seen on many
factors like age, sex, nutritional status, environment, and degree of stress (Lucas and
Jamroz 1961; Sturkie and Griminger 1976; Campbell 1988). Since all chickens in this
study were hatched on the same day and housed in similar isolators and fed the same
feed, some of those factors may have little influence on the variation seen. Sex and
degree of stress may have a strong influence on the outcome in addition to genetics. It
should be noted that the time of sampling may be considered stressful for the subjects due
to handling, restraint, and catching the chickens in the isolators. Although there were
significant statistical differences noted, these cannot be explained by the viral infection

alone. One interesting observation is that with all the chickens that died with LL induced

136

by ALV-J or ALV-A, there were no lymphoblastic cells in blood smears even though
there was lymphoblastic cell infiltrates in various tissues.

Although ALV-J is mainly encountered in broiler breeder chickens, this study
along with earlier work by Payne et al (1992) demonstrates that white leghorn chickens
are susceptible to infection and tumor formation under experimental conditions. The main
tumors encountered in this study were LL and hemangiomas, while Payne did not report
the presence of LL in the leghoms or meat type chickens. The differences were probably
not due to genetic differences among experimental birds. Three lines of white leghorn
chickens that were common to both experiments have very similar genetic backgrounds.
Differences may be due to the use of antigenically different viruses. Determining the
receptor on the target cells may offer additional insight to explain the different tumor

manifestations seen (myeloid versus lymphoid).

137

REFERENCES

1. Bacon, L. D., H. D. Hunt and H. H. Cheng. A review of the developement of chicken
lines to resolve genes determining resistance to diseases. Poult Sci 79: 1082-1093.
2000.

2. Campbell, T. W. Avian Hematology and Cytology. Ames, IA, Iowa State University
Press. 1988

3. Carson, F. . In Histotechnology: A Self-Instructional Text. ed.S. Borysewicz. Chicago,
American Society Clinical Pathologists Press. pp108-109. 1990.

4. Crittenden, L. B. Retroviral elements in the genome of the chicken: Implications for
Poultry Genetics and Breeding. Critical Review of Poultry Biology(No. 3): 73-
109. 1991.

5. Crittenden, L. B., S. McMahon, M. S. Halpem and A. M. F adly. Embryonic infection
with the endogenous avian leukosis virus Rous- associated virus-O alters

responses to exogenous avian leukosis virus infection. J Virol 61(3): 722-725.
1987.

6. Fadly, A. M. . Subgroup J avian leukosis virus infection in meat-type chickens: A
review. IV Asian Pacific Conference on Poultry Health.1998.

7. Fadly, A. M., L. B. Crittenden and E. J. Smith. Variation in tolerance induction and
oncogenicity due to strain of avian leukosis virus. Avian Pathol: 665-677. 1987.

8. Fadly, A. M. and E. J. Smith . An overview of subgroup J-like avian leukosis virus
infection in broiler breeder flocks in the United States. Avian Tumor Viruses
Symposium, Reno, Nevada, American Association of Avian Pathologists. 1997.

9. Fadly, A. M. and E. J. Smith. Isolation and some characteristics of a subgroup J -like
avian leukosis virus associated with myeloid leukosis in meat-type chickens in the
United States. Avian Dis 43(3): 391-400. 1999.

10. Fadly, A. M. and R. L. Witter. Oncomaviruses: leukosis/sarcoma and
reticuloendotheliosis. In A Laboratory Manuel for the Isolation and Identification
of Avian Pathogens. eds,D. E. Swayne, J. R. Glisson, M. W. Jackwood, J. E.
Pearson and W. M. Reed. Kennett Square, PA., American Association of Avian
Pathologists. pp185-196. 1998.

1 1. Lewin, B. Genes. New York, Oxford University Press. 1994

12. Lucas, A. M. and C. J arnroz. Altas of Avian Hematology. Washington DC, United
States Department of Agriculture. 1961

13. Payne, L. N., S. R. Brown, N. Bumstead, K. Howes, J. A. Frazier and M. E.

Thouless. A novel subgroup of exogenous avian leukosis virus in chickens. J Gen
Virol 72(Pt 4): 801-807. 1991.

138

14.

15.

l6.

17.

18.

19.

20.

21.

22.

23.

24.

Payne, L. N. and A. M. Fadly. Leukosis/Sarcoma Group. In Diseases of Poultry.
eds,B. W. Calnek, H. J. Barnes, C. W. Beard, L. R. McDougald and Y. M. Saif.
Ames, Iowa, Iowa State University Press. le4-466. 1997.

Payne, L. N., A. M. Gillespie and K. Howes. Myeloid leukaemogenicity and
transmission of the HPRS-103 strain of avian leukosis virus. Leukemia 6(11):
1167-1176. 1992.

Purchase, H. G., D. G. Gilrnour, C. H. Romero and W. Okazaki. Post-infection
genetic resistance to avian lymphoid leukosis resides in B target cell. Nature
270(5632): 61-62. 1977.

Rubin, H., A. Cornelius and L. Fanshier. The pattern of congenital transmission of an
avian leukosis virus. Proc. Natl. Acad. Sci. USA 47: 1058-1060. 1961.

Shen, P. F., E. J. Smith and L. D. Bacon. The ontogeny of blood cells, complement,
and irnmunoglobulins in 3- to 12-week-old 15 5 -B congenicwhite leghorn

chickens. Poult Sci 63: 1083-1093. 1984.

Siccardi, F. J. and B. R. Burmester (1970). The differential diagnosis of lymphoid
leukosis and Marek's disease, USDA. 1412.

Silva, R. F ., A. M. Fadly and H. D. Hunt. Hypervariability in the envelope genes of
subgroup J avian leukosis viruses obtained from different farms i n the United
States. Virology 272: 106-111. 2000.

Smith, E. J ., A. Fadly and W. Okazaki. An enzyme-linked immunosorbent assay for
detecting avian leukosis- sarcoma viruses. Avian Dis 23(3): 698-707. 1979.

Smith, L. M., S. R. Brown, K. Howes, S. McLeod, S. S. Arshad, G. S. Barron, K.
Venugopal, J. C. McKay and L. N. Payne. Development and application of
polymerase chain reaction (PCR) tests for the detection of subgroup J avian
leukosis virus. Virus Res 54(1): 87-98. 1998.

Sturkie, P. D. and P. Griminger. Blood: Physical characteristics, formed elements,
hemoglobin, and coagulation. In Avian Physiology. ed.P. D. Sturkie. New York,
Springer-Verlag. 1. pp53-75. 1976.

Venugopal, K., L. M. Smith, K. Howes and L. N. Payne. Antigenic variants of J

subgroup avian leukosis virus: sequence analysis reveals multiple changes in the
env gene. J Gen Virol 79(Pt 4): 757-766. 1998.

139

CHAPTER 3
Influence of Endogenous Virus 21 (EV21) on the Response of White Leghorn

Chickens to Infection with Subgroup J Avian Leukosis Virus

140

1. Abstract:

The influence of endogenous virus 21 (EV21) on the response of White Leghorn
chickens to infection with ALV-J was studied. F1 progeny from the cross EV21+ males
with 15B] females were hatched, characterized as late feathering or early feathering by
the length of the primary feathers, and inoculated at day of hatch with the U. S. prototype
of ALV-J, ADOL Hc-l. Confirmation of feathering typing was performed at 10 weeks of
age by the R2 alloantisera assay. Whole blood samples for virus isolation and
neutralization assays were collected at 4, 10, 18 and 31 weeks of age. Necropsy was
performed on all birds that died during the experiment and at termination. Gross tumor
samples were collected for microscopic examination. Birds harboring EV21 were more
likely to be ALV-J virus positive at all sampling periods than birds lacking EV21. The
incidence of ALV-J antibody in chickens lacking EV21 was higher than in chickens
harboring EV21. Birds harboring EV21 were also more Viremic tolerant at the end of the
experiment than birds lacking EV21. Tumor development was minimal for both groups.

LL was the most common tumor type.

11. Introduction

There are many avian retrovirus-like elements in the normal chicken genome.
They include the endogenous virus (ev) loci, avian retrotransposon-chickens or ART-C H,
and endogenous avian virus or EAVs (Dunwiddie et al. 1986; Boyce-Jacino et al. 1992;
Nikiforov and Gudkov 1994). Recently, a novel element has been reported in the
literature, ev/J by Benson et al (1998). However, later work demonstrated that ev/J and

EAV-HP were virtually identical (Sacco et al. 2000). The ev loci are related to subgroup

141

E avian leukosis virus (ALV) and are either complete and infectious or defective viruses
(Robinson 1978; Crittenden 1981; Crittenden and Astrin 1981; Smith 1987). Twenty-
nine ev loci have been located and identified thus far, (Gudkov et al. 1986; Smith 1987).
Rovigatti and Astrin (1983) determined that a normal chicken averages about five ev loci
in its genome. Depending on whether the ev loci are complete or defective, and what
defects are present, these birds may react positively to various diagnostic tests without
being infected with exogenous retrovirus.

One such important ev locus is the ev21 locus. It encodes for a complete
infectious subgroup E ALV known as EV21. EV21 has been mapped to the Z
chromosome and is linked to the dominant sex linked gene K which regulates slow or late
feathering (Warren 1925). Late feathering can be differentiated fiom early feathering and
is widely used for sex determination of chicks in the poultry industry (Warren 1930).
However, there have been reports of reduced production and increased leukosis mortality
in female progeny from dams with the K gene (Lowe and Garwood 1981; Harris et al.
1984). In studies by Bacon et a1 (1988) and Harris et al (1984), the ev21 gene is
expressed as a complete infectious endogenous virus, which can be transmitted to the
progeny congenitally and result in increased susceptibility to infection by exogenous
ALVs because of immunologic tolerance.

Subgroup J ALV, first described in the early 19905 by Payne et a1 (1991), mainly
causes myelocytomatosis in broiler breeder chickens. Although meat type chickens are
mainly affected by ALV-J, there have been reports of white leghorn chickens developing
myelocytomatosis due to subgroup J -like infection (Fadly 1998). Experimental studies

using leghorn chickens inoculated with subgroup J have demonstrated that they are

142

susceptible to infection and can develop tumors (Payne et al. 1992; chapter2). The
objective of this study was to determine the role of EV21 on the response of white

leghorn chickens to infection with ALV-J using the U. S. prototype virus ADOL-Hcl.

III. METHODS AND MATERIALS

A. Chickens: Two lines of chickens fi'om the Avian Disease and Oncology
Laboratory in East Lansing, M1 were used. These were males of the Regional Poultry
Research Laboratory (RPRL) line 0.44-EV21+ (a late feathering line) and females of the
early feathering RPRL 15B1 line (Bacon et al. 2000). The F1 progeny are either late
feathering chicks (both male and female) that are EV21+ or early feathering chicks (also
both male and female) that are EV21-. The breeder line 0.44-EV21+ (EV21+) male
chickens are detected by feather phenotype and blood typing using the R2 alloantiserum
assay (Bacon et al. 1996). The breeder flocks for the two lines are free fi'om many avian
pathogens, including exogenous ALV, as determined by routine serological surveys.
Chicks were separated into early or late feathering groups by length of primary feathers
on both wings. Birds were housed in plastic isolators according to feather phenotype for

31 weeks and given food and water ad libitum.
B. Virus: Strain ADOL-Hcl, the U. S. prototype, of ALV-J was used (F adly and Smith

1999). The virus titer of the virus stock was determine to be 105 infectious units (IU)/ml.

Each treatment group received 104 IU/bird intraperitoneally at day of hatch.

143

C. Experimental Design: 82 day-old chicks from the previously mentioned cross were
separated into early or late feathering groups according to feather phenotype. All birds
were inoculated at day of batch intraperitoneally with 104 IU/bird with a 30g-V2 inch
needle. At 4, 10, 18 and 31 weeks of age, whole blood samples were collected and the
plasma was used for V1 and VN assays. At 10 weeks of age red blood cells and/or plasma
was used to perform the R2 assay for endogenous virus to confirm feather phenotype
(Bacon et al. 1996; Bacon 2000). Termination of the study was conducted at 31 weeks of
age. All birds that died during the experiment and those that survived to the end of the

experiment were necropsied.

D. Virological and serological assays: Virus isolation (VI) and virus neutralization
(VN) assays were conducted on all plasma samples at each sampling period using line 0
(GE) CEF. Briefly, VI was performed using 100 microliters of plasma added to 4% calf
serum Leib McCoy media containing penicillin, streptomycin and amphotericin B plus
0.04IU of heparin per plate and CEFs in suspension. Media was changed the following
day to 1% calf serum Leib McCoy media containing above antibiotics and fimgicide.
Cultures were allowed to grow for 7- 9 days. Plates were frozen and thawed twice (F adly
and Witter 1998). Cell lysates from CEF cultures was tested by the gs antigen p27
ELISA (Smith et al. 1979). VN assays were performed according to procedures by Fadly
and Witter (1998). Briefly, test samples were diluted 1:5 with Leib McCoy media without
serum and heat inactivated for 30 minutes at 56° C. Dilute virus stocks ofADOL-Hcl
was added such that each well of a 96 well microtiter plate received 500-1000 viral

particles/ml. Equal volumes of test samples were added to the microtiter plates and

144

incubated for 45 minutes. Appropriate positive and negative control samples were
included. Cells (5X104 cells/well) were added in 4% calf serum Leib McCoy containing
antibiotics and fungicide, and incubated 7-9 days at 37° C. Cultures were frozen and
thawed twice. The cell lysates were tested for the presence of p27 by ELISA. The R2

alloantiserum assay was performed as described by Bacon et a1 1996, 2000.

E. Pathology: Necropsy was performed on all birds that died during the experiment and
at termination. Tissue samples for histology were taken from birds with gross tumor
formation. All samples were fixed in 10% neutral buffered formalin, embedded in

paraffin, sectioned, and stained with hematoxylin and eosin.

F. Statistical Analysis: Statistical analysis was performed using the SAS 7.0 program.
Viremia and antibody data were analyzed with a chi-square test. Results were considered

significant at a level of P < 0.05.

IV. RESULTS

A. Virological and serological assays: The results of the serologic assays performed at
the various sampling periods are presented in Table 3.1. The first sampling period, 4
weeks post inoculation, demonstrated that 93% of the chickens harboring EV21 were
Viremic whereas only 43% of the chickens lacking EV21 were virernic. This difference
continued at all sampling periods for birds harboring EV21 compared to birds lacking
EV21 where viremia was present at a much lower level. A difference in antibody

production occurred at 10 weeks with 16% of the chickens harboring EV21 having

145

detectable levels of antibody versus 83% of the chickens lacking EV21 having antibody
to ALV-J. The 18 weeks sampling period had the highest percentage of chickens with
antibody to ALV-J with 54% chickens harboring EV21with detectable antibody and 98%

of chickens lacking EV21 with detectable antibody.

Table 3.1: Development of ALV-J induced viremia and antibody in white leghorn

chickens harboring or lacking EV21.

Age at Testing -—No. +/No. Tested (%)

 

 

 

 

 

 

 

 

 

 

 

 

4 weeks 10 weeks 18 weeks 31 weeks
Type of VI“ AbJ" v1 AbJ VI AbJ v1 AbJ
chicken
Harboring 37/40 0/40 37/40 6/38 20/37 19/37 21/35 15/35
EV21 (93)1 (0)3 (93)‘ (16)3 (54)1 (51 )3 (60)1 (43)3
Lacking 17/40 0/40 9/40 33/40 2/40 39/40 5/38 28/38
EV21 (43)2 (0)3 (23)2 (83)“ (5)2 (98)“ (13)2 (74)“

 

 

avirus isolation performed on lineO (c/e) CEF for ALV-J detection only
bantibody presence to ALV-J as determined by VN assays

12 Numbers change when results are significant at a level of P5 0.05 for virus isolation at
that time period.

3’4 Numbers change when results are significant at a level of P: 0.05 for antibody
detection at that time period.

Table 3.2 contains the virus positive birds fi'om Table 3.1 and displays them in
regards to antibody status. In chickens harboring EV21, the number of birds
characterized as virus positive, antibody negative (V+, A-) decreased over the time
course of the study. However, at 31 weeks there were still 19/35 (54%) V+, A- chickens.
These chickens were considered Viremic tolerant. With regards to chickens lacking

EV21, there was a sharp decrease from 4 to 10 weeks (45% to 5%) in V+, A- chickens

146

which should be expected as birds seroconvert and clear the virus. Interestingly, two

birds (5%) were V+, A- at 31 weeks in this group and can be considered Viremic tolerant.

The R2 alloantisera assay performed at 10 weeks of age for the detection of

endogenous ALV virus revealed that 100% of chickens characterized as early feathering

by feather phenotype were negative for endogenous virus. Thirty-nine out of 40 chickens

characterized as late feathering by feather phenotype were positive for endogenous ALV

virus. The one chicken mis-categorized by feather phenotype was euthanized afier the 10

week sampling due to severe leg paralysis.

Table 3.2: Characterization of ALV-J positive chickens with regards to antibody status

Age at Testing—No. +/No. Tested (%)

 

 

 

 

 

 

 

 

 

 

 

Type of Serological 4 weeks 10 weeks 18 weeks 31 weeks
chicken status
Harboring V+, A-al 37/40(93) 25/38(66) 18/37(49) 19/35(54)
EV21
V+, A+° O/40(O) l/38(3) 2/37(5) 2/35(6)
Lacking V+, A- 17/40(43) 2/40(5) 1/40(3) 2/38(5)
EV21
V+, A+ O/40(0) 7/40(18) l/40(3) 3/38(8)

 

aVirus positive, antibody negative
l’Virus positive, antibody positive

A. Pathology: Four birds were diagnosed with LL on gross examination. One

additional bird was diagnosed with LL microscopically. Two other birds had suspect

microscopic lesions of LL in the spleen. These two birds were not included in the

analysis. One other bird was diagnosed with hemangioma by gross examination. In

regards to EV21 status, 1/38 (2.6%) at risk chickens lacking EV21 developed tumors

while 5/36 (13.8%) at risk chickens harboring EV21 developed tumors. At risk is defined

 

as the number of chickens that died with tumors plus the number of chickens that

survived to termination.

V. DISCUSSION

Endogenous viruses have been known to interfere with antibody development to
exogenous leukosis viruses (Halpem et al. 1975; Crittenden et al. 1982; Crittenden and
F adly 1985). There are several reports regarding EV21 virus and its influence on the
response to exogenous ALV infection in chickens exposed afier hatch (Smith and
Crittenden 1988; Fadly and Smith 1991; Gavora et al. 1995; F adly and Smith 1997).
Most of these reports used a subgroup A ALV as the exogenous virus for inoculation
followed by serological assessment for antibody production. The purpose of this study
was to determine the influence of EV210n the response of white leghorn chickens to
infection with ALV-J.

The data presented (Table 3.1) demonstrated that birds harboring EV21 were
significantly more likely to be Viremic at the 4 time periods tested than birds lacking
EV21. The largest difference in the level of viremia occurred at 10 weeks of age. These
findings are consistent with other reports (Smith and Crittenden 1988; Gavora et a1. 1995;

F adly and Smith 1997) that examined the differences in viremia between birds harboring
and lacking EV21 following infection with subgroup A ALV. The delayed ALV
antibody response seen at 10, 18 and 31 weeks of age in chickens harboring EV21
supported earlier studies by Crittenden (1982), Crittenden and Fadly (1985), and Halpem

(1975) that indicated that endogenous viruses interfere with antibody production.

148

A large percentage of ALV-J Viremic tolerant chickens were observed in this
study (Table 3.2) with most occurring in chickens harboring EV21. This characteristic
occurred at the 10, 18 and 31 weeks of age sampling periods. The marked difference in
response by the two groups of chickens can be attributed to the presence or lack of EV2 1.
Smith and Crittenden (198 8) also noted the tolerance inducing effect of EV21 on RPL-40
(ALV-A field isolate) viremia, cloacal shedding, and neutralizing antibodies in chickens
congenitally infected with EV21. Crittenden et al (1982, 1984) demonstrated that Rous-
associated virus 0 (RAV-0) which is encoded at the ev2 locus, can also induce
immunologic tolerance along with the ev3 locus with subgroups A and B ALV. The data
presented here can further substantiate the effects of EV21 on responses of white leghorn
chickens infected with exogenous ALVs. The data suggests that the use of antibody tests
for ALV surveillance may not detect the true ALV-J infection status in late feathering
leghoms due to the lack of antibody production.

The lack of tumor formation in this experiment was unexpected. Explanations as to
why this occurred include some resistance of the genetic line used in this study, the
genetic make up of the ALV-J virus, or the combination of both factors. White leghorn
chickens can develop tumors when infected with ALV-J but at a lower rate than meat
type chickens (Payne et al. 1992). Previous experiments using the same genetic cross
and subgroup A ALV yielded tumors in up to 32% of infected birds regardless of EV21
status (F adly and Smith 1997). In that report, only the genetic transmission of EV21
increased the incidence of tumors following ALV infection at hatch, not contact exposure
to EV21. Smith and F adly (1988) evaluated the influence of congenital transmission of

EV21 on the incidence of tumors and concluded that congenital transmission of EV21 did

149

have an effect on tumor formation. Another report by Smith and Crittenden (1988)
examined genetic resistance and the susceptibility of late feathering females on
transmission of EV21 to the progeny congenitally. They determined that cellular
resistance to subgroup E ALV in the late feathering dams limited congenital transmission
of EV21 to the progeny. Due to the fact that the EV21+ males used in the cross for the
progeny used in this study have a line 0 genetic background, they may have contributed
some cellular resistance to the progeny (Smith and Crittenden 1988). ALV-J has EAV
viral sequences in the envelope gene (Bai et al. 1995; Benson et al. 1998) and together
with the genetic resistance acquired from the male parent, it may have influenced the
manner in which ALV-J induces tumor formation. Although tumor formation was low,
there was correlation with previous reports (Smith and Crittenden 1988; Smith and Fadly
1988; Fadly and Smith 1997) in that the chickens lacking EV21 had fewer tumors than
chickens harboring EV21, 2.6% versus 13.8%, respectively.

Examination of the data herein clearly indicates that EV21 influences ALV-J
viremia and antibody production in white leghorn chickens. How EV21 may influence
ALV-J infection in meat type birds is unknown. However, caution is warranted in using
only serological assays to detect ALV-J infection because of the high frequency of the

development of tolerance that would result in false negatives test results.

150

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plasma using R2 antiserum. Avian Pathol 29: 153-164. 2000.

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lines to resolve genes determining resistance to diseases. Poult Sci 79: 1082-1093.
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. Bacon, L. D., E. J. Smith, L. B. Crittenden and G. B. Havenstein. Association of the
slow feathering (k) and endogenous viral (ev21) gene on the Z chromosome of
chickens. Poult Sci 67: 191-197. 1988.

. Bacon, L. D., E. J. Smith, A. M. F adly and L. B. Crittenden. Development of an
alloantiserum (R2) that detects susceptibility of chickens to subgroup E
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. Bai, J ., L. N. Payne and M. A. Skinner. HPRS-103 (exogenous avian leukosis virus,
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E51 and an E element found previously only in sarcoma viruses. J Virol 69(2):
779-784. 1995.

. Benson, S. J ., B. L. Ruis, A. M. Fadly and K. F. Conklin. The unique envelope gene of
the subgroup J avian leukosis virus derives from ev/J proviruses, a novel family of
avian endogenous viruses. J Virol 72(12): 10157-10164. 1998.

. Boyce-Jacino, M. T., K. O'Donoghue and A. J. F aras. Multiple complex families of
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. Crittenden, L. B. Exogenous and endogenous leukosis virus genes-a review. Avian
Pathol 10: 101-112. 1981.

. Crittenden, L. B. and S. M. Astrin. Genes, viruses and avian leukosis. Bioscience 31:
305-310. 1981.

10. Crittenden, L. B. and A. M. Fadly. Responses of chickens lacking or expressing

endogenous avian leukosis virus genes to infection with exogenous virus. Poult
Sci 64(3): 454-463. 1985.

11. Crittenden, L. B., A. M. Fadly and E. J. Smith. Effect of endogenous leukosis virus

genes on response to infection with avian leukosis and reticuloendotheliosis
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and ALV infection and shedding in chickens. Avian Dis 28(4): 1037-1056. 1984.

Dunwiddie, C. T., R. Resnick, M. T. Boyce-Jacino, J. N. Alegre and A. J. Faras.
Molecular cloning and characterization of gag-, pol-, and env-related genes
sequences in the ev-chicken. J Virol 59: 669-675. 1986.

Fadly, A. M. . Subgroup J avian leukosis virus infection in meat-type chickens: A
review. IV Asian Pacific Conference on Poultry Health.1998.

Fadly, A. M. and E. J. Smith. Influence of maternal antibody on avian leukosis virus
infection in White Leghorn chickens harboring endogenous virus-21 (EV21).
Avian Dis 35(3): 443-451. 1991.

Fadly, A. M. and E. J. Smith. Role of contact and genetic transmission of
endogenous virus-21 in the susceptibility of chickens to avian leukosis virus
infection and tumors. Poult Sci 76(7): 968-973. 1997.

Fadly, A. M. and E. J. Smith. Isolation and some characteristics of a subgroup J-like
avian leukosis virus associated with myeloid leukosis in meat—type chickens in the
United States. Avian Dis 43(3): 391-400. 1999.

Fadly, A. M. and R. L. Witter. Oncomaviruses: leukosis/sarcoma and
reticuloendotheliosis. In A Laboratory Manuel for the Isolation and Identification
of Avian Pathogens. eds,D. E. Swayne, J. R. Glisson, M. W. J ackwood, J. E.
Pearson and W. M. Reed. Kennett Square, PA., American Association of Avian
Pathologists. pp185-196. 1998.

Gavora, J. S., J. L. Spencer, B. Benkel, C. Gagnon, A. Emsley and A. Kulenkamp.
Endogenous viral genes influence infection with avian leukosis virus. Avian
Pathol 24: 653-664. 1995.

Gudkov, A. V., E. Korec, M. V. Chemov, A. T. Tikhonenko, I. B. Obukh and I.
Hlozanek. Genetic structure of the endogenous proviruses and expression of the
gag gene in Brown Leghorn chickens. Folia Biol 32(1): 65-72. 1986.

Halpem, M. S., D. P. Bolognesi, R. R. Friss and W. S. Mason. Expression of the
major glycoprotein of avian tumor virus in cells of chf(+) chicken embryos. J
Virol 15: 1131-1140. 1975.

Harris, D. L., V. A. Garwood, P. C. Lowe, P. Y. Hester, L. B. Crittenden and A. M.
Fadly. Influence of sex-linked feathering phenotypes of parents and progeny upon
lymphoid leukosis virus infection status and egg production. Poult Sci 63(3): 401-
413. 1984.

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Lowe, P. C. and V. A. Garwood. Independent effects of K and k+ alleles and
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1123-1126. 1981.

Nikiforov, M. A. and A. V. Gudkov. ART_CH: a VL30 in chickens? J Virol 68: 846-
853. 1994.

Payne, L. N., S. R. Brown, N. Bumstead, K. Howes, J. A. Frazier and M. E.
Thouless. A novel subgroup of exogenous avian leukosis virus in chickens. J Gen
Virol 72(Pt 4): 801-807. 1991.

Payne, L. N., A. M. Gillespie and K. Howes. Myeloid leukaemogenicity and
transmission of the HPRS-103 strain of avian leukosis virus. Leukemia 6(11):
1167-1176. 1992.

Robinson, H. L. Inheritance and expression of chicken genes that are related to avian
leukosis sarcoma virus genes. Curr Top Microbiol Irnmunol 83: 1-36. 1978.

Rovigatti, U. G. and S. M. Astrin. Avian endogenous viral genes. Curr Top
Microbiol Irnmunol 103: 1-21. 1983.

Sacco, M. A., D. M. Flannery, K. Howes and K. Venugopal. Avian endogenous
retrovirus EAV-HP shares regions of identity with avian leukosis virus subgroup J
and the avian retrotransposon ART-CH. J Virol 74(3): 1296-1306. 2000.

Smith, E. J. Endogenous avian leukemia viruses. In Avian Leukosis. ed.G. F.
DeBoer. Boston, Martinus Nijhoff. 1. pp101-120. 1987.

Smith, E. J. and L. B. Crittenden. Genetic cellular resistance to subgroup E avian
leukosis virus in slow- feathering dams reduces congenital transmission of an

endogenous retrovirus encoded at locus ev21. Poult Sci 67(12): 1668-1673. 1988.

Smith, E. J., A. Fadly and W. Okazaki. An enzyme-linked immunosorbent assay for
detecting avian leukosis- sarcoma viruses. Avian Dis 23(3): 698-707. 1979.

Smith, E. J. and A. M. Fadly. Influence of congenital transmission of endogenous
virus-21 on the immune response to avian leukosis virus infection and the
incidence of tumors in chickens. Poult Sci 67(12): 1674-1679. 1988.

Warren, D. C. Inheritance of rate of feathering in poultry. J Hered 16: 13-18. 1925.

Warren, D. C. Crossbred poultry. Kansas Agr Exp Sta Bull 252. 1930.

153

CHAPTER 4

Tissue Tropism of Avian Leukosis Virus Subgroup J in White Leghorn Chickens

t

154

1. Abstract

Distribution of ALV-J in various tissues of embryonally inoculated white leghorn
chickens with strain ADOL Hcl of ALV-J was studied. At 2 and 6 weeks of age, various
tissues including thymus, spleen and brusa of Fabricius from infected and phosphate
buffered saline inoculated controls chickens were tested for the presence of ALV-J by
immunohistochemistry using a monoclonal antibody to the envelope glycoprotein (gpSS)
of ALV-J. Fonnalin-fixed tissues from infected chickens exhibited a reddish staining
indicating the presence of ALV-J in tissue specific cells as well as other widely
distributed cells such as endothelial cells and smooth muscle cells. Tissues expressing
gp85 included adrenal gland, bursa of Fabricius, gonads, heart, kidney, liver, bone
marrow, nerve, pancreas, proventriculus, spleen and thymus at both 2 and 6 weeks of age.
This distribution is different than previously reported by Arshad et al (1997) which may
be due to the type of chickens used (Brown leghorn versus white leghorn), the antigenic
differences between the European prototype and the U. S. prototype of ALV-J, or
sensitivity of antibody. Bursal transformation can be documented with ALV-J using the
methyl green pyronine staining procedure. However, it does not occur with the same
frequency as RAV-l, ALV-A prototype virus, in the highly susceptible chicken cross
1515 X 71. Although myelocytomatosis is the main tumor manifestation, bursal follicle

transformation that results in LL can occur with ALV-J infection.

155

11. Introduction

The tissue tropism of ALVs has been documented by many different researchers
over the years employing various methods of detection (Di Stefano and Dougherty 1969;
Di Stefano et al. 1973; Spencer and Gilka 1982; Gilka and Spencer 1984; Spencer et al.
1984; Robinson et al. 1993). These previous reports dealt with ALVs that would cause
LL regardless of subgroup. In many tissues varying degrees of antigen was detected and
it was noted that RAV-l, a subgroup A ALV virus, was detected in bone marrow and
bursal tissues more than in spleen and thymus tissue (Baba and Humphries 1986). A
report by Brown and Robinson (1988) determined that RAV-l had high tropism for bursa
of F abricius and muscle versus RAV-2, a subgroup B ALV, which had high tropism for
bursa and thymus (Brown and Robinson 1988). Other reports examined site specific
replication of ALVs. Macrophages harvested from the yolk sac of chicken embryos that
are resistant to subgroup E infection were found to be resistant to subgroups A and D but
susceptible to subgroups B and C (Gazzolo et a1. 1974; Gazzolo et al. 1975). Gazzolo et
al (1979) also determined that ALV can be present in macrophages up to three years
while avian sarcoma viruses cannot. Robinson et a1 (1993) determined that RAV-l
proviral DNA can integrate in various tissues but the various tissue express gag and
envelope proteins in different amounts.

Avian leukosis virus subgroup J (ALV-J) was first described in the early 19905
(Payne et al. 1991) in Europe and then described in the United States during the mid
1990s (F adly and Smith 1997). The predominate tumor described in the literature and in
the field is myelocytomatosis (Payne et al. 1991; Payne et al. 1992; F adly and Smith

1997; Fadly 1998; Fadly and Smith 1999). Recently, Arshad et a1 (1997) described the

156

tissue tropism of HPRS-103 using gag expression of p27 by immunohistochemistry.
Examination of previous reports on tissue tropism of subgroups A, B and J indicate that
there are different levels of expression depending on the subgroup. Due to
immunological differences noted between HRPS-103 and ADOL-Hcl (Fadly and Smith
1997), tissue tropism of ADOL-Hcl by immunohistochemistry was studied in white
leghorn chickens that are highly susceptible to ALV induced tumor formation, using a
monoclonal antibody against gp85 of ADOL-Hcl.

Results fi'om Chapter 2 with regards to bursal follicle transformation demonstrate
that ALV-J can cause transformation. To more accurately assess the transformation
abilities of the virus, bursal follicle transformation induced by ADOL-Hcl was studied in
white leghorn chickens that are highly susceptible to ALV induced tumor formation and

compared with RAV-l , an ALV-A virus.

III. Methods and Materials

A. Chickens: The highly susceptible cross 1515 X 71 chickens from the Avian Disease
and Oncology Laboratory, East Lansing, MI, were used: Sixty 7-day-old embryos for the
tissue tropism experiment and 50 day-old chicks for the bursa] follicle transformation
experiment. The breeder flocks for this cross are free from many avian pathogens,
including ALV, as determined by routine serological surveys. Birds were house by
treatment group in Horsfall isolation units up to 10 weeks and given food and water ad

libitum.

157

B. Viruses: Strain ADOL-Hcl, the U. S. prototype, of ALV-J and RAV-l, the
prototype for ALV subgroup A, were used. The virus titer for ADOL-Hcl was
determined to be 105 infectious units (IU)/ml. The virus titer for RAV-l was determined

to be 105 IU/ml. Each treatment group was given 104 IU/bird of the assigned virus.

C. Experimental Design: Tissue tropism experiment: Two groups of 30, 7-day-old
embryos were incubated in separate units. One group was inoculated with ADOL-Hcl
via the yolk sac with a 30g ‘/2 inch needle and the other group served as controls and was
inoculated with sterile phospate buffered saline (PBS) (0.1ml) injected via the yolk sac
with a 30g 1/2 inch needle. All chicks were sampled for viremia at hatch. At 2 and 6
weeks of age, whole blood samples for virus isolation assays were collected. Ten birds
were randomly chosen fiom each group and euthanized at each time period. Tissues
harvested included adrenal glands, kidney, pancreas, spleen, heart, proventriculus,
skeletal muscle, bone marrow, thymus, bursa of F abricius, gonads and sciatic nerve.
Tissues were fixed in 10% neutral buffered formalin over night and transferred to 60%
methanol until processed for immunohistochemistry.

Bursal follicle transformation experiment: Fifty, day old chicks were randomly
separated into three groups. One group (19 chicks) was inoculated with ADOL-Hcl, one
group (19 chicks) with RAV-l and one group served as uninoculated controls (12
chicks). All inoculations were intraperitoneal (1P) using a 30g 1/2 inch needle. At 4 and 10
weeks of age blood samples were collected for virologic assays. Bursal transformation
was determined by MGP staining. At 4 and 10 weeks of age, 5 chickens were randomly

chosen from each group and euthanized. Bursal tissue was harvested and plica separated

158

and placed flat in cassettes, usually 2 to 3 cassettes per bird. Fixation occurred in 4%
paraforrnaldehyde in PBS. Tissues were embedded in paraffin, sectioned and stained
with MGP stain (Siccardi and Burmester 1970; Carson 1990) and examine histologically

for evidence of transformation.

D. Virological and serological assays: Virus isolation (V I) and VN were conducted on
samples at each sampling period using line 0 (C/E) CEF as directed in experimental
design. Briefly, VI was performed using 100 microliters of plasma added to 4% calf
serum Leib McCoy media containing penicillin, streptomycin and amphotericin B plus
0.04IU of heparin per plate and CEFs in suspension. Media was changed the following
day to 1% calf serum Leib McCoy media containing the above antibiotics and fimgicide.
Cultures were allowed to grow for 7- 9 days. Plates were fiozen and thawed twice (Fadly
and Witter 1998). Cell lysates from CEF cultures was tested by the gs antigen p27
ELISA (Smith et al. 1979). VN assays were performed according to procedures by
Fadly and Witter (1998). Briefly, test samples were diluted 1:5 with Leib McCoy media
without serum and heat inactivated for 30 minutes at 56° C. Dilute virus stocks of RAV-
1 or ADOL-Hcl were added such that each plate of a 96 well microtiter plate received
500-1000 viral particles/ml. Equal volumes of test samples were added to the rrricrotiter
plates and incubated for 45 minutes. Appropriate positive and negative control samples
were included. Cells were added (5X104 cells/well) in 4% calf serum Leib McCoy
containing antibiotics and fungicide, and incubated 7-9 days at 37° C. Cultures were

frozen and thawed twice. The cell lysates were tested for the presence of p27 by ELISA.

159

VN were performed separately with both RAV-l and ADOL-Hcl for indication of

accidental cross infection in the bursal transformation experiment.

E. Immunohistochemistry: Tissue sections were removed from 60% methanol and
embedded in paraffin and sectioned at 415m. The monoclonal antibody used was
developed by A. J. Cui (Cui, Avian Dis, in press) against gpSS of ALV-J in mice. The
following procedure was used for staining the tissues. Slides were deparaffinized,
hydrated with distilled water and placed in tris buffered saline (TBS) buffer for 5
minutes. Next, 3% H202 in methanol was applied for 60 minutes to block endogenous
peroxidases. Slides were rinsed in running tap water for 5 minutes, placed in TBS plus
Tween 20 for 5 minutes and placed on an automatic stainer (LEICA St5050 Stainer) with
2, 2 minutes rinses in TBS plus Tween 20 between the following steps. Super Block, 3
non-species specific protein blocking agent, (Scytek, Logan, UT) was applied to the
slides for 5 rrrinutes, followed by application of a blocking agent for endogenous
avidin/biotin for 5 minutes. The primary antibody was diluted at 1:200 in normal
antibody diluent (Scytek) (from concentrate) and applied by hand and incubated for 60
minutes. A Vector (Burlingame, CA) biotinylated anti-mouse/anti-rabbit (in horse)
antibody diluted at 1:136 in normal antibody diluent for 30 minutes was applied followed
by application of a Vector Ready to Use (R.T.U.) ABC (avidin-biotin complex) for 30
minutes. The final step in the procedure was application of Vector Nova Red for 15
minutes. The slides were removed from the automatic stainer and were counterstained
with Lerner 2 Hematoxylin for 1 minute, then placed in running water, with 1% Glacial

acetic acid blue for 2 minutes. Slides were dehydrated, cleared and coverslipped with a

160

synthetic mounting media. Several negative controls included a system control that had
normal horse sera diluted at 1:28 instead of primary antibody for 60 minutes on a section
of infected tissues, uninfected tissues treated with primary antibody, and uninfected
tissues were treated with normal horse sera. All controls were included each time a new
set of slides was placed on the automatic slide stainer. Tissues were examined

microscopically for red pigment within the cell cytoplasm.

F. Scoring of Tissues: Tissue tr0pism experiment: Tissue sections were scored for the
presence or absence of gpSS and number of positive cells within a tissue. Tissue sections
were scored by counting the tissue specific cells (kidney tubules, pancreatic cells, etc)
that were stained. Widely distributed cells such as connective tissue, smooth muscle
components in the gastrointestinal tract, endothelial cells, etc. were not counted. Each
tissue on a slide was examined at 40X and in four randomly chosen fields and tissue
specific cells were counted. The average number of cells of the four fields was
determined and a positive cell staining average was given to the tissue for that bird. The
averages were combined for all tissue samples and divided by the respective number of
tissues scored to obtain an overall average score for each tissue. Presence of gp85
positive cells specific to a certain tissue were scored as none (no staining), mild (1-25
positive cells), moderate, (26-75‘positive cells) and marked (76 and up positive cells).
Bursal transformation scoring: All sections of bursal plica were examined
microscopically. Evidence of transformation included increased pyroniphilia as

compared to surrounding follicles and/or normal architecture was disrupted such that the

161

cortex and medulla were indistinguishable from each other. If one follicle was positive

for transformation, the tissue was considered positive.

IV. Results:

A. Tissue tropism experiment: The distribution of gp85 in tissue specific cells is
summarized in Table 4.1. Skeletal muscle taken from the superficial pectoral muscle was
not scored because of the high background staining. All other tissue examined were
scored. Tissues with the highest average number of cells staining for antigen expression
at 2 weeks of age were proventriculus (glandular epithelial cells) with 91 cells, spleen
(reticuloendothelial cells and lymphocytes) with 79 cells, and adrenal glands (cortical and
medullary) with 74 cells. Bone marrow had the least amount of cells staining for the
antigen expression with an average of 16 cells. There were no tissues in the “none”
category, 1 in the mild category, 10 in the moderate category and 2 in the marked
category. Tissues with the highest average number of cells staining for antigen at 6 weeks
of age were adrenal gland (139 cells), proventriculus (137 cells), pancreas (125 acinar
and islets of Langerhans cells), kidney (121 distal tubule epithelial cells), and spleen (l 16
cells). Bone marrow had the least amount of cells staining for the antigen with an average
of 36. There were no tissues into the “none” and mild categories. 6 in the moderate
category, and 6 in the marked category. Figures 4.1-4.13 illustrate various tissues with
positive staining along with the lack of staining in uninfected tissues. Images in this
chapter are presented in color. Results of VI were as follows: At 2 weeks of age, 20/20

ADOL Hcl inoculated group tested positive and 0/20 were positive in the control group.

162

At 6 weeks of age, 9/9 ADOL-Hcl inoculated group, tested positive and 0/ 10 were

positive in the control group.

Table 4.1 Distribution of gpSS expression“ in 1515 X 71 Chickens Inoculated as Embryos

 

 

 

 

 

 

 

 

 

with ALV-J.
Tissue 2 weeks 6 weeks Tissue 2 weeks 6 weeks
Adrenal gland 7/8(74) 9/9(139) Muscle nt nt
Bursa 6/9(47) 8/9(73) Nerve 7/10(53) 9/9(68)
Gonad 7/8(73) 9/9(67) Pancreas 9/10(62) 9/9(125)
Heart 9/10(57) 9/9(83) Proventriculus 10/ 10(91) 9/9(l 37)
Kidney 9/10(69) 9/9(121) Spleen 8/10(79) 9/9(116)
Liver 8/9(46) 9/9(46) Thymus 9/10(30) 9/9(60)
Bone marrow 5/ 8(1 6) 6/6(36)

 

 

 

 

 

 

* No. birds positive/No. birds examined (average number of trssue specific cells).
Nt not tested due to high background staining.

 

B. Bursal transformation experiment: The results for serological and bursal
transformation experiment are summarized in Tables 4.2 and 4.3. There was no evidence
of cross infection between the RAV-l and ADOL-Hcl groups or infection of the control
group. Transformation was seen in only the virus inoculated groups. At 4 weeks of age,
the RAV-l infected group had a 40% transformation rate while the ADOL-Hcl infected
group had a 20% transformation rate. At 10 weeks of age, the RAV-linfected group had

100% transformation while the ADOL-Hclinfected group had 20%.

163

Table 4.2: Development of ALV-J or ALV-A induced viremia and antibody in chickens

of a highly susceptible cross to ALV induced tumor formation.

 

 

 

 

4 weeks 10 weeks
Group VI + AbJ+ AbRAV-H VI + AbJ+ AbRAV-l+
Controls 0/12* 0/ 12 0/ 12 0/7 0/7 0/7
RAV-l 19/19 0/19 0/19 2/13 0/13 11/13
ADOL-Hcl 18/18 0/18 0/18 13/13 6/13 0/13

 

 

 

 

 

 

 

 

 

AbJ+: Antibody to J
AbRav-l+: Antibody to RAV-l
*No+/No tested

Table 4.3: Bursal follicle transformation induced by ALV-A or ALV-J in a highly

susceptible cross of chickens to ALV induced tumor formation.

 

 

 

 

 

 

V Group 4 weeks 10 weeks
Control 0/5“ 0/5
RAV-l 2/5 5/5
ADOL-Hcl 1/5 1/5
*No+/No tested

 

 

164

 

 

Figure 4.1: Immunohistochemistry on bursa of Fabricius sections. A.
Uninfected bird. 20X. B. Infected bird. Overall distribution of staining. 4X.
C. Infected bird. Staining at intersection of cortex and medulla of bursal
follicle. B cells and macrophages are staining positive. 40X.

165

 

Figure 4.2: Immunohistochemistry on spleen sections. A. Uninfected bird.
10X B. Infected bird, overall distribution of staining.10X C. Infected bird,
staining of reticuloendothelial cells.40X

33..

.47.

4., .1
.. HIV...

.u.

1:.

 

Immunohistochemistry on nerve sections. A. Uninfected

Figure 4.4

sciatic nerve. 20X. B. Infected sciatic nerve section. Staining present in

axons and surrounding the nuclei. 40X.

168

 

Figure 4.5: Immunohistochemistry on proventriculus sections. A.
Uninfected proventriculus. 10X. B. Infected proventriculus. Staining
present in glandular epithelial cells. 20X. C. Infected proventriculus.
Stain present in cytoplasm of glandular epithelium. 40X.

169

 

Figure 4.6: Immunohistochemistry on liver sections. A. Uninfected liver
section. 10X. B. Infected liver section. Individual hepatocytes are
staining positively. Positive staining occurring in sinusoids consisting of
red blood cells and possibly Kupfi‘er cells. 20X.

170

 

Figure 4.6: Immunohistochemistry on liver sections. A. Uninfected liver
section. 10X. B. Infected liver section. Individual hepatocytes are
staining positively. Positive staining occurring in sinusoids consisting of
red blood cells and possibly Kupfi‘er cells. 20X.

170

 

Figure 4.7: Immunohistochemistry on testis sections. A. Uninfected testis section.
20X. B. Infected testis section. Staining is present in the interstitial cells. 20X.

17]

 

Figure 4.8: Immunohistochenristry on pancreas sections. A. Uninfected pancreas
section. 10X. B. Infected pancreas section. Staining is present in both acinar cells and
islets of Langerhans. 20X.

172

 

Figure 4.9: Immunohistochemistry on adrenal gland sections. A. Uninfected adrenal
gland section. 20X. B. Infected adrenal gland section. Staining is present in both
cortical and medullary tissue. 20X.

173

 

Figure 4.10: Immunohistochemistry on bone rmrrow sections. A. Uninfected bone
marrow section. 40X. B. Infected bone marrow section. Staining in erythrocytic cell
lines and granulocytic cell lines. 40X.

174

 

Figure 4.11: Immunohistochemistry on ovary sections. A. Uninfected ovary section.
40X. B. Infected ovary section. Stromal cells staining along with endothelial cells. 40X.

175

 

Figure 4.12: Immunohistochemistry on heart sections. A. Uninfected heart section.
10X. B. Infected heart section. 10X. C. Infected heart section. Myofibers are staining
positive. 40X.

176

 

Immunohistochemistry on thymus sections. A. Uninfected thymus. 10X.

4.13

B. Infected thymus. Most staining occurs in the medulla. Some staining present in
cortical cells. 20X. C. Infected thymus. Cortical lymphoid cells staining. 40X.

Figure

177

V. Discussion

Tissue tropism for avian leukosis virus has been previously studied and first
reported in1967 (Dougherty and Di Stefano 1967). Dougherty and Di Stefano (1967),
using a subgroup A virus (ALV-F42) reported that viral budding could be seen by
electron microscopy in all three embryonal germ layers and in all tissues examined
except nervous tissue in congenitally infected chickens. Although they were able to
detect virus in the brain by the COFAL method, it was determined by careful
examination of various nervous tissue samples that the source of the virus was blood and
was not virus replicating in the cells of nervous tissues.

Other researchers have used immunohistochemical staining techniques to study
ALV distribution in tissue in order to gain insight about pathogenesis of the infection
(Dougherty et al. 1972; Di Stefano et al. 1973; Spencer and Gilka 1982; Spencer et al.
1983; Gilka and Spencer 1984; Spencer et al. 1984; Gilka and Spencer 1985; Gilka and
Spencer 1987; Arshad et al. 1997). All of these reports used an antibody directed against
the gs antigen (p27) for detection of ALV. Using the p27 antibody allows for detection
of all subgroups of ALV, including endogenous ALVs. Because p27 antibody can detect
endogenous ALVs, some caution must be used when interpreting results fi'om chickens
that have endogenous virus loci that could express the p27 protein. Careful examination
of control birds and/or uninfected tissues must be carried out to determine how much
endogenous p27 can be detected as compared to the infected tissues. Using p27 does
have advantages in that» it will detect all subgroups of ALV and therefore only one

antibody is needed for diagnostic purposes.

178

Other methods for detecting ALV include using Southern Blot analysis (Baba and
Humphries 1986) and in situ hybridization (Arshad et al. 1999). Both of these methods
use DNA as the basis of detection by using various probes. Southern Blot analysis can
determine if there is proviral genomes in the tissue but does not indicate specifically
which cells are positive because of the DNA extraction procedures. In situ hybridization
allows for the detection of either DNA or RNA in fixed tissues so that specific cell types
can be identified as containing the DNA or RNA in question.

Results presented in Table 4.1 demonstrated that a monoclonal antibody to gp85
can be successfirlly used to detect ALV-J in various tissues without the concern of
detection of endogenous viral antigen. Detection of endogenous viral antigen was a
concern because of the high number of endogenous viral loci in the genetic cross of
chickens used in this study. Using only a p27 based antibody could possibly detect both
ALV-J and ALV-E and make it difficult to determine what is background staining
(endogenous p27) and what is exogenous p27 staining.

Previous work by Arshad et a1 (1997) evaluated the distribution of ALV-J gs
antigen in tissues of various lines of chickens including a Brown Leghorn line, line 21 (a
meat-type chicken), and four white leghorn lines (0, 151, 61, and N). Tissues with the
highest expression of ALV antigen in tissue specific cells were adrenal gland, heart,
kidney, proventriculus, and pancreas. These same tissues were the same highest tissue
specific staining tissues reported herein except heart which was replaced by spleen in this
report. Prior work by others also noted that these tissues contained large amounts of
ALV antigen (Dougherty and Di Stefano 1967; Di Stefano and Dougherty 1969; Di

Stefano et al. 1973; Spencer et al. 1984). However, sciatic nerve tissue tested positive

179

for antigen quite often which has been infrequently reported before (Arshad et al. 1997 )
or not reported at all in previous reports where different detection techniques were used
(Dougherty and Di Stefano 1967; Dougherty et al. 1972; Di Stefano et al. 1973).

Interestingly, some differences were observed between the embryo inoculated
birds in Arshad’s work and in this study. Bursal expression of ALV-J in this study was
seen in most bursal sections examined with a pattern of distribution of more intense
staining of gp85 at the cortical-medullary junction of the follicles with much less staining
of the medulla in some follicles. Previous work only demonstrated bursal staining in the
medulla. Another difference between Arshad’s work and this study was staining of
sciatic nerve tissue and bone marrow. In the current study, staining was detected in the
bone marrow which had the least number of cells staining positive. Staining was
detected in both red and white blood cell lineages. Arshad et al (1997) suggested that the
reason for his inability to detect gs antigen in the bone marrow was due to the low
sensitivity. Another possible explanation could have been due to the location in the cells
where the various proteins are expressed. GAG proteins are responsible for the physical
structure of the virion and are assembled in the cytosol near the cell membrane (Coffin
1992; Coffin 1996). Env proteins, mainly gp85, are expressed on the cell surface
membrane where budding occurs (Coffin 1992; Coffin 1996). This difference in location
of these viral proteins may explain why antibody to gp85 may react when antibodies to
p27 may not.

Other possible explanations for the differences between this study and Arshad’s
may be due to the genetics of the chickens used for the experiments. Examination of the

results from the leghorn chickens inoculated as embryos revealed that certain tissues

180

(gonads, liver, and pancreas) were not consistently positive for ALV-J in brown leghoms
or white leghorn lines. In this study, the majority of tissues examined were positive at 2
weeks and all tissues except one bursa of Fabricius section was positive at 6 weeks. To
infect a cell, the retrovirus uses a cell surface receptor that normally is expressed by the
cell. In the case of subgroup A, the receptor is known to be related to the human low-
density lipoprotein receptor or LDLR (Bates et al. 1993; Young et al. 1993). The receptor
for ALV-J is unknown. It may be possible that in the genetic cross chosen for this
experiment, the ALV-J receptor is expressed in greater abundance than in the white
leghorn lines used by Arshad, thereby increasing the amount of antigen detected.

Previous work demonstrated the ability of ALV-J to transform bursal follicles in
four different lines of chickens but the design of the study did not allow a direct
comparison to ALV-A (see Chapter 2). Using the same genetic lines of chickens allowed
for better evaluation of the ability of ALV-J to transform B cells and thus, bursal
follicles, as compared to ALV-A. In this study, a clear difference in the rate of bursal
follicle transformation between ALV-J and ALV-A was demonstrated; 20% for ALV-J
infected group and 100% for ALV-A infected group at 10 weeks of age, in a line of
chickens that is highly susceptible to ALV-induced tumors

Previous publications on ALV tissue tropism for various subgroups reveals that a
variety of methods can be used for detection of antigens and that some differences can be
noted in the overall distribution of the virus among subgroups A, B and now J. New
information regarding detection of viral antigen in bursal follicles and the ability of ALV-
J to cause follicle transformation are presented herein begins to explain the outcome of

ALV-J infection in experimentally infected white leghoms (see Chapter 2). Previous

181

work where white leghoms were experimentally infected with ALV-J did not produce LL
(Payne et al. 1991; Payne et al. 1992). The differences seen suggest that both host and

viral factors are important in the outcome of infection and in tumor development.

182

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. Arshad, S. S., K. Howes, G. S. Barron, L. M. Smith, P. H. Russell and L. N. Payne.
Tissue tropism of the HPRS-103 strain of J subgroup avian leukosis virus and of a
derivative acutely transforming virus. Vet Pathol 34(2): 127-137. 1997.

. Arshad, S. S., L. M. Smith, K. Howes, P. H. Russell, K. Venugopal and L. N. Payne.
Tropism of subgroup J avian leukosis virus as detected by in situ hybridization.
Avian Pathol 28: 163-169. 1999.

. Baba, T. W. and E. H. Humphries. Selective integration of avian leukosis virus in
different hematopoietic tissues. Virology 155(2): 557-566. 1986.

. Bates, P., J. A. Young and H. E. Varmus. A receptor for subgroup A Rous sarcoma
virus is related to the low density lipoprotein receptor. Cell 74(6): 1043-1051.
1993.

. Brown, D. W. and H. L. Robinson. Influence of env and long terminal repeat
sequences on the tissue tropism of avian leukosis viruses. J Virol 62(12): 4828-
4831. 1988.

. Carson, F. . In Histotechnology: A Self-Instructional Text. ed.S. Borysewicz. Chicago,
American Society Clinical Pathologists Press. pp108-109. 1990.

. Coffin, J. M. Structure and Classification of Retroviruses. In The Retroviridae. ed.J.
A. Levy. New York, Plenum Press. 1. pp19-49. 1992.

. Coffin, J. M. Retroviridae: The Viruses and Their Replication. In Fields Virology.
eds,B. N. Fields, D. M. Knipe, P. M. Howley and e. al. Philadelphia, Lippincott-
Raven Publishers. 2. pp1767-1847. 1996.

. Di Stefano, H. S. and R. M. Dougherty. Multiplication of avian leukosis virus in

endocrine organs of congenitally infected chickens. J Natl Cancer Inst 42(1): 147-
154. 1969.

10. Di Stefano, H. S., A. A. Marucci and R. M. Dougherty. Immunohistochemical

demonstration of avian leukosis virus antigens in paraffin embedded tissue. Proc
Soc Exp Biol Med 142(4): 1111-1113. 1973.

ll. Dougherty, R. M. and H. S. Di Stefano. Sites of avian leukosis virus multiplication in

congenitally infected chickens. Cancer Res 27(2): 322-332. 1967.

12. Dougherty, R. M., A. A. Marucci and H. S. Distefano. Application of

immunohistochemistry to study of avian leukosis virus. J Gen Virol 15(2): 149-
162. 1972.

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F adly, A. M. . Subgroup J avian leukosis virus infection in meat-type chickens: A
review. IV Asian Pacific Conference on Poultry Health.1998.

Fadly, A. M. and E. J. Smith . An overview of subgroup J -like avian leukosis virus
infection in broiler breeder flocks in the United States. Avian Tumor Viruses
Symposium, Reno, Nevada, American Association of Avian Pathologists.1997.

Fadly, A. M. and E. J. Smith. Isolation and some characteristics of a subgroup J-like
avian leukosis virus associated with myeloid leukosis in meat-type chickens in the
United States. Avian Dis 43(3): 391-400. 1999.

Fadly, A. M. and R. L. Witter. Oncomaviruses: leukosis/sarcoma and
reticuloendotheliosis. In A Laboratory Manuel for the Isolation and Identification
of Avian Pathogens. eds,D. E. Swayne, J. R. Glisson, M. W. J ackwood, J. E.
Pearson and W. M. Reed. Kennett Square, PA., American Association of Avian
Pathologists. ppl 85-196. 1998.

Gazzolo, L., C. Moscovici and M. G. Moscovici. Persistence of avian oncoviruses in
chicken macrophages. Infect Immun 23(2): 294-297. 1979.

Gazzolo, L., M. G. Moscovici and C. Moscovici. Replication of avian sarcoma
viruses in chicken macrophages. Virology 58(2): 514-525. 1974.

Gazzolo, L., M. G. Moscovici and C. Moscovict. Susceptibility and resistance of
chicken macrophages to avian RNA tumor viruses. Virology 67(2): 553-565.
1975.

Gilka, F. and J. L. Spencer. Immunohistochemical identification of group specific
antigen in avian leukosis virus infected chickens. Can J Comp Med 48(3): 322-
326. I984.

Gilka, F. and J. L. Spencer. Viral matrix inclusion bodies in myocardium of
lymphoid leukosis virus- infected chickens. Am J Vet Res 46(9): 1953-1960.
1985.

Gilka, F. and J. L. Spencer. Importance of the medullary macrophage in the
replication of lymphoid leukosis virus in the bursa of Fabricius of chickens. Am J
Vet Res 48(4): 613-620. 1987.

Payne, L. N., S. R. Brown, N. Bumstead, K. Howes, J. A. Frazier and M. E.
Thouless. A novel subgroup of exogenous avian leukosis virus in chickens. J Gen
Virol 72(Pt 4): 801-807. 1991.

Payne, L. N., A. M. Gillespie and K. Howes. Induction of myeloid leukosis and other
tumours with the HPRS-103 strain of ALV. Vet Rec 129(20): 447-448. 1991.

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Payne, L. N., A. M. Gillespie and K. Howes. Myeloid leukaemogenicity and
transmission of the HPRS-103 strain of avian leukosis virus. Leukemia 6(11):
1167-1176. 1992.

Robinson, H. L., L. Rarnamoorthy, K. Collart and D. W. Brown. Tissue tr0pism of
avian leukosis viruses: analyses for viral DNA and proteins. Virology 193(1):
443-445. 1993.

Siccardi, F. J. and B. R. Burmester (1970). The differential diagnosis of lymphoid
leukosis and Marek's disease, USDA. 1412.

Smith, E. J., A. F adly and W. Okazaki. An enzyme-linked immunosorbent assay for
detecting avian leukosis- sarcoma viruses. Avian Dis 23(3): 698-707. 1979.

Spencer, J. L. and F. Gilka. Lymphoid leukosis: detection of group specific viral
antigen in chicken spleens by immunofluorescence and complement fixation. Can
J Comp Med 46(4): 370-375. 1982.

Spencer, J. L., F. Gilka and J. S. Gavora. Detection of lymphoid leukosis virus
infected chickens by testing for group specific antigen for virus in feather pulp.
Avian Pathol 12: 83-99. 1983.

Spencer, J. L., F. Gilka, J. S. Gavora and P. F. Wright. Distribution of lymphoid
leukosis virus and p27 group-specific antigen in tissues from laying hens. Avian
Dis 28(2): 358-373. 1984.

Young, J. A., P. Bates and H. E. Varmus. Isolation of a chicken gene that confers
susceptibility to infection by subgroup A avian leukosis and sarcoma viruses. J
Virol 67(4): 1811-1816. 1993.

185

CHAPTER 5

Conclusions and Future Directions

186

Avian leukosis is an old disease of poultry dating back to its first description in
1865 by Roloff. Termed lymphoid leukosis in 1961, the disease causes decreased egg
production, lower performance, and tumors of mainly B cell origin. There are six
subgroups of leukosis viruses that affect chickens and four that affect other avian species.
The most recently described of the six subgroups affecting chickens is subgroup J. ALV-
J infection causes myelocytomatosis in meat-type chickens. Reports of infection of egg
laying chickens have occurred in North America and Europe. Because of the possible
threat to the egg laying industry, the overall goal for this research was to determine the
pathogenicity and the pathogenesis of ALV-J in white leghorn chickens. The objectives
were: 1) Compare pathogenicity of ALV-J in various lines of white Leghorn chickens; 2)
Determine the effects of endogenous virus 21 on the pathogenesis of ALV-J in white
leghoms; and 3) Determine the tissue tropism of ALV-J and the susceptibility of the B

cell to ALV-J induced transformation in white leghoms.

OBJECTIVE l. The most significant findings for objective 1 were that white leghorn
chickens can develop LL when infected with ALV-J as embryos or at day of hatch, the
genetics of the chicken will influence how the bird responds to infection after hatch, and
Line 63 exhibits resistance to tumor formation. Previous research by Payne and others
(Payne et al. 1991; Payne et al. 1992) did not report the development of LL with
infection of leghorn chickens with ALV-J. This report demonstrates that LL can be an
end result of ALV-J infection in egg-laying chickens. This is an important finding for the
industry because producers may believe that they have a subgroup A or B infection if LL

tumors develop, when in fact, it may be subgroup J. Serological tests performed on these

187

flocks would indicate a lack of antibody response to ALV-A or B. A second serological
test for ALV-J antibodies should be included to determine exposure of the flock to ALV-
J.

The next significant finding is that the genetic constitution of the bird influences
the outcome of infection with ALV-J after hatch. Line 0 chickens, which lack
endogenous virus loci, were able to seroconvert very quickly with ALV-J infection, as
compared to a subgroup A infection. The other three lines were not able to clear the
viremia despite the presence of neutralizing antibodies at thirty weeks of age. These
other lines have various endogenous viral loci in their genomes, which traditionally
delays the antibody response to ALV-A or -B. However, they eventually do seroconvert
by 30 weeks of age. This was not the case with ALV-J infection after hatch.

The final finding is that Line 63 exhibits resistance to tumor formation when
infected with ALV-J. Line 63 is known to be resistant to tumor formation when infected
with subgroup A (Bacon et al. 2000). Approximately 0.5% of Line 63 chickens will
develop LL when infected with ALV-A (Crittenden et al. 1964; Crittenden et al. 1972).
In this study, Line 63 chickens only developed 1 tumor and it was classified as LL.
Bursal transformation results indicated again that only 1 bird had evidence of
transformation. The lack of tumor formation in Line 6 following ALV-J infection is

important information for researchers in planning future experiments with ALV-J.

OBJECTIVE 2. The most significant results gained from this objective was that EV21

influences the serological outcome of ALV-J infection after hatch. The poultry industry

uses early and late feathering phenotypes to sex chicks at hatch instead of vent sexing for

188

some genetic lines. EV21 is associated with the K gene and is responsible for the late
feathering phenotype. The cross of chickens used in this study had exactly the same
genetic information in each group except for the presence or absence of EV21. The
mating was conducted in such a manner to cause both males and females to be early
feathering or late feathering, thereby removing the factor of sex in the outcome of
infection. It is well documented that females are more susceptible to tumor formation
than males with ALV infection. The serological results demonstrated that an unusually
high percentage of slow feathering EV21+ chickens remained Viremic tolerant for the
entire experiment (54%). Previous research by Smith and Fadly (Smith and Fadly 1988;
F adly and Smith 1997) reported a delayed immune response in chickens harboring EV21
when infected with ALV-A after hatch. However, the percentage of Viremic tolerant
birds at the end of the experiments was small. The unusually high percentage of Viremic
tolerant chickens in this study would escape detection using serological basis tests only
for flock monitoring and would serve as a source of congenitally infected progeny. The
elimination of vertical transmission between hens and progeny is the most important
aspect in eradication programs for leukosis. Viremic tolerant birds would cause

significant delays in eradication programs.

OBJECTIVE 3. The most significant finding for this objective was that some tissues
determined to be positive for ALV-J had not been previously reported as susceptible to
ALV-J infection. Previous reports that determined tissue tropism of ALV-J report
variable to no detectable levels of p27 antigen in peripheral nerve, spleen, thymus, and

bone marrow in a variety of different chicken lines (Arshad et al. 1997). This study

189

demonstrated ALV-J antigen in all tissues examined, including peripheral nerve, spleen,
thymus and bone marrow. By six weeks of age, all but one section of tissue examined
was positive for tissue specific staining of gpSS antigen. Traditionally,
immunohistochemistry has used a monoclonal antibody to p27, the group specific antigen
of ALVs. Using p27 permits detection of all subgroups and can be used for diagnostic
purposes. However, the possibility of detecting endogenous virus is present because
some subgroup E loci express p27. The genetic cross of chickens used in this study has a
high endogenous viral loci load, which could have interfered with detection of ALV-J
using a p27 based assay. Using a monoclonal antibody to gp85 that is subgroup specific
removes the possible detection of endogenous virus. The tissues that had not been
previously reported as containing ALV-J in white leghoms were detected using the gp85
monoclonal antibody and included peripheral nerve, spleen, thymus and bone marrow.
Using the gp85 antibody may increase the sensitivity of the detection method compared

to using the antibody to p27.

FUTURE DIRECTIONS

From the results gained from these studies, additional questions were raised about
ALV-J infection in egg laying chickens. One question was, how is the virus escaping the
immune system in those chickens with endogenous virus loci, causing the animals to
become Viremic tolerant instead of mounting a delayed immune response? Experiments
designed to look at the interactions of endogenous virus and ALV-J could answer this
question. By examining the MHC class I and II expression from chickens with ev loci

infected with ALV-J versus line 0 chickens which lack ev loci, may give some indication

190

of the interactions of exogenous and endogenous viral proteins and host proteins that
allow viremia to continue. Work by Stedman et a1 (2000) demonstrated that the
phagocytic functions of macrophages and heterophils are not decreased and lymphocytic
functions are not impaired in broilers infected with ALV-J. Therefore, the virus is
apparently not immunosuppressive and does not reduce the ability of the bird to respond
to infection with other pathogens. Functional assays on white leghorn white blood cells
may reveal a difference and help explain the increased Viremic tolerance seen with white
leghoms with ev loci.

Another question arose from the Line 6 tumor results. To confirm the tumor
resistance of Line 63 chickens seen in objective 1, a separate experiment using Line 63
chickens infected as embryos and at day-of-hatch with more birds is necessary. With
only 10 at risk chickens remaining at the end of 30 weeks, the conclusion that Line 63
birds are resistant to ALV-J induced tumor formation is tenuous. An experiment similar
to that in objective 1 more at risk birds would be necessary.

One other question that could be addressed is the effect of B haplotype on ALV-J
infection in both broilers and layers. The B haplotype is well characterized in leghorn
chickens. Recent work by Bacon (yet unpublished data) examined the effect of B
haplotype in aged hens and their ability to mount an antibody response. There is some
evidence that B21 homozygotes have higher neutralizing antibody titers compared to B2
or B5 homozygotes. By examining the effect of B haplotypes in day-of-hatch inoculated
chicks, information regarding antibody response and tumor incidence can further support
Bacon’s findings. Additionally, examination of B haplotype in broiler chickens, which is

not well characterized, may provide new information on the effect of genotype on disease

191

outcome, and may allow breeder production companies to consider such information in

their genetic selection process.

192

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. Arshad, S. S., K. Howes, G. S. Barron, L. M. Smith, P. H. Russell and L. N. Payne.
Tissue tropism of the HPRS-103 strain of J subgroup avian leukosis virus and of a

derivative acutely transforming virus. Vet Pathol 34(2): 127-137. 1997.

. Bacon, L. D., H. D. Hunt and H. H. Cheng. A review of the developement of chicken
lines to resolve genes determining resistance to diseases. Poult Sci 79: 1082-1093.
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sarcoma and strain RPL 12 viruses in the cells, embryos, and chickens of two
inbred lines. J Natl Cancer Inst Monograph 17: 161-177. 1964.

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. Payne, L. N., A. M. Gillespie and K. Howes. Induction of myeloid leukosis and other
tumours with the HPRS-103 strain of ALV. Vet Rec 129(20): 447-448. 1991.

. Payne, L. N., A. M. Gillespie and K. Howes. Myeloid leukaemogenicity and
transmission of the HPRS-103 strain of avian leukosis virus. Leukemia 6(11):
1167-1176. 1992.

. Smith, E. J. and A. M. Fadly. Influence of congenital transmission of endogenous
virus-21 on the immune response to avian leukosis virus infection and the
incidence of tumors in chickens. Poult Sci 67(12): 1674-1679. 1988.

. Stedman, N. L., T. L. Brown and D. I. Bounous . Function of Heterophils,
macrophages, and lymphocytes isolated from broilers naturally infected with
avain leukosis virus subgroup J. International symposium on ALV-J and other

avian retroviruses, Rauischholzhausen, Germany, Institut fur
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