EFFECT 8F FLUOROPHEWLALANWE
0N THE REFLECATQON GF AVIAN
INFECTSGUS BROHCHETES M9
NEWCASTLE BiSEASE VERUSES 5N
CHECKEN EfiiBRYO RENEE" CELLS

Thesis for the Degree of M. S.

MICHEGAN STATE UNWERSITY

LORELL HENRY ANGELETY, SR.
1970

   
  

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THESIS

ABSTRACT

EFFECT OF FLUOROPHENYLALANINE ON THE REPLICATION OF
AVIAN INFECTIOUS BRONCHITIS AND NEWCASTLE DISEASE
VIRUSES IN CHICKEN EMBRYO KIDNEY CELLS

BY
Lorell Henry Angelety, Sr.

Synthesis of avian infectious bronchitis (IBV) and
of Newcastle disease (NDV) viruses in chicken embryo kidney
cells was effectively inhibited by p-DL-fluorOphenylalanine
(FPA). The amount of extracellular virus was not related
to efficiency of adsorption to or entry of the host by the
virus. Extracellular virus was 1.6 and 3.0 logs less for
IBV and NDV, respectively, when compared with virus in the
absence of the inhibitor. Inhibition of viral synthesis
was reversible by the addition of DL-phenylalanine to the
cultural medium.

Differences in sensitivity of viral syntheses to
inhibition by FPA may reflect a difference in the ability
of either structural (capsid) or non-structural (enzymes)
proteins to retain functional integrity following incor-

poration of the analogue, FPA.

EFFECT OF FLUOROPHENYLALANINE ON THE REPLICATION OF
AVIAN INFECTIOUS BRONCHITIS AND NEWCASTLE DISEASE

VIRUSES IN CHICKEN EMBRYO KIDNEY CELLS

BY

Lorell Henry Angelety, Sr.

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology
and Public Health

1970

6 (7H4;
.Ev/wa’c

DEDICATION

I dedicate this thesis to those who made it

possible, my wife and my parents.

ii

ACKNOWLEDGMENTS

I wish to express my sincere gratitude to
Dr. Charles H. Cunningham, Professor of Microbiology and
Public Health, for his guidance and encouragement through-
out this investigation.

I also wish to extend my sincere thanks to
Mrs. Martha P. Spring, Department of Microbiology and
Public Health and Dr. Mark F. Stinski, formerly of the
Department, for their kind and invaluable assistance and
instruction in the proper virologic and cell culture
techniques.

I am especially grateful to Dr. George G. Wright,
Branch Chief, Immunology Branch, Fort Detrick, for his
assistance in the preparation of this manuscript.

In addition, the author is indebted to the Depart-
ment of the Army, Fort Detrick for the training grant
which made possible my study at Michigan State University.

This study was supported in part by the Michigan
Agricultural Experiment Station and by a grant-in aid from

the United States Department of Agriculture.

iii

TABLE OF CONTENTS

Page

LIST OF TABLES O O O O O O O O O O O I O O O O O 0 Vi
LIST OF FIGURES O I O I O O O O O O I O O O I O O Vii
INTRODUCT ION O O O O O O I O O O O O O O O O O O O 1

LITERATURE REVIEW . . . . . . . . . . . . . . . . 2

(I)

MATERIALS AND METHODS . . . . . . . . . . . . . .

Viruses . . . . . . . . . . . . . . . . . . . .
Cell Cultures . . . . . . . . . . . . . . .
p-DL- Fluorophenylalanine and
DL-phenylalanine . . . . . . . . . . . . . .
Antiserum . . . . . . . . . . . . . . . . . .
Inoculation of cells with virus and their
treatment with p-DL- Fluorophenylalanine . . . 10

00 0000

RESULTS 0 O O O O I O O O O O O O O O O O O O O O 12

Toxicity of p-DL-Fluorophenylalanine for

Cells . . . . . . . . . . . . . . . . . . . . 12
Effect of p-DL-Fluorophenylalanine on

the Synthesis of Infectious Bronchitis

and Newcastle Disease Viruses . . . . . . . . 12
Effect of P-DL-Fluorophenylalanine on

the Adsorption of Infectious Bronchitis

and Newcastle Disease Viruses to Cells . . . l4
Reversal of P-DL-Fluorophenylalanine

Inhibition of Infectious Bronchitis

and Newcastle Disease Viral Syntheses

by DL- -Phenylalanine . . . . . . . . 14
Effect of Pre-Treatment of Cells with

p-DL- FluorOphenylalanine on the Entry

of the Host Cell by Infectious Bronchitis

Virus . . . . . . . . . . . . . . . . . . . . 17

iv

Page

Effect of the Time of Addition of p—DL-
Fluorophenylalanine on the Synthesis
of Infectious Bronchitis and Newcastle
Disease Viruses . . . . . . . . . . . . . . . 19

DISCUSSION 0 O O O O O O O O O O O O O O O O O O O 23

LITEMTURE CITED . . . O . . . O O . O O O . . O . 26

LIST OF TABLES

Table Page

1. Toxicity of p-DL-fluorophenylalanine
for cells on the basis of
cytopathogenicity . . . . . . . . . . . . 13

2. Effect of p-DL-fluorophenylalanine on
the adsorption of avian infectious
bronchitis and Newcastle disease
viruses to cells . . . . . . . . . . . . l6

3. Reversal of p-DL-fluorophenylalanine
inhibition of avian infectious
bronchitis and Newcastle disease

viral synthesis by DL-phenylalanine . . . 18

vi

LIST OF FIGURES

Figure Page

1. Effect of p-DL-fluorophenylalanine on
the synthesis of avian infectious
bronchitis and Newcastle disease
viruses . . . . . . . . . . . . . . . . . 15

2. Effect of pre—treatment of cells with
p—DL—fluorophenylalanine on the
entry of the host cell by avian
infectious bronchitis virus as
measured by the inability of anti-
body to neutralize intracellular
virus . . . . . . . . . . . . . . . . . . 20

3. Effect of the time of addition of
p-DL-fluorophenylalanine on the
synthesis of avian infectious
bronchitis and Newcastle disease
viruses . . . . . . . . . . . . . . . . . 22

vii

INTRODUCTION

It has been demonstrated that viral isolates from
apparently typical clinical cases of infectious bronchitis,
were in fact mixtures of avian infectious bronchitis virus
(IBV) and other viruses, the most common of which was
avian adenovirus (IO). Recently it has been demonstrated
(21) that laboratory stocks of IBV may contain latent
Newcastle disease virus (NDV).

The present study was initiated to determine the
effect of p-DL-fluorophenylalanine (FPA) on the replication
of IBV and NDV in chicken embryo kidney cells as a possible

means for selective separation of the two viruses.

LITERATURE REVIEW

Infectious bronchitis virus has a diameter of 83-100
mp (29) and bears club-shaped spikes 20 mp long arranged in
clusters (11). The virion possesses an ether—sensitive
lipoprotein envelope (27), and a ribonucleic acid core (3).
The specific gravity of the particle is 1.24 in CsCl and
1.19 in sucrose. The sedimentation constant of the virus
is 344 S which corresponds to a 80-100 mu sphere (l7), and
is in agreement with dimensions obtained by electron micro-
scopy. The virion does not normally agglutinate chicken
erythrocytes, unless it has been modified by trypsin, ether
or DEAE-cellulose treatment (12,13).

The virus has been cultivated in a variety of avian
cell and tissue cultures derived from the embryonating
chicken egg and the chicken but mammalian cell cultures are
generally reported not to support the growth of the virus
(16). The cells of choice for propagation of the virus are
chicken embryo kidney cells (CEKC)(24).

After attachment of the virion to specific receptor
sites at the surface of the host cell membrane, there is an
eclipse period of four hours with the maximum titer produced

after 36-48 hours (14,24)

Newcastle disease virus is roughly spherical,
with a diameter of 100-180 mu, a particle weight of 800
x 106 daltons, and a sedimentation constant of 1,100 S
(31). The inner ribonucleoprotein is surrounded by a
spiked-armed envelope. The spikes are 8 mu long and 8-10
mu apart (39). The inner component has a diameter of 17-
18 mp with a central channel of 5 mu in diameter (19).

Newcastle disease virus is readily propagated in
the chicken embryo (23) and in a wide variety of avian
and mammalian cells. There is an eclipse period of two
hours with the maximum titer produced after 24 hours in
chicken lung cells (18).

Virus specific soluble antigens have been reported
for both IBV and NDV. Infectious bronchitis virus pos-
sesses at least three antigens separable on the basis of
size, buoyant density, thermostability and sensitivity to
enzymes (38).

Newcastle disease virus has at least two antigens,
which corresponds to a surface component (hemagglutinin)
and an inner component (nucleoprotein). The antigens are
differentiated by their complement-fixing, hemagglutining
and enzymatic activity (31).

Newcastle disease and infectious bronchitis
viruses, although similar morphologically and chemically,
are members of two distinct groups of viruses. Newcastle

disease virus is grouped with the paramyxoviruses by

virtue of its chemical and biophysical properties (42).
Infectious bronchitis virus is at present unclassified,
but recognition of the similarity between IBV and human
respiratory viruses, B814 and 229E, indicated the possi-
bility of a new and previously unrecognized virus group
(4,9,26). The morphologic features of these viruses and
that of murine hepatitis virus resembles a solar corona,
which has prompted the suggestion that the group be termed
coronavirus, with IBV as the prototype (5).

Fluorophenylalanine is useful in the study of
viral replication as this amino acid analogue is incor-
porated in place of phenylalanine during the synthesis
of protein and causes the production of aberrant and non-
functional proteins (28). Inhibition by FPA of the syn-
thesis of functional proteins has permitted study of the
sequential events of viral replication that would be
difficult to follow by conventional methods such as one-
step growth curves and others.

Synthesis of poliovirus in HeLa cells is inhibited
by the presence of FPA in the growth medium (1). The
inhibition is not the result of a toxic effect of FPA
on the cells.

The effect of FPA on the replication of polio and
Western equine encephalitis (WEE) viruses is a gradient
effect as the proteins involved in the maturation of the

virus are affected by low concentrations of FPA. The

proteins involved in the replication of the RNA are af-
fected by high concentrations of the inhibitor (22).

Fluorophenylalanine is capable of interrupting
RNA synthesis at any time during the multiplication cycle
of polio and WEE viruses. The protein affected is RNA
dependent RNA polymerase. The polymerase of poliovirus
is a labile protein that uses the RNA of poliovirus as a
template for the replication of viral nucleic acid (6).
The enzyme is rendered non-functional through incorpora-
tion of the analogue into it. Decay of functional poly—
merase, leads to an interruption of RNA synthesis 45
minutes after the addition of FPA to the growth medium.

There is a change in the capsid protein of polio-
virus following addition of low concentrations of FPA to
the medium of infected HeLa cells. The virus particles
produced resemble poliovirus, but have obvious irregulari-
ties as determined by electron microsc0py. The virus
particles are heat labile and are antigenically similar
to heat-denatured virions. The general character of the
virus particle is thought to be the result of the forma-
tion of fraudulent capsid protein, which is incapable of
forming the proper three dimensional configuration neces-
sary for production of infectious virus (20).

Inhibition of the replication of influenza virus
by FPA, may be reversed by the addition of phenylalanine

to the growth medium (2). There is no detectable delay

in viral production if the phenylalanine is added more
than seven hours after infection, lack of a delay is not
due to the accumulation of mature virus in the host cell,
it is due to the accumulation of viral precursors.

Inhibition of influenza virus by FPA has allowed
the separation of the replication cycle into two phases.
The first phase is inhibited by high concentrations of
FPA, whereas low concentrations inhibits a phase follow-
ing the first by 1-1 1/2 hours (41).

A random delay in the release of infectious virus,
the duration of which varies inversely with the multiplic-
ity of infection, occurs early in the replication cycle.
Synchronization of viral replication may be accomplished
by inhibiting replication and subsequently reversing the
inhibition by the addition of phenylalanine to the growth
medium (41).

Replication of fowl plague virus in whole chicken
embryo and chicken lung cells is inhibited by FPA (45),
which does not interfere with the adsorption of the virus
to or penetration of the host cell, nor does it alter
irreversibly the ability of the host cell to support repli—
cation of the virus.

Multiplication of the virus is blocked by FPA in
at least two stages. The first stage occurs between ad-
sorption and production of the S-antigen (ribonucleoprotein),

and the second stage occurs between production of the

S-antigen and the hemagglutinin and infectious virus.

In the presence of FPA the S-antigen accumulates in the
nucleus with subsequent inhibition of the production of
the hemagglutinin and infectious virus. This separation
of the assembly stages is dependent upon the level of
the inhibitor and the time at which FPA is added to the
growth medium.

Fluorophenylalanine inhibits the synthesis of
fowl plague virus RNA, when added to infected cells
prior to initiation of nucleic acid synthesis, but not
when added later (29). In contrast, synthesis of the RNA
of picorna (22) and arboviruses (40) can be inhibited by
FPA at anytime during replication of the viruses.

The inhibitor does not interfere with the incor-

14

poration of C labeled leucine into virus protein, but

rather the l4C-leucine labeled proteins are incorporated
at about 15% of the normal rate into intact particles.
Multiplication of NDV in chicken embryo fibroblast
cells is nearly completely inhibited by FPA at 200 ug/ml.
Production of a functional early protein, presumably RNA
dependent RNA polymerase, is inhibited when FPA is added
up to three hours after infection. After this period RNA
synthesis is not affected by the addition of the inhibitor.
According to Akers (3), 600 ug FPA/ml reduced by
2-3 logs the yield of IBV in CEKC, when compared to the

virus titer in the absence of the inhibitor.

MATERIALS AND METHODS

Viruses

The Beaudette strain of IBV (IBV-42) adapted to
CEKC was used and assayed on CEKC in petri dishes as
plaque-forming units (pfu) by the agar overlay method
(15). Cells were inoculated with 6.0 x 105 pfu of the
128th passage of the virus. The extracellular fluid was
harvested at 48 hours, pooled and centrifuged at 10,000 g
for 30 minutes. The supernatant fluid, which contained
2.2 x 107 pfu/ml, was dispensed in 1 m1 volumes and
stored at -90°C until used.

The Texas Gilbert-Boney (GB) strain of NDV, 1.0 x
107 pfu, was used to inoculate CEKC. After 24 hours the
extracellular fluid was harvested and processed as des-

cribed above for IBV. There were 3.1 x 107 pfu of virus/

m1 when assayed on CEKC.

Cell Cultures

 

Primary CEKC were suspended in appropriate con-
centrations of Medium 199 (Grand Island Biological Company,
Inc.) containing 2 mM glutamine, and supplemented with
vitamins and amino acids of Eagle's essential medium, 100

units/ml penicillin, 100 g/ml dihydrostreptomycin, 50

8

 

(I‘ll-l (It-(‘1‘!

 

units/ml Mycostatin (Squibb) and 0.1% sodium bicarbonate.
Newborn calf serum (Grand Island Biological Company, Inc.)
was then added to a final concentration of 5%.

Tube cultures were prepared by dispensing 1 ml

(1:300 dilution of packed cells) 3.3 x 106

cells/ml into
16 x 125 mm tissue culture tubes (Rochester Scientific
Company, Inc.). Cells in petri dishes were used to study
the effect of FPA on adsorption and the penetration of

IBV and NDV and for the assay of the viruses were prepared
by dispensing 4 m1 (1:100 dilution ofpacked cells), 107
cells/ml into 15 x 60 mm plastic petri dishes (Falcon
Plastics). All cell cultures before and after inoculation
were incubated at 37°C in an atmosphere of 8% C02, 80-85%
relative humidity. Confluent monolayers of cells were
formed in tubes and petri dishes after 48 hours.

p-DL-Fluorgphenylalanine and
DL-Phenylalanine

 

p-DL-Fluorophenylalanine (Sigma Chemical Company)
and DL-phenylalanine (Laboratory Park) were prepared as
10 mg/ml stocks in glass double distilled water, steri-

lized by filtration, and stored at 4°C.

Antiserum

 

Anti-IBV-41 chicken serum (36) kindly supplied by
Dr. Mark F. Stinski, formerly of the Department of Micro-

biology and Public Health, Michigan State University,

10

East Lansing, Michigan, was heated at 56°C for 30 minutes

prior to use.

Inoculation of Cells with Virus and their
Treatment with prL—Fluorophenylalanine

 

 

After the extracellular fluid was decanted from
tube and petri dish cultures of CEKC, the cells were
washed with Hanks' balanced salt solution (HBSS). Sus-
pensions of viruses were diluted to the desired pfu/ml
at 4°C in phosphate buffered saline (PBS) without Ca++
or Mg++ and containing 3% new born calf serum.

Cultures of CEKC in tubes were inoculated with
IBV, 5.0 x 105 pfu/0.2 ml, or NDV, 6.0 x 105 pfu/0.2 ml,
which represents a multiplicity of infection of 1.
Incubation for adsorption of the virus was at 37°C for 20
minutes.

Cultures of CEKC in petri dishes were inoculated
with 0.5 m1 of the viruses and incubated at either 37°C
for 90 minutes or 4°C for 30 minutes for certain experiments.

To determine the toxicity of FPA for CEKC without
virus, tube cultures were treated with the cultural medium
containing graded concentrations of the analogue and were
observed microscopically for any cytopathic effects after
24 and 48 hours.

The inhibitory activity of the analogue on viral
replication was determined using graded concentrations of
the analogue in the cultural medium on IBV and NDV infected

cells.

11

Details as to the treatment of CEKC in petri
dishes will be described under Results.

The effect of FPA on cells (toxicity) and on viral
synthesis was determined using two and three tube cultures
of CEKC, respectively, for each test concentration of the
inhibitor.

The effect of FPA on adsorption to or penetration
of the host cell, as well as virus concentration was de-
termined using three petri dish cultures of CEKC for each

virus sample or test condition.

RESULTS

Toxicity_ofyp—DL-Fluorophenylalanine
for Cells

 

 

The cells tolerated as much as 600 ug FPA/ml for
24 hours, but they were able to tolerate 300 mg for 48
hours based on cytopathogenicity (Table 1). On the above
basis and that the maximum production of IBV occurs 36
and 48 hours (14,24), 300 ug FPA/ml was selected as the
maximum concentration to be used to determine the effect
of FPA on the replication of IBV and NDV in CEKC.
Effect of p-DL-Fluorophenylalanine
on the Syntheses of Infectious

BronChitis and Newcastle Disease
Viruses

 

 

 

Cells were inoculated with IBV or NDV, washed
with HBSS and then Medium 199 containing 0-300 ug FPA/ml
was added to appropriate groups of tube cultures. Non-
infected cell cultures were treated with PBS without Ca++
or Mg++ containing 3% serum, instead of virus during the
adsorption period and they served as controls. After 48
hours the extracellular fluids were harvested and assayed
for virus. The titer of virus in the absence of FPA was

considered to be the normal yield of virus. The titer of

virus in the various concentrations of FPA was compared

12

13

TABLE 1

Toxicity of p-DL-fluorophenylalanine for CEKC
on the basis of cytOpathogenicity

 

 

ug/ml FPA 24 Hours 48 Hours
0.0 Normal cells confluent Normal cells confluent
sheet sheet
100.0 Normal cells confluent Normal cells confluent
sheet sheet
200.0 Normal cells confluent Normal cells confluent
sheet sheet
300.0 Cell rounding Cell rounding
confluent sheet confluent sheet
400.0 Cell rounding Extensive cell rounding
confluent sheet with detachment of cells
500.0 Cell rounding Extensive cell rounding
confluent sheet with detachment of cells
600.0 Pronounced cell Extensive cell rounding

rounding and granular

with detachment of cells

 

14

to those in the absence of FPA and expressed as the per
cent of normal yield.

The yield of IBV was rapidly reduced as a function
of the FPA concentration in the growth medium. As little
as 50 pg FPA/ml, which is equal to the concentration of
phenylalanine in Medium 199 resulted in a 30% reduction
in the titer of IBV. The maximum concentration of FPA
employed (300 ug/ml) reduced the titer of IBV by 99.9%.
In contrast, 150 ug FPA/m1 was required to reduce the NDV
titer by 30%, whereas 300 ug FPA/ml, the maximum concen-
tration tested reduced the titer by 96% (Fig. 1).

Effect of p-DL-Fluorophenylalanine on

the Adsorption of Infectious Bronchitis
and Newcastle Disease Viruses to Cells

 

 

 

Dilutions of IBV and NDV suspensions were prepared
in PBS without Ca++ or Mg++ but with 3% serum. The PBS
contained either no FPA or 300 ug FPA/ml. Cultures of
CEKC in petri dishes were then inoculated for assay by
the plaque method (15).

The efficiency of adsorption of both viruses was

not reduced by the inhibitor (Table 2).

Reversal of p-DL-Fluorophenylalanine
Inhibition of Infectious Bronchitis and
Newcastle Disease ViralTSyntheses 5y
DL-Phenylalanine

 

 

 

 

To determine the specificity of reversal by

phenylalanine (PA) of the observed inhibition of IBV and

15

   

 

'00 O“"'O = NDV
H = IBV
“0‘
\
\
\
\
\
\
O\ O
\
\
\

a. 10 :-

35 .

1: Z

3 _

)-

§ _ ‘0

g
g Lot

0.] 1 1 1 1 1

 

     

 

50 100 150 200 250 300

p-Fluorophenylalanine (,qg/ml)

 

#—

Fig. l.--Effect of FPA on the synthesis of IBV and NDV.

16

TABLE 2

Effect of FPA on the adsorption of IBV
and NDV onto CEKC monolayers

 

 

Titer (pfu/m1)

 

 

Diluent
IBV NDV
* 7 7
PBS 2.1 x 10 2.9 x 10
PBS + FPAI 2.2 x 107 3.1 x 107

 

*
FPA-free (control) + 3% nbcs

2|:

300 ug/ml FPA + 3% nbcs

l7

NDV syntheses by FPA, Medium 199 containing 50 ug PA/ml,
300 ug FPA/ml, or 300 ug FPA/ml or 600 ug PA/ml was added
to tube cultures to which IBV and NDV, respectively, had
been adsorbed. After 48 hours the extracellular fluids
were assayed for virus.

Phenylalanine reversed the inhibition of viral
synthesis induced by FPA. The titers of IBV and NDV were
3.1 and 1.5 logs greater, respectively, in the presence
of the higher concentration of PA, than the titer in the
absence of high concentrations of PA (Table 3).

Effect of Pre-Treatment of Cells with
p-DL-FluorOphenylalanine on the Entry

of the Host Cell byIfifectious BronChitis
Virus

 

 

 

Intracellular IBV is not neutralized by extra-
cellular specific antibody. The virion adsorbs to but
does not penetrate the cell at 4°C (37). Accessibility
of virus to antibody was studied in cells treated with
FPA and the rate of penetration of the cells was compared
with that obtained under FPA-free conditions.

Medium 199 which was either FPA-free or contained
300 pg FPA/ml was added to petri dish cultures of CEKC.
After incubation at 37°C for 2 hours, the cells were washed
with cold PBS, re-incubated at 4°C for 30 minutes, and

3

then inoculated with 8.4 x 10 pfu per culture. After

adsorption at 4°C for 30 minutes the inoculum was decanted

18

TABLE 3

Reversal of FPA inhibition of IBV and NDV synthesis
by phenylalanine

 

 

Loglo Titer

 

Growth Medium

 

IBV NDV

*
Medium 199 6.2 7.1
Medium 199 + 300 ug/ml FPA 2.2 5.2
Med1um 199 + 300 ug/ml PFA 5.3 6.7

+ 600 ug/ml PA

 

*
Medium 199 contains 50 ug/ml PA

19

and the cells were washed with cold PBS. After incubation
at 37°C for 5, 15, 30, and 45 minutes, the cells were
washed with cold PBS and treated with 0.5 ml of anti-IBV-4l
antiserum (1:20). The antiserum treated cells were then
incubated at 4°C for 30 minutes and then overlaid with
Medium 199 containing 0.9% agar (Difco-Noble) and 5% serum.
The plates were processed as outlined in the plaque assay
method (15).

Pre-treatment of the host cell with FPA did not
alter the efficiency of the virion to adsorb to or pene-
trate CEKC (Fig. 2).

Effect of the Time of Addition of
p-DL-Fluorophenylalanine on the

Synthesis of Infectious Bronchitis
and Newcastle DiSease Vifuses

 

 

 

 

The synthesis of mature virus is dependent upon
the synthesis of early and late proteins. An experiment
was performed to determine the latest time at which FPA
could be added to infected cell cultures and achieve
maximum inhibition of viral synthesis.

Tube cultures of CEKC infected with IBV or NDV
were treated after 0, 2, 4, 6, 8, 12 and 24 hours at 37°C
with Medium 199 which contained 300 ug FPA/ml. A dupli-
cate set of infected cell cultures were processed in an
identical manner, with FPA-free medium. The extracellular

fluid was collected after 48 hours and assayed for virus.

20

 

 

 

IOOO ;'
C
I H =FPA-lru CEKC
. H =FPA-Irootod
: ‘ csxc
\
.3 100':- A
7., .
\. .
.2 .
a b
c
1 a b
. o
, 2 b
A
IO :
l _L i l J I
5 15 25 35 45 55

Incubation Time (37 C)

 

_’ " i-“ —— s L,“ 1, __..-,-.

Fig. 2.--Effect of pre-treatment of CEKC cultures with
FPA on the penetration of the host cell by IBV
as measured by the inability of antibody to
neutralized intracellular virus.

21

Virus titers in the presence of the inhibitor at
the various time periods, were compared to those in the
absence of FPA and expressed as the per cent of normal
yield.

Maximum inhibition of IBV and NDV occurred when
the inhibitor was added to infected cultures no later
than 4-6 hours (Fig. 3), which is approximately the eclipse
period reported for the synthesis of these viruses. In-
corporation of the FPA probably interfers with the forma-
tion of early proteins that are essential precursors
necessary for the synthesis of mature virus.

The amount of virus produced when the inhibitor
was added after 4-6 hours, was only about 0.5% and 3% for
IBV and NDV, respectively, when compared to the controls.
The quantity of virus produced when the inhibitor was
added as late as 24 hours was only about 50% and 3% for

IBV and NDV, respectively, when compared to the controls.

22

0—0 :NDV

10 O——-. =IBV

jTIIITU]

 

LO

7. Normal Yield

1’ W Irtvlr (f

I

0.1

IOFr

 

 

l l l L l I

O 4 8 12 16 2O 24
Time of Addition of FPA (hows)

 

 

 

__._—

Fig. 3.—-Effect of the time of addition of FPA on the
synthesis of IBV and NDV.

DISCUSSION

Adsorption of IBV and NDV to the host cells was
not affected by the FPA. The results clearly indicate
pronounced differences in the effect of FPA on the syn-
thesis of IBV and NDV, which might be utilized for the
differential separation of these two viruses in mixed
populations.

Inhibition of viral replication by FPA appears
to be due to the production of aberrant or non-functional
proteins following incorporation of the analogue (20).

Of particular importance is the report that incorporation

of FPA into a amylase of Bacillus subtilis does not change

 

its biophysical properties, e.g. sedimentation velocity,
but there was a reduction in its enzymatic activity (44).
Proteins participating in the synthesis of mengo and
polio viruses differ in their ability to remain functional
following the incorporation of FPA (8,22). The capsid
protein(s) become non-functional in the presence of low
concentrations of FPA without the cessation of RNA syn-
thesis; RNA synthesis is inhibited and RNA-dependent RNA
polymerase is rendered non-functional in the presence of
high concentrations of the inhibitor. In the presence of

low concentrations of the analogue protein synthesis is

23

24

not affected but the production of infectious virus is
inhibited (20).

It is probable that the differences in sensitivity
of IBV and NDV to FPA during replication are related to
the ability of proteins of the viruses to maintain enzy-
matic and structural integrity following incorporation of
the analogue.

Replication of viruses requires the synthesis of
structural (capsid) and non-structural (enzymes) pro-
teins. A non-structural protein (RNA Nucleotidyl Trans-
ferase) necessary for the synthesis of RNA, has been
reported for mengo (7), polio (6), Semliki Forest (25),
foot-and-mouth disease (30), vesicular stomatitis (43),
fowl plague (34), and ND (35) viruses. Although evidence
of this enzyme has not been reported for IBV, the fact
that it is an RNA virus would suggest the existence of
the enzyme. Synthesis of RNA of NDV is inhibited by FPA
no later than two hours after infection (33). This would
indicate that RNA synthesis requires an early protein,
presumably RNA-dependent RNA polymerase.

If the differences in the sensitivity of IBV and
NDV is reflected in the polymerases of the viruses, it
would appear that the enzyme of NDV is able to maintain
its activity to a greater degree following incorporation

of FPA residues into the molecule.

25

Similar considerations may also apply to the
structural proteins of the virus particle. Proteins of
fowl plague virus formed in the presence of FPA are in-
corporated into intact particles at only 15% of the nor-
mal rate (32). This would indicate that the capsid
proteins containing FPA do not have the proper configura-
tion necessary for ready incorporation into the viral
particle. Incorporation of capsid proteins containing
FPA into virions without the pr0per configuration may
result in a virion that is more sensitive to heat or
nuclease inactivation, as proposed for poliovirus (20).

Critical evaluation of these hypotheses for the
observed difference in the ability of the proteins of
IBV and NDV to retain enzymatic or structural integrity
must await the isolation and analysis of amino acids of

the individual proteins of the viruses.

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