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This is to certify that the
thesis entitled
Structure and Organization of the Genome

of Feline Herpesvirus—l

presented by

Paul A. Rota

has been accepted towards fulfillment

of the requirements for
Ph. D. , Microbiology
degree in

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STRUCTURE AND ORGANIZATION OF THE GENOME OF FELINE HERPESVIRUS-l
BY

Paul A. Rota

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1986

ABSTRACT
STRUCTURE AND ORGANIZATION or THE canons or FELINE HERPESVIRUS-l
by

Paul A. Rota

The structure and organization of the genome of feline herpesvirus-l
(FHV-l) were studied using a variety of biochemical techniques. The genome
of FHV-l was found to be similar in structure and general organization to
the genomes of other alpha herpesviruses. The FHV-l genome is
approximately 134 kilobase pairs (kb) in size and is composed of two
covalently joined segments. The longer segment is 103 kb in size and
composed of unique DNA. The shorter segment is 31 kb in size and is
composed of an 8 kb unique region which is flanked by 11 kb inverted
repeat regions. The short region can invert relative to the long giving
the genome two possible isomeric forms. A set of recombinant bacteriophage
clones containing most of the FHV-l genome was isolated and used to
construct Sal I, Hind III and Eco RI restriction endonuclease cleavage
maps of the viral genome. These clones and restriction maps were used in
subsequent studies to map the immediate early and late genes of FHV-l.

Radiolabeled FHV-l immediate early RNA hybridized to restriction
fragments of FHV-l DNA which are located primarily within the inverted
repeat regions in the short segment of the viral genome. Two RNA species
with sizes of 5 and 1.8 kilobases were transcribed from the FHV-l

immediate early gene region. In addition, two immediate early polypeptides

with molecular weights of 155 and 120 kilodaltons (kd) were identified by
in yitgg and in yiyg translation of FHV-l immediate early RNA.

FHV-l late RNA was hybrid selected by the individual cloned
restriction fragments and the in yitrg translation products of the eluted
RNA were analyzed by SDS-PAGE. The results indicated that FHV-l late genes
are distributed throughout the genome but are primarily found in the
unique long region. The region of the genome contained within Sal I
fragment E contained a cluster of 3-4 late genes two of which may code for
precusors to viral glycoproteins.

Five herpesvirus strains which were isolated from dogs and were
antigenically related to FHV-l were analyzed by restriction endonuclease
digestion, Southern blotting and immunoprecipitation. The results
indicated that the canine isolates were antigenically and genetically very
similar to FHV-l. Comparison of the restriction digest patterns of DNA
from the C-27 prototype strain of FHV-l, FHV-l field isolates, and
FHV-l-like canine isolates indicated that the genome of FHV-l is very
stable. Most of the restriction fragments showing size heterogeneity were

located at the viral termini and within the inverted repeat sequences.

to Susan
and

my family

ACKNOWLEDGEMENTS

I am extremely grateful to my advisor, Dr. Roger K. Maes, for his
help, advice, and encouragement during my stay in his laboratory and
especially during the preparation of this thesis. I am also thankful for
Dr. Maes' friendship and concern for both my personal and professional
development. I wish that we had a little more time to explore the
intracacies of bass fishing. I would also like to thank the members of my
guidance committee, Drs. Dodgson, Sadoff, Silva, Miller, and Conrad, for
their help and advice.

I would like to thank Debby Verkamp and Anke Klotsek for help with
plaque purifications and Steve Spatz for technical assistance.

Special thanks to Dr. Velicer and his staff for the use of their cell
culture facilities and equipment, to Dr. Magee and his staff for frequent
use of their equipment, to Idah Sithole and Bob Isfort for help with in
yiggg translations, to Paul Bates and Jerry Dodgson for advice on cloning,
and to Drs. Patterson and Verner for advice on handling RNA.

Finally I would like to thank my fellow graduate students at Giltner
for their friendship and support during my stay here. I wish them luck in
their future endeavors both on and off the softball diamond. A wise
philosopher once said, "Happiness is found along the way, not at the end

of the road."

TABLE. OF CONTENTS

List of Tables ......................................................... iii
List of Figures ......................................................... iv
Introduction ............................................................. 1
Chapter 1: Literature Review ............................................. a
Part 1: Feline Herpesvirus-l .................................. 5
Part 2: The Herpesviruses: An Overview ....................... 18
References ................................................... 36

Chapter 2: Physical Characterization of the Genome of Feline

Herpesvirus-l ................................................ 43
Chapter 3: The Immediate Early Genes and Polypeptides of

Feline Herpesvirus-l ......................................... 93
Chapter 4: Mapping the Late Genes of Feline Herpesvirus-l .............. 126
Chapter 5: Biochemical and Antigenic Characterization of Feline

Herpesvirus-l-Like Isolates From Dogs ....................... 158

Summary ................................................................ 184

ii

LIST OF TABLES

Chapter 2

Table 1. Restriction enzyme digestion patterns of FHV-l DNA .......... 57

Table 2. Hybridization and restriction enzyme digestion patterns of
FHV-l Hind III fragment A .................................... 63

Table 3. Hybridization of FHV-l Sal I fragments to Hind III fragments.75

Table 4. Hind III digestion patterns of FHV-l Sal I fragments ......... 76

iii

Chapter 1

Figure
Figure

Figure

l.

2.

3.

Chapter 2

Figure
Figure
Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

l.
2.

3.

10.

LIST OF FIGURES

Organization of the genomes of herpesviruses ................. 23
Models for the equalization of repeats in herpesviruses ...... 26

Location of HSV mRNA species abundant at different stages

of replication ............................................... 34
Restriction endonuclease digestion patterns of FHV-l DNA ..... S3
FHV-l DNA restriction fragment molarity determination ........ SS
Terminal restriction fragment identification ................. S9

. Blot hybridization patterns of FHV-l Sal I terminal

fragments .................................................... 61

. Electron microscopic analysis of FHV-l DNA ................... 65
. Electron microscopic analysis of the S region of FHV-l DNA...67
. Analysis of EMBL-3 clones containing FHV-l DNA inserts ....... 7O

. Linkage relationships between FHV-l Sal I and Hind III

fragments .................................................... 73

. Blot hybridization analysis of the 8 region of the FHV-l

genome ....................................................... 80
Physical map of the FHV-l genome for Sal I, Hind III

and Eco R1 ................................................... 82

iv

Chapter 3

Figure 1. Hybridization of IE RNA to FHV-l DNA ........................ 104
Figure 2. Hybridization of IE RNA to DNA from recombinant clones

containing FHV-l DNA inserts ................................ 106
Figure 3. Hybridization of IE RNA to SalI/Eco R1 digestion fragments

of bacteriophage clones containing FHV-l IE genes ........... 108
Figure 4. The immediate early region of FHV-l DNA ..................... 111
Figure 5. Northern blot analysis of FHV-l IE RNA ...................... 114
Figure 6. SDS-PAGE analysis of FHV-l IE polypeptides .................. 116
Chapter 4
Figure 1. In 31252 translation products of FHV-l late RNA ............. 135
Figure 2. In yiggg translation products selected for by FHV-l

Sal I fragments ............................................. 139
Figure 3. Map locations of FHV-l late gene products ................... 142
Figure 4. Analysis of EM-S ............................................ 145
Figure 5. Northern blot analysis of FHV—l late RNA .................... 147
Figure 6. In yigrg translation products selected for by subfragments

of EM-S ..................................................... 149
Chapter 5
Figure 1. Restriction enzyme digest patterns of C-27 DNA and DNA

from five canine isolates ................................... 167
Figure 2. Ban HI digestion patterns of C-27 DNA and five isolates

of FHV~1 of feline origin ................................... 170
Figure 3. Southern blot analysis of C-27 DNA and DNA from five canine

isolates .................................................... 172

Figure 4. SDS-PAGE and immunoprecipitation analysis of C-27 and
canine isolate purified virions ............................. 174
Figure 5. Restriction enzyme digest patterns of C-27 DNA and canine

herpesvirus DNA ............................................. 177

vi

INTRODUCTION

Feline herpesvirus-l is a significant cause of upper respiratory
tract disease in cats. Because of the worldwide distribution and severity
of FHV-l infections, the biological properties of FHV-l have been well
studied and several modified live and attenuated virus vaccines have been
developed. Few studies, however, have been conducted to investigate the
biochemical structure and function of this virus. The goal of the research
presented in this thesis was to characterize the basic structure and
organization of the FHV-l genome. The experimental approaches to achieve
this have been established in the analysis of other herpesviruses.

There are several reasons that justify the initiation of detailed
biochemical studies on this new herpesvirus system. The first reason is
that FHV-l is a significant pathogen of cats. Increased understanding of
the biochemical structure of FHV-l will allow for a more complete
understanding of the pathogenesis of this virus. For example, once the
basic structure of the viral genome has been established, studies can be.
conducted to detect FHV-l DNA in tissues from latently infected cats and
to determine the physical state of the latent DNA. Restriction digests of
DNA from a number of field isolates and vaccine strains could be analyzed
to identify the sites of potential virulence determinants. This type of
information would be useful in the development of new attenuated virus
vaccines and in the evaluation of new strains. Also, FHV-l antigens can be
expressed via recombinant DNA techniques and used for the construction of

more effective and economical vaccines against FHV-l.

l

2

The second reason is that the information obtained in these studies
will contribute to the rapidly expanding pool of information available
concerning the molecular biology of herpesviruses. There is evidence that
the genomes of various members of this important group of viruses are
arranged in a similar manner. Some highly conserved genes are located at
similar postions on the genomes of different herpesviruses. To expand
these studies, gene mapping data is needed for a variety of herpesviruses
rather than from a few well-characterized viruses.

Finally, the FHV-l infection of the cat may provide a way to study
herpesvirus infections in the natural host. Herpesviruses which cause
disease in humans are usually studied in experimental animals which are
not the natural hosts of these viruses. The results obtained in these
animal models might not reflect the true nature of the virus-host
interaction. It will be especially important to test the efficacy of new
vaccines and antiviral agents in natural host system where all aspects of
the infection can be measured.

Each of these points is discussed in greater detail in the Literature
Review (Chapter 1). Chapters 2-5 contain the research data and are
presented in manuscript form. Unfortunately, this format results in some
repetitiveness since certain information is included in the Introductions
and Methods sections of all of the manuscripts.

Chapter 2 describes the physical structure of the FHV-l genome, the
isolation of the recombinant bacteriophage clones, and the construction of
restriction maps of the viral genome. This information forms the basis for
the work completed in Chapters 3 and 4. Chapter 3 describes the
identification of the immediate early genes and polypeptides of FHV-l

while Chapter 4 contains an analysis of FHV-l late gene organization.

3

In Chapter 5, several herpesvirus isolates that were obtained from
clinically ill dogs were examined and found to be nearly identical to
FHV-l. The experiments in this chapter provided an interesting way to
compare the restriction digest patterns of a number of FHV-l isolates and
investigate genetic heterogeneity in FHV-l. The locations of the
restriction fragments showing size variation could be determined using the

map generated in Chapter 2.

Chapter 1

Literature Review

Paul A. Rota

Part I. Feline Herpesvirus-l

Introduction

 

Feline herpesvirus-l (FHV-l) was first isolated by Crandell and
Maurer (1958) from kittens with acute upper respiratory tract disease. The
disease was later designated feline viral rhinotracheitis and the viral
agent referred to as feline rhinotracheitis virus (Crandell and Despeaux,
1959). Subsequent work by Ditchfield and Grinyer (1965) revealed that this
virus was a herpesvirus. Based on its biological properties, feline
rhinotracheitis virus was placed in the alpha herpesvirus subfamily and
classified as feline herpesvirus-l (Roizman, 1980). FHV-l has been shown
to be antigenically distinct from FHV-Z, a herpes-like virus that causes
urethral obstruction in cats (Fabricant, 1984). For simplicity, the term
FHV-l will be used throughout this review.

FHV-l has a worldwide distribution and is responsible for a large
proportion of upper respiratory tract disease in cats (Povey, 1976). The
other virus which is a major cause of respiratory disease in cats is
feline calicivirus (a picornavirus) (Studdert, 1978).

The most recent review of FHV-l was written by Povey (1979). This
article provides a very complete source of information concerning the
morphology and physical properties of FHV-l and gives an excellent
overview of FHV-l induced disease in cats. The most interesting research
on FHV-l since this review has focused on the characterization of the
biochemical structure of FHV-l, identification of the tissue or tissues of
FHV-l latency, and evaluation of the immune response to FHV-l in cats at

defined times post vaccination and during reactivation. My goal in this

5

6

literature review is to discuss both the earlier work on FHV-l and

incorporate the more recent research about this virus.

Viral Morphology, Growth Characteristics and Host Range

The morphology of FHV-l is similar to that of most herpesviruses
(Ditchfield and Grinyer, 1965). The viral capsid is hexagonal and has an
average diameter of 108nm. A membranous envelope surrounds the capsid
giving the complete virion an average diameter of 178 nm. The envelope
contains viral glycoprotein antigens that are important in virus-host cell
interaction. As is the case for other herpesviruses, the infectivity of
FHV-l is greatly reduced by exposure to lipid solvents such as ether,
chloroform (Johnson, 1966), and sodium deoxycholate (Bartholomew and
Gillespie, 1968). The infectivity of FHV-l is also destroyed by treatment
with proteases such as trypsin (Johnson, 1966).

FHV-l is very sensitive to heat and humidity (Povey, 1979). Cell
culture fluids lose viability rapidly at ambient temperaturers. At 23°C,
the virus losses 99% of is viability in 5 minutes if the relative humidity
is greater than 30%. Virus stored at -50°C will still lose 90% of its
viability in 5 months.

FHV-l will replicate in primary, secondary or established cells lines
of feline origin (Lee, et al., 1968). Cells of non-feline origin will not
support the growth of FHV-l. For most in yiggg studies, FHV-l has been
grown in Crandell-Rees feline kidney (CRFK) cells. At an input
multiplicity of 1 pfu/cell, infective virus was first detected within CRFK
cells at 6 to 8 hours post-infection and in the extracellular fluid at 9
hours post-infection. Direct cell to cell spread of infectious virus was

detected at 6 to 7 hours post-infection (Wardley, et al., 1976). By 30

7

hours, the average yield was 200 virions per infected cell (Tegtmeyer and
Enders, 1969) and most of this progeny virus remained cell associated. The
growth characteristics of FHV-l were different in tracheal ring organ
cultures (Milek, 1976). In this system, the viral growth rate was slower
and the final viral titer was lower than in cell culture monolayers. This
indicates that growth studies using monolayer cultures might not
accurately reflect the in yiyg growth situation. It would be interesting
to compare the in 21239 growth characteristics of field strains to
attenuated vaccine strains. Unfortunately, at this time, no studies of
this type have been attempted.

The cytopathic effect induced by FHV-l is similar to that of other
herpesviruses. Changes in infected cells include syncytia formation and/or
a rounding of cells followed by degeneration. The nature of these virus
induced changes depends on the input multiplicity of virus. FHV-l can
spread from cell to cell by the intracellular route (Wardley, 1976).
Infected cells may detach from the monolayer leaving holes in the
monolayer which can reach 1 cm in size (Crandell et al., 1960). Plaque
assays have been developed for FHV-l. Differences in plaque morphology in
CRFK cells have been observed and can be used to differentiate between
vaccine and field strains of virus (Gaskell, et al., 1985).

The only species susceptible to FHV-l infection is the cat.
Experimental infections of many other mammals have failed to produce
disease or evidence of virus replication (Povey, 1979). Because of its
limited in yiyg and in 21552 host range FHV-l is often compared with
varicella-zoster virus (VZV), the causitive agent of chicken pox in

humans. In contrast, other alpha herpesviruses, such as pseudorabies virus

8

and herpes simplex virus will infect a wide variety of cells in culture
and experimental animals (Roizman, 1980).

Serological studies using polyclonal antisera on a number of FHV-l
isolates from different parts of the world revealed that the virus has
only one serotype. All of the test isolates were serologically identical
to the C-27 prototype strain of FHV-l (Metianu and Viart, 1974). Strains
of modified virulence do exist and several attenuated virus vaccines have
been developed (Slater and York, 1976) . These vaccine strains have no
observable antigenic differences from field isolates based on the
serological methods used. Serological studies have also shown that there
is no cross reactivity between FHV-l and FHV-2 (Fabricant, 1984), herpes
simplex, pseudorabies (Crandell and Weddington, 1967), and infectious
bovine rhinotracheitis virus (Johnson and Thomas, 1966). However, Evermann
et a1. (1982) has reported that antiserum against FHV-l was able to
neutralize the infectivity of canine herpesvirus. The biochemical basis

for this cross reactivity has not been defined.

Biochemical Properties

 

While the biological properties of FHV-l have been studied in great
detail, little is known about the biochemical structure of the virus.
Recently, two groups have reported on the analysis of FHV-l virion
structural components. Maes et a1. (1984) identified at least 17 virion
associated polypeptides on the C-27 strain of FHV-l. Three of these
polypeptides were glycosylated and had approximate molecular weights of
105, 68 and 60 kilodaltons (kd). These glchproteins were present on the
membranes of infected cells and the 105 kd glycoprotein was detected in

the supernatant medium. All three glycoproteins were recognized by a goat

9

anti-FHV-l serum which had a high virus neuralization titer. In another
study, Fargeaud et a1. (1984) used gradient gel analysis to detect 23
polypeptides on virions of the IFFA RTF-CO strain. FITC-labeled Con A was
used to identify 6 glycosylated peptides on FHV-l virions with sizes of
125, 116, 112, 83, 70, and 62 kd. The sizes of the glycoproteins detected
on virions by Fargeaud et a1 generally correspond to the sizes detected by
Maes et a1. with the exception of the 83 kd molecule. It is possible that
this molecule is a breakdown product of one of the larger glycoproteins or
represents an antigen unique to the strain studied. It is unclear, as to
whether the largest glycoprotein band or bands identified in both studies
(125-105 kd) contains only one or a mixture of several different
glycoproteins. Often, variations in glycosylation cause individual
glycoproteins to migrate as more than one band in SDS-PAGE analysis.
Further studies using monospecific or monoclonal antibodies directed
against individual viral antigens will be needed to construct a more
complete antigenic profile of FHV-l. Generally, the number and size
distribution of FHV-l virion proteins and glycoproteins is consistent with
the antigenic profiles of other herpesviruses (Spear, 1976).

The DNA of FHV-l has been found to have a density of 1.705 g/cm3
corresponding to a G+C content of 46% (Roizman, 1980). Only one report has
been published on the molecular biology of FHV-l (Hermann et al., 1984).
In this study, restriction enzyme digestion patterns of DNA from a number
of FHV-l clinical isolates and vaccine strains from Europe, the United
Kingdom and the United States were compared. The results showed that there
was little difference in the restriction digestion patterns of the DNA
from various isolates. The restriction patterns of DNA from virus isolated

from tissues other than the upper respiratory tract were not different

10

from the digestion patterns observed for DNA from respiratory isolates.
A130, no major differences were found between the restriction patterns of
DNA from field isolates and DNA from a temperature sensitive vaccine
strain. The restriction patterns of DNA from a trigeminal ganglion isolate
showed no differences when compared to DNA from the original input virus
suggesting that the virus is not altered during latency. The results of
this study indicated that FHV-l has a very stable genome and this is
another characteristic which it shares with VZV (Martin et al., 1982).
It appears that it may be difficult to differentiate between various
strains of FHV-l based on restriction endonuclease analysis as is possible
with other herpesvirsus such as bovine herpesvirus (Engels et al., 1981)
and pseudorabies virus (Paul et al., 1982). However, more studies on the
molecular epidemiology of FHV-l need to be conducted using a variety of
restriction enzymes on a large number of isolates.

From the results of restriction enzyme analysis of Hermann et a1.
(1984), the size of the FHV-l genome was estimated to be approximately 80
megadaltons. It is obvious that further basic research is needed to

characterize the physical structure of the FHV-l genome.

The Disease

FHV-l occurs on a worldwide basis and 50-70% of cats in Europe, North
America and Asia have virus-neutralizing antibodies to FHV-l (Povey and
Johnson, 1971, Ellis, 1981). The clinical syndrome produced in cats
infected with FHV-l was recently reviewed by Gaskell and Goddard (1984).
Upper respiratory tract disease in cats is generally uniform and the
clinical signs include depression, sneezing, fever, and copious ocular and

nasal discharge. Conjunctivitis may also develop. The severity of the

11

clinical signs and the length of the incubation period ( usually 2-6 days)
depend on the infecting dose (Gaskell and Povey, 1979). The disease is
usually resolved within 10-20 days but may become chronic in some cats.
The mortality rates are high among kittens and debilitated cats which may
develop a generalized infection (Gaskell and Goddard, 1984). Less common
clinical signs include skin ulcerations and nervous system disorders
(Flecknell et al., 1979).

Cats are highly susceptible to FHV-l infection and the virus is
naturally spread by direct contact via the oral, intranasal and
conjunctival route. The intranasal route is most often used to induce
experimental infections. Congential infection of kittens following vaginal
instillation of virus has also been demonstrated (Bittle and Peckham,
1971). Abortions associated with FHV-l, however, are most likely secondary
effects of the severe debilitation caused by the upper respiratory disease
since virus was not isolated from aborted fetuses (Hoover and Griesemer,
1971).

During the acute phase of the disease, virus can be readily isolated
from the nasopharynx, tonsils and upper trachea at titers of up to 106
TCIDso/ml. Only rarely has FHV-l been found in the serum (Gaskell and
Povey, 1979).

FHV-l infection has more severe osteolytic effects in kittens which
often suffer necrosis of the long bones in addition to the necrosis of the
turbinate bones which frequently occurs as a result of infections in adult
cats (Povey, 1979).

Despite its susceptibility to the environment, FHV-l remains the most
significant viral upper respiratory tract pathogen of cats. Indirect

transmission through contact with infected material probably only occurs

12

in areas where with high cat density such as boarding catteries (Gaskell
and Goddard, 1984). The majority of viral spread occurs as a result of
close contact bewteen animals. Like for many other herpesviruses, the
successfull persistence of FHV-l in the cat population is partially
dependent upon the ability of the virus to establish latent infections.
Susceptible cats are infected horizontally either by virus shed from cats
with acute disease or by asymptomatic carrier cats which are shedding
virus from a reactivated latent infection. Cats with latent FHV-l
infection shed virus intermittently (Gaskell and Povey, 1977) and this
shedding is not always accompanied by a recrudescence of clinical signs
(Gaskell and Goddard, 1984). During such a reactivation phase, virus can
be isolated from the oro-phanyngeal secretions though at lower titers
(10S TCIDso/ml) than during acute disease (Gaskell and Goddard,

1984).

FHV-l Latency

 

The presence of latent FHV-l infections has been demonstrated only in
a very preliminary way. Latent FHV-l virus has been experimentally
reactivated in 2132 following administration of corticosteroids. A recent
study (Ellis, 1981) revealed that virus could be reactivated from 25.8% of
healthly cats by this method. Reactivation can also be triggered by stress
and experimenatlly initiated rehousing stress was sufficient to induce
virus shedding in 18% of carrier cats (Gaskell and Povey, 1982). In this
same study, corticosteroid treatment was able to induce reactivation in
64% of these carrier cats. Approximately 40% of latently infected queens
shed virus during the post partum period. Though none of the kittens

examined in this study developed clinical signs, 50% developed latent

13

infections. Since there was no evidence for in 25352 infection, it appears
that the kittens were infected by virus shed by the queen during the
immediate post partum period and that passive antibody was able to prevent
the disease in the kittens but not the establishment of a latent
infection.

Current research efforts have focused on attempting to identify the
anatomical site of FHV-l latency. Since herpesviruses frequently establish
latent infections in neural tissue (Hill, 1985), these tissues were the
first to be examined for FHV-l latency. Gaskell and Povey (1979a) were
able to isolate FHV-l from homogenized trigeminal ganglia or olfactory
bulbs (Gaskell and Goddard, 1984) of shedding carrier cats. However, Ellis
(1982) examined a number of tissues, including trigeminal ganglia, from
carrier cats using homogenization, explant and co-cultivation techniques
and was unable to recover FHV-l from any of these tissues even though the
cats were shedding at the time of sampling. More recently, Gaskell et a1.
(1985) have been able to recover FHV-l from the trigeminal ganglia of 3 of
17 cats latently infected with field virus using a tissue fragment culture
technique. Interestingly, these nervous tissue isolates had plaque
morphologies similar to a vaccine strain of FHV-l and not the input virus.
Though similar results have been reported for bovine herpesvirus
(Nettleton et al., 1984), Gaskell's study was complicated by the fact that
the cats from which the virus was isolated were being treated with BVdU
((E)-S-(2-bromovinyl)-2'—deoxyuridine).

The inability to consistently detect latent FHV-l by explantation or
co-cultivation techniques may reflect a deficiency of these methods to
recover FHV-l rather than an absence of virus in the explanted tissue.

Only a small proportion of the cells in a tissue sample may contain virus

14
and the amount of virus shed from these tissues during reactivation may be
very small. This low virus yield combined with the added problem of the
extreme labilty of FHV-l may result in levels of virus that are below the
detection limits of a cell culture system. More sensitive techniques such
as in sign hybridization with nucleic acid probes need to be done on a
variety of tissues from carrier cats before the site of FHV-l latency can

be unequivocally determined.

FHV-l Vaccines

Because of the prevalence and severity of FHV-l infections in cats, a
large effort has been directed towards the development of vaccines. One of
the most useful attenuated vaccines was developed by Slater and York
(1976) by serial low temperature passage (25°C) of FHV-l in CRFK cells.
Cats intranasally exposed to vaccine virus at the 171th passage level did
not develop clinical signs and were clinically protected from challenge
with a virulent strain of the virus. This vaccine also gave protection
when administered by the intramuscular route. Later work (Orr et al.,
1978) revealed that cats given the same type of vaccine by the
intramuscular route did not develop clinical signs but replicated
challenge virus and a proportion of them became carriers. In contrast,
when the vaccine was administered via the intranasal route (Orr et al.,
1980), there was little replication of a virulent challenge virus given 5
days post-vaccination. In addition, virus was not recovered from the
intranasally vaccinated, challenged cats after corticosteroid treatment.
Though only a small number of cats were tested for latent infection, the
results of this study indicated that vaccine given by the intranasal route

was able to prevent the disease and, more importantly, may have prevented

15
the cats from becoming carriers. This work also suggested that local
immunity which was elicited by antigen stimulation of mucosal surfaces may
have an important influence in determining the outcome of a herpesvirus

infection and the frequency of latent infection in the natural host.

Immunity

Researchers in the United Kingdom have attempted to examine the
nature of the immune response elicited by FHV-l in cats. Challenge studies
had shown that full protection was not achieved with an intranasal vaccine
until 6 days post-vaccination (Cocker et al., 1984a). Later work was aimed
at characterization of this response (Cocker et al., 1984b). Even though
vaccinated cats were resistant to FHV-l challenge by 6 days
pest-vaccination, analysis of the serum and nasal secretions of these cats
revealed only low levels of neutralizing antibodies and interferon. This
resistance was specific for FHV-l since FHV-l-vaccinated cats challenged
with feline calicivirus were infected and developed clinical signs.
Lymphocytes from both blood and tonsil showed no proliferative response to
FHV-l antigens. It was concluded that "a local cytotoxic cell" response in
the tonsil, an important site in primary virus replication (Gaskell and
Povey, 1979a), was able to prevent viral dissemination and was responsible
for the observed protection against FHV-l. Tonsils have been shown by
other workers to be important sites of local immunity against infectious
agents (Drucker et al., 1979).

In a study of the mechanisms involved in immunity to herpesviruses,
Wardley et al. (1976) demonstrated that during the acute phase of the

disease the spread of FHV-l was limited by antibody-complement mediated

16

lysis and antibody directed cellular cytotoxicity (ADCC) mediated by both
lymphocytes and macrophages. These responses were detectable by 6 hours
post-infection when intracellular spread of the virus begins. Direct
cytotoxicity was also noted but the responses were variable and did not
occur early enough in the infection to limit viral spread. These workers
postulated that defects in the immune responses needed to control viral
dissemination within the host may lead to the establishment of latent
infections and to more severe recrudescent disease.

A similar study by Goddard and Gaskell (1984) attempted to evaluate
immune function in cats during reactivation of latent FHV-l infections. A
natural stimulus, rehousing stress, was used to induce reactivation. No
significant suppression of specific or non-specific immunity was
associated with FHV-l reactivation and subsequent shedding. In the shedder
cats, reactivation served to boost specific immunity. The shedder cats
could be differentiated from the non-shedders in that the shedders had a
lower baseline cytotoxic antibody levels, a greater stress response to
rehousing and had experienced more severe primary disease episode than
non-shedders. These results indicate that specific resistance mechanisms
may not be important in controlling herpesvirus reactivation.

It has been noted in the study of other herpesvirus infections that
the levels of effector functions may be more important than memory
functions. For example, in HSV systems, levels of lymphokines and
interferon changed markedly during virus reactivation (Sheridan et al.,

1982). Such parameters still need to be examined in FHV-l infections.

l7

FHV-l as a Natural Host Model

 

It is important to define all of the immune responses that affect the
outcome of a herpesvirus infection (Nahmias and Coleman, 1984). Frequently
there are diffuculties in establishing the validity of observation made in
experimental animals. Therefore, there is great interest in developing
suitable natural host models for herpesvirus infections. These systems
could be used to fully evaluate all phases of the viral infection process
including the establishment, maintenance and reactivation of latent
infections. Also, the ability of candidate vaccines and vaccine delivery
procedures to prevent clinical disease and latent infections could be
tested in these systems. In the near future, it will be especially
important to test the effectiveness of immunity induced by herpesvirus
vaccines containing isolated viral antigens produced via recombinant DNA
techniques in natural host systems. For example, a recombinant
glycoprotein vaccine against pseudorabies produced protective immunity in
mice but not in pigs (Robbins et al., 1984).

At this time, the major drawback to using the FHV-l infection of the
cat as a natural host model is a lack of information about the system.
This can only be remedied by continuing research efforts. It will be
important to have a detailed understanding of the biochemical and
antigenic structure of FHV-l. Fortunately the methodology for completing
these studies has been well established by other herpesvirus workers.

One of the major advantages to using FHV-l as a natural host model is
that reactivation of latent infections can be induced by natural,
non-pharmacological means. This would allow for an accurate evaluation of
immune function in cats during the reactivation process and would be

completed without the complications of general immunosuppression caused by

l8

corticosteroid treatment. Hopefully, this system will allow the molecular
biological events responsible for the induction of virus reactivation in a
natural host to be defined as well. Finally, FHV-l causes widespread
disease in cats. Therefore, any novel vaccine strategies can be tested in
a large, naturally susceptible population. An added incentive is that
these novel vaccines will have obvious commercial potential.

A few similarities can be found beteween FHV-l infections of cats and
HSV infections of humans. Both viruses cause severe disease in newborns
exposed when the virus is shed from a reactivated infection in the
mother. Both viruses also have been shown to cause conjunctivitis and
severe encephalitis in newborns (Orr, 1983). FHV-l may therefore be a good
model for research on neonatal herpesvirus infections.

The first evidence for interest in FHV-l as a natural host model for
human herpesvirus infections is provided by a very recent paper in which
FHV-l was used to study inner ear infections (Falser, 1984). This is
interesting since inner ear infections in humans are sometimes caused by

reactivation of latent herpesvirus infections (Davis et a1. , 1981).

Part II. The Herpesviruses: An Overview

Introduction

The purpose of the following section is to provide a general
introduction to selected characteristics of herpesviruses. The topics
covered are those which will be examined for FHV-l in this thesis.

Treatment of these topics can not be exhaustive because of the

19

considerable amount of research that has been published about
herpesviruses. Rather, important principles will be illustrated using
examples from well characterized herpesvirus systems such.

The herpesviruses include a vast family of DNA viruses which infect a
large number of eucaryotic species. Herpesviruses have been observed to
infect fungi, fish, oysters, amphibians, birds and many mammals including
most domestic animals, monkeys and humans. The common features of the
herpesviruses are: identical virion construction, a large double-stranded
DNA genome, and a regulated reproduction cycle (Feldman, 1980). Another
important property of this virus group is their ability to persist in the
natural host for long periods of time after primary infection. These
latent asymptomatic infections can periodically reactivate allowing virus
to spread horizontally through direct contact with the individual in which
reactivation occurred.

Herpesviruses have been divided into three subfamilies based on their
biological properties (Honess and Watson, 1977). FHV-l along with
pseudorabies virus (PRV), herpes simplex virus (HSV) and varicella-zoster
virus (VZV) belong to the alpha herpesviruses. Viruses in this subfamily
cause self-limiting skin or upper respiratory tract infections. The site
of virus persistence is usually the central nervous system. Beta
herpesviruses are more commonly referred to as the cytomegaloviruses. They
cause little overt disease except in neonates or immunocompromised adults.
The gamma herpesviruses are composed of lymphotropic viruses such as
Epstein-Barr virus and Marek's disease virus. Members of this group cause
lymphoproliferative diseases and can persist in lymphocytes. They may

cause fatal leukemias and lymphomas.

20

Morphological Characteristics

 

Early electron microscopic studies (Wildy, 1960) revealed that
herpesvirions contained linear double-stranded DNA packaged within a
hexagonal capsid composed of 162 capsomeres. This nucleocapsid is
surrounded by tegument proteins which are contained within a lipid
envelope (Wildy, 1971). Later, electrophoretic analysis showed that HSV
had a total of 25-30 peptides present in the virion. As much as 40% of
virion protein was found to be part of the viral envelope (Kaplan and
Ben-Porat, 1970). Many of the envelope proteins are glycoproteins (Choppin
and Scheid, 1980, Sormiento et al., 1979), and have important functions in
viral infectivity. These molecules are involved in the attachment to and
penetration of host cell membranes (Spear, 1979). In addition, because
glycoproteins are expressed on the surface of the infected cell, they are
important targets of the host's cellular and humoral immune response to
viral infection. For this reason, there is a great deal of interest in
developing glycoprotein vaccines against herpesviruses by various
biochemical and molecular biological techniques. The most advanced work in
this regard has centered on the use of glycoprotein D (gD) as a vaccine
for herpes simplex infections in humans (Watson et al., 1982).

Herpesvirus capsid proteins can be purified from the virion envelope
for biochemical analysis. Herpesviruses have one major capsid protein with
a molecular weight between 110 and 150 kilodaltons. In HSV and PRV the
capsid comprises 28% of total virion protein (Courtney et al., 1971).

Contained within the capsid are core proteins which are phosphorylated
and associate with the viral DNA. These proteins are unstable and
diffucult to purify once the capsid has been removed. HSV has been found

to contain two core proteins (Olshevshi and Becker, 1970).

21
The capsid of HSV is surrounded by a series of eight globular proteins
referred to as tegument proteins (Roizman, 1980). The function of these

molecules is not well defined.

Viral DNA

Herpesvirus DNA is associated with the core proteins and located
within the viral capsid. The physical properties of many herpesvirus DNA’s
have been determined using modern molecular biological techniques. The
entire genomes of many herpesviruses have been cloned in plasmid or
bacteriophage vectors. The availability of cloned viral DNA has allowed
detailed restriction endonuclease cleavage maps and extensive DNA sequence
data to be obtained for a number of important herpesviruses (Sullivan et
al., 1984, Davison and Scott, 1983, Mayfield et al., 1983, Spector et al.,
1982, Henry et al., 1981, Goldin et al., 1981, Hayward et al., 1975).

The G+C content of herpesvirus DNAs, as determined by isopycnic
density gradient centrifugation, has been shown to vary considerably.
Values range from a low of 33% for canine herpesvirus to a high of 77% for
monkey B-virus DNA (Roizman, 1980). The molecular weights of the genomes
of many herpesviruses have been determined by equilibrium sedimentation,
electron microscopy, and restriction enzyme analysis. These values range
from approximately 120 kilobase pairs (kb) for channel catfish virus DNA
(Honess and Watson, 1977) to 230 kb for cytomegalovirus DNA (DeHarchi et
al., 1977).

Varying amounts of genetic relatedness have been found between the
genomes of various herpesviruses. HSV 1 and 2 share 50% homology (Feldman,
1980). Marek's disease virus has been found to have extensive homology

with the vaccine virus, herpesvirus of turkeys, when lower stringency

22
hybridization conditions are used (Gibbs, et a1, 1984). Limited regions of
homology have been noted between the DNA's of other herpesviruses. Davison
and Wilkie (1983) used molecular hybridization to identify regions of
homology between the genomes of HSV-1, HSV-2, PRV, VZV, and equine
herpesvirus (EHV). Their results indicated that herpesvirus genes are
highly conserved and that their organization on the viral genome is
similar. This homology has been useful in gene mapping studies. For
example, a glycoprotein gene on the pesudorabies virus genome was recently
identified by hybridization with a HSV DNA probe containing the HSV
glycoprotein B gene (Robbins et al., 1985).

Herpesvirus genomes were placed into three classes depending upon the
arrangement of unique and repeat DNA sequences on the molecule (Ben-Porat
and Kaplan, 1982). These arrangements are shown in Figure 1. HSV has a
class 3 genome which contains two sets of inverted repeats. One set of
repeats brackets a long unique sequence and the other brackets a short
unique region. In this arrangent, both the long and short unique regions
can invert relative to each other giving rise to a genome with with four
possible isomeric forms. PRV, VZV, and equine herpesvirus-l have class 2
genomes. In this configuration, only the unique short region is bracketed
by inverted repeats. The short region can invert relative to the long
producing two possible genomic isomers. Class 1 genomes are found in
Epstein-Barr virus and channel catfish virus. These molecules contain long
and short unique DNA regions interspersed with repeat regions. These
repeats are not in the inverted orientation and genomes with this
structure have only one isomer. The location and arrangement of the repeat
sequences within a herpesvirus genome can be determined by electron

microscopy (Ruyechan et al., 1983) of denatured and reannealed DNA

23

Figure 1. Organization of the genomes of herpesviruses. Us and U1
designate the unique short and unique long sequences, respectively. Repeat
sequences are designated by rectangles; for repeats with inverted
complementarity rectangles are cross-hatched. Arrows indicate possible

orientations of repeat sequences. (adapted from: Ben-Porat, 1982)

 

 

24

 

 

1“!

 

_: I. Class 1

—’-
‘———-

 

Ul

 

' Class 3
Us

—-)
‘—

 

01

Figure 1.

Us

25
molecules and by restriction endonuclease digestion and blot hybridization
studies (Sullivan et al., 1984, Henry et al., 1981).

Recent studies have indicated that some herpesviruses might have
genome structures belonging to more than one class. A vaccine strain of
PRV has been shown to have inverted repeats bracketing the long unique
region as well as the short (Lominiczi et al., 1984a). This strain has a
four isomer genome. Kinchington et al., (1985) found that a small
population of VZV genomes have inverted long unique regions. It will be
interesting to determine if such novel genomic structures exist in other
herpesviruses.

Although the issue is still being debated, most studies with HSV and
PRV have shown that the different isomers of the genome are functionally
equivalent (Roizman, 1979, Ben-Porat and Kaplan, 1985). Virions containing
the different isomers were isolated from infected cells in equimolar
amounts. Each isomer of HSV and PRV has been shown to be infectious since
infection of cells with a single isomer yields progeny virus with
equimolar amounts of the possible isomers.

Two models for isomerization of PRV based on crossing over between
different viral DNA molecules have been proposed by Ihara et al., (1982)

and Ben-Porat (1982). These models are diagrammed in Figure 2.

Genetic Variability

The molecular epidemiology of herpesvirus infections has been studied
using restriction endonuclease digestion analysis. This method is useful
to accurately trace the source and monitor the spread of infections as
well as provide a measure of genetic variability in herpesviruses.

Varying amounts of restriction fragment heterogeneity have been found

26

Figure 2. Models for equalization of repeats in herpesviruses. Crosses
indicate sites of recombination. Rectangles with and without

cross-hatching indicate inverted repeats. (adapted from: Wagner, 1985)

 

 

27

 

 

 

 

 

Figure 2.

28

between different isolates of a given herpesvirus. For example, VZV has a
very stable genome and restriction digest analysis of DNA from a number of
strains revealed very similar fragment patterns (Martin et al., 1982,
Richards et al., 1979). The genomes of PRV (Paul at al., 1982) and HSV
(Buchman et al., 1980) contain more variability between isolates. Most of
the variability is limited to size differences in the restriction
fragments which map within the inverted repeat regions of the genome
(Ben-Porat and Kaplan, 1985, Roizman, 1979) rather than a gain or loss of
partcular restriction sites. The genome of herpes saimiri has been shown
to be even more variable than the viruses mentioned above (Desrosiers et
al., 1982).

Restriction endonuclease digestion analysis of herpesvirus DNA has
been used with varying amounts of success to evaulate the pathogenicity of
various isolates and to map virulence determinants. A deletion in the
unique short region of the genome of avirulent vaccine strains of PRV can
be identified by restriction digest analysis (Lomniczi et al., 1984a).
This deletion maps to a site which codes for a major glycoprotein antigen
(Mettenleiter et al., 1985). However, further studies (Lomniczi et al.,
1984b) revealed that attenuation in PRV is a multigenic event and that
these vaccine strains had genetic alterations in other regions of the
genome that were not evident by restriction digestion analysis and could
only be detected using complementation analysis.

However, in some cases herpesvirus strains of varying pathogenicity
can be identified using restriction digest analysis. For example,
consistent differences between infectious bovine rhinotracheitis (severe
infections including abortions) and infectious pustular vulvovaginitis

(harmless infections) strains of bovine herpesvirus-l have been detected

29

by restriction enzyme analysis (Engels, 1981) even though these strains
appear anitgenically identical. In a similar study of this type, fetal and
respiratory isolates of equine herpesvirus were differeniated based on

restriction digest patterns (Studdert et al., 1981).

Herpesvirus Gene Mapping

 

A wide variety of techniques has been used to determine the locations
of herpesvirus genes. Several of these methods will be discussed briefly
in this section.

HSV 1 and HSV 2 can be differentiated by restriction endonuclease
digestion analysis and by the SDS-PAGE migration patterns of viral
glycoproteins. Ruyechan et al. (1979) were able to use this information to
map the locations of several glycoproteins by analyzing the DNA and
glycoproteins of intertypic recombinants between HSV l and 2.

Gene mapping studies in HSV were facilitated by the availibility of
cloned restriction fragments of the viral genome. These fragments were
used to specifically select infected cell RNA which hybridized to defined
regions of viral DNA. Lee et al. (1982) were able to map HSV gD and gE
genes within the S region of the genome by translating selected RNA i9
yiggg and analyzing the translation products in immunoprecipitation assays
with monoclonal antibodies directed against the glycoproteins. Anderson et
a1. (1980) purified individual mRNA species by a combination of
hybrid-selection and gel electrophoresis and, by translating these RNAs i2
yiggg, were able to precisely map HSV immediate early genes.

Other methods of gene mapping use the biological activity of the gene
product of interest as an assay. The alkaline exonuclease of HSV was

mapped by microinjecting hybrid-selected infected cell RNA or cloned viral

30
DNA into Xenopus oocytes (Preston and Cordingley, 1982). The injected
oocytes were then assayed for exonuclease activity. The HSV thymidine
kinase gene was mapped by both microinjection of oocytes (McKnight and
Gavis, 1980), as described above, and by transfection assays (Wigler et
al, 1977). In the transfection assays, cloned restriction fragments were
tested for their ability to restore tk activity to tk- cells or used in
marker rescue experiments with tk- mutant viruses.

Recently, herpesvirus researchers have coupled the availability of a
large battery of monoclonal and monospecific antibodies with the use of a
number of procaryotic expression vectors containing either random viral
DNA fragments or viral cDNA to map viral genes. To map the g0 gene of VZV
to the Us region of the viral genome, Ellis et a1. (1985) expressed
randomly generated DNA fragments from a VZV plasmid clone library in an
open reading frame (ORF) vector. Clones expressing the glycoprotein were
identified using a monoclonal antibody and gene identity was confirmed by
demonstrating that DNA from the positive clones could select RNA which
coded for gC. In similar studies, a bacteriophage expression vector,
!lgtll (Young and Davis, 1983), was used to map genes of CMV (Mocarski et
al. 1985) and PRV (Petrovskis et a1. 1985). An interestng aspect of the
latter study was that only antibody prepared against denatured antigens
was able to detect the expressed peptides.

Gene mapping strategies often employ a combination of these
techniques (Rea et al., 1985). For example, the gene for a PRV
glycoprotein that is found in the cell culture medium was mapped to a
specific restriction fragment by translating hybrid-selected RNA in the
presence and absence of a dog pancreas microsome extract. Peptides

requiring futher processing, such as glycosylation, were identified by

31
comparing the translation products from the two reactions. The restriction
fragment was then expressed in an ORF vector and antibody to the expressed
peptide was prepared in mice. This antibody was used in

immunoprecipitation studies to confirm the gene identification.

Herpesvirus Gene Expression and Regulation

 

Transcription in herpesviruses has recently been reviewed by Wagner
(1985). In general, herpesvirus mRNA's appear to be unspliced and many
mRNA species are transcribed from overlapping regions of the genome.

Honess and Roizman (1975) placed herpes simplex genes and gene
products into three classes based on the temporal order of their
expression. This "cascade" pattern of regulation is similar in other
herpesviruses. Immediately upon viral entry into the host cell, immediate
early genes are expressed. This expression occurs in the absence of pp
3222 viral protein synthesis. The immediate early gene products function
as positive regulators of early gene expression. The early gene products
inhibit the expression of immediate early genes while stimulating the
expression of late genes. In turn, expression of early genes is inhibited
by the late genes products. To complete the cycle a late gene product .
which is a virion component has been shown to stimulate the expression of
immediate early genes (O'Hare and Hayward, 1984).

The cascade is more complex than originally proposed, however, as some
late genes are expressed at early times of infection but become more
abundant at late times. These genes are referred to as "leaky late" genes

(Spear and Roizman, 1980).

32

Immediate Early Genes

Immediate early genes can be more easily identified since they are
the only viral genes transcribed in the presence of cycloheximide.
Immediate early RNA is made up of only a few species. These all map in a
limited area of the viral genome. The immediate early genes of HSV and PRV
are clustered within the inverted repeat sequences (Wagner, 1985). In HSV,
5 immediate early peptides with molecular weights of 175, 110, 68, 64, and
12 kd have been identified (Wagner, 1985) while in PRV only a single
peptide with a molecular weight of 180 kd was found (Ihara et al., 1983).

Immediate early peptides are often phosphorylated and though their
function is still unclear, these peptides appear to function in the shut
off of host cell protein synthesis through a mechanism that is not
understood. HSV immediate early proteins have DNA binding activity
(Freeman and Powell, 1982) and have been shown to effect the expression of
heat shock peptides within the infected cell (Notarianni and Preston,
1982). Immediate early proteins may also function in the regulation of
latency. Expression of HSV immediate early genes has been found in
latently infected tissues from experimental animals (Green et al., 1981)
but not in human tissues (Galloway et al., 1982). Recently, a model was
proposed by O'Hare and Hayward (1985) in which HSV immediate early genes
function to regulate their own expression as well as early gene

expression.

Early and Late Genes

 

The early genes map throughout the genome but only a limited number
of mRNA species are recognized. Early gene products function primarily in

viral DNA replication. In HSV early genes have been found to code for a

33

thymidine kinase, alkaline exonuclease, DNA binding proteins, DNA
polymerase, ribonucleotide reductase and deoxypyrimidine triphosphatase
(Wagner, 1985).

"Leaky late" gene expression is detectable in infected cells in the
absence of viral DNA replication while true late gene expression is
sensitive to inhibitors of viral DNA replication. Both "leaky late" and
true late genes map throughout the viral genome. Many of the late genes
code for structural components of the virion such as the major capsid
antigen and viral envelope glycoproteins (Wagner, 1985).

Figure 3 is a HSV transcript map (Wagner, 1985). This figure is
presented to show the distribution of the three classes of genes on the
HSV genome. There is increasing evidence that herpesviruses with similar
genomic structure may have conserved the locations of important viral
genes (Davis and Wilkie, 1983, Culliane and Davison, 1985). To further
explore colinear genome organization in herpesviruses, the location of

viral genes needs to be determined in a number of different herpesviruses.

34

Figure 3. Location of HSV mRNA species abundant at different stages of
replication. Figure shows size of RNA transcripts and, if known, size of
polypeptide coded for by the RNA transcript. (reproduced with permission

from: Wagner, 1985)

35

 

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7'; g2 '85 "k
mu Matte)
20' ID ‘20 «210 .100
M ms «79“ “211119
haw-'10)
20 42. Q5 «6 133420 52. 10 G7 20 II 3010”)
I5 I21 I“! a no man “I “21 .5 so
I» I. u
no no: .,
Wham—.1370?)
so .11» «o «no: u on «same a is
an nmt¢n s, in 44 can»: eeuutniis
., ounce as» ., 23 u no Aaron
:2. Q! on d: ' I! (II ea: N“
to» as . as IJ‘rflv- n» lane:
2.7. H U 64) 1.1 so:
Hahn ‘
ml?) and M mm . . all go: In
M I _ m W W
m WI. Mm 05(2)
In! an an!
m

Figure 3.

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Research Data

42

Chapter 2

Physical Characterization of the Genome of Feline Herpesvirus-1

Paul A. Rota and Roger R. Maes

43

Abstract

The physical structure of the genome of feline herpesvirus-1, a major
upper respiratory tract pathogen of cats, was studied. Restriction
endonuclease analysis with Bam HI, Eco R1, Hind III, and Sal I indicated
that the size of the genome was approximately 134 kilobase pairs (kb).
Restriction fragments containing the viral termini were identified by
lambda 5' exonuclease digestion of intact viral DNA prior to restriction
endonuclease digestion. Blot hybridizations using the Sal I terminal
fragments as probes indicated that one end of the viral genome contained
both terminal and internal repeat sequences. Electron microscopic analysis
of denatured and reannealed FHV-l DNA revealed a single-stranded loop and
double-stranded stem at only one end of the molecule and confirmed the
presence of inverted repeat sequences. The size of the single-stranded
loop region was determined to be approximatly 8 kb. To facilitate further
studies, nine bacteriophage clones were isolated which contained 85% of
the viral genome as Sal I inserts. These clones were used in blot
hybridization experiments and as substrates for restriction digest
analysis. Data from these studies permitted construction of Sal I and
partial Hind III and Eco R1 restriction maps of the viral genome. FHV-l
DNA is composed of a long (L) and a short (S) segment. The long segment (
U1 ) is 103 kb in size and is composed of unique DNA. The adjacent S
segment is approximately 31 kb in size and contains a central portion of
unique DNA ( Us ) which is approximately 8 kb in size. The Us region is
bounded by inverted repeat sequences which are 11 kb in size. The entire
S region of the genome can invert relative to the L resulting in a genome

with two isomeric forms.

44

Introduction

Feline herpesvirus-l (FHV-l) is a major viral pathogen of cats. In
adult cats, the virus causes upper respiratory tract disease. In kittens,
the infection can generalize resulting in mortality rates of up to 50%.
FHV-l can also cause genital tract infections and has been associated with
abortions (Povey, 1979, Pratt, 1983). Like other herpesviruses, FHV-l can
cause latent infections, and the site of latency appears to be the
trigeminal ganglia (Gaskell et al., 1985). The prevalence and seriousness
of FHV-l have lead to development of both modified-live and inactivated
virus vaccines. While these vaccines can prevent disease, they are unable
to prevent exposed cats from becoming infected (Povey and Wilson, 1978).
Therefore, vaccinated cats still develop latent FHV-l infections, and
periodic reactivation of these latent infections allows the virus to
spread to both vaccinated and non-vaccinated cats (Gaskell and Povey,
1978).

FHV-l has been classified as a member of the subfamily
alphaherpesvirinae. Its host range i2 yiyg is limited to Felidae and i2
yiggg to cells of feline origin. The virus has a short reproductive cycle.
The infection spreads rapidly in cell culture leading to mass destruction
of cells (Roizman, 1980). Most isolates of FHV-l are of uniform
pathogenicity, although strains of modified virulence have been developed
for vaccine use (Slater and York, 1976). Serological studies indicate that
various clinical isolates have similar antigens (Johnson and Thomas,
1966). More recently, restriction enzyme analysis of viral DNA from a

number of clinical isolates has shown that the genome of FHV-l is

45

46
remarkably stable in comparison to that of other herpesviruses (Herrmann
et al., 1984).

The biological properties of FHV-l have been well studied but little
is known about the biochemical structure of this virus. Two recent studies
have characterized the major virion-associated structural molecules and
identified three major antigenic glycoproteins on the virion and the
surface of infected cells (Fargeaud et al., 1984, Maes et al., 1984).

We are interested in studying the molecular biology of FHV-l as a
first step in developing FHV-l as a natural host model for herpesvirus
infections. This report describes an initial characterization of the
physical structure of the FHV-l genome. Various biochemical methods were
used to determine the size and isomeric arrangement of the genome. In
addition, a number of recombinant bacteriophage clones containing most of
the genome as inserts have been isolated. These clones were used in blot
hybridization and double digestion experiments to generate the first

restriction map of the viral genome.

Methods and Materials

Virus and Cells. The C-27 prototype strain of FHV-l was obtained from the

 

American Type Culture Collection, Rockville, MD (#VR 636). The virus was
propagated in Crandell-Rees Feline Kidney (CRFK) cells which were
maintained in Eagle's Minimum Essential Medium (EMEM) (K.C. Biologics,
Lenexa, KS.) with 10% fetal bovine serum (Hyclone defined, Sterile

Systems, Logan, UT).

Isolation of Viral DNA. Ten tissue culture roller bottles (490 cm, Corning

 

Glass Works, Corning, NY) containing confluent monolayers of CRFK cells
were infected with virus at a multiplicity of infection of .01. When the
cytopathic effect was advanced, infected cells were shaken from the
culture vessel surface and pelleted by low speed centrifugation (500 rpm,
10 min). Washed cells were resuspended in 10 ml of TE (lOmM Tris-HCl pH
7.5, 1 mM EDTA) and left on ice for 15 minutes. NP-40 (Sigma, St. Louis,
MO.) was added to a final concentration of .5%, and the cells were
incubated for another 15 minutes on ice. Cell nuclei were pelleted by
centrifugation at 2,000 x g for 10 minutes, and the supernatant fluid
containing the viral nucleocapsids was adjusted to 1% SDS and 25mM EDTA.
Pre-digested (80'C., 15 min.) pronase was added to a final concentration
of 1 mg/ml and the virus preparation was incubated overnight at 37’C.
Saturated sodium iodide (1.5 volumes) containing 1 ug/ml ethidium bromide
was then added and the mixture was centrifuged for 48 hours at 44,000 rpm
at 20’C. in an SW 50.1 Rotor (Beckman Instruments, Palo Alto, CA.).

Viral DNA bands were visualized by u.v. illumination and harvested by

side-puncturing the tube with a 20 ga. needle. DNA-containing samples

47

48
were extracted three times with isoamyl alcohol (5 volumes) before being

dialyzed extensively against TE.

Restriction Enzyme Analysis. Viral DNA was incubated with 10 U/ug of
either Bam HI, Eco Rl, Sal I or Hind III (Bethesda Research Laboratories,
Gaithersburg, MD.) using the reaction conditions recommended by the
manufacturer. When the digestion was complete, the fragments were
separated by agarose gel electrophoresis (20 volts, 20 hours in
Tris-acetate buffer: .O4M Tris-acetate, .002M EDTA) and the fragments were
visualized by ethidium bromide staining and u.v. illumination. In some
experiments, the fragments were end-labeled prior to electrophoresis by
adding 1 uCi of the appropriate a32P-1abeled trinucleotide (Bam HI:
dGTP, Eco R1 and Hind III: dATP, Sal I: dTTP, Amersham, Arlington Heights,
111., sp. act. 800 Ci/mmole) to the reaction along with 1 unit of the
Klenow fragment of DNA polymerase (BRL). Samples were incubated at room
temperature for 45 minutes, heated to 68'C to inactivate the polymerase,
and precipitated twice with ethanol to remove unincorporated label before
being subjected to electrophoresis. When electrophoresis was complete, the
gel was dried on a gel dryer for 45 minutes without heat and exposed to
Kodak X-AR film. To determine fragment molarity, films exposed for various
lengths of time were scanned with a densitometer equipped with an
integrator (Ortec 4310, Oak Ridge, TN).

Double restriction digests with either Sal I and Eco R1 or Sal I and

Hind III were carried out simultaneously in Sal I buffer (BRL).

Southern Blotting. After electrophoresis, restriction fragments were

transferred to nitrocellulose filters by the method of Southern (1975).

49

Filters were prehybridized for 4 hrs at 42°C. in 20% formamide, .6M
NaCl, .O6M sodium citrate, .OlM EDTA, .1% SDS, 5X Denhardt's solution (.1%
ficoll, .l% polyvinylpryollidone, .1% bovine serum albumin), and 50 ug/ml
denatured salmon sperm DNA. Hybridizations were performed at 42°C. in
50% formamide, 1X Denhardt's, 4X SSC (1X SSC: .15M NaCl. .OlSM sodium
citrate, pH 7.0), .lM EDTA, .1% SDS, 50 ug/ml denatured salmon sperm DNA.
After hybridization, filters were washed twice for 15 minutes in 2X SSC at
room temperature followed by two 1 hr washes in .1X SSC at 68°C. The
filters were allowed to dry and exposed to X-ray film.

32P-labeled probes were prepared from either total viral DNA,
total recombinant phage DNA or agarose gel purified restriction fragments
by nick translation using standard methods (Maniatis et al., 1982).
Restriction fragments for use as probes were purified from agarose gels by
electrophoresis onto nitrocellulose filters (NA-45, Schleicher and
Schuell, Keane, NH). Fragments were eluted from the filters by incubation
at 68°C for 25 minutes in TE with 1M NaCl. Recovered fragments were
extracted twice with water-saturated butanol and precipitated twice with
ethanol. Usually, fragments were subjected to a second electrophoretic

purification before being nick translated.

Terminal fragment identification. Identification of fragments containing
the viral termini was done by two methods. First, 1 ug aliquots of viral
DNA were incubated at 37°C. for 10, 20, 30, 40 and 60 minutes with

lambda 5' exonuclease (BRL, 1.5U/ug) in 67 mM glycine pH 9.5, 2.5 mM MgCl.
Reactions were stopped by heating the samples to 65°C. for 15 minutes
followed by extraction with phenolzchloroform: isoamyl alcohol (25:24:1).

After precipitation with ethanol, viral DNA was resuspended in the

50

appropriate restriction enzyme buffer and digested with the enzyme
overnight. Fragments were end-labeled and separated by agarose gel
electrophoresis. Untreated viral DNA was digested, end-labeled and run in
an adjacent lane on the gel as a control.

In the second approach, synthetic Bam HI linkers (BRL) were
end-labeled with gamma32P ATP using polynucleotide kinase and ligated
to intact viral DNA using the methods described by Spector et al.(1982).
Unattached linkers were removed from the DNA by electrophoresis through
.8% low melting point agarose (Seakem, FMC, Rockland, ME). The viral DNA
band was visualized by staining the gel with ethidium bromide and the DNA
was recovered by melting the excised band in TE at 65°C. followed by
repeated phenolzchloroform extractions and ethanol precipitation. Labeled
DNA was digested with restriction enzymes and the fragments were resolved
by agarose gel electrophoresis. After electrophoresis, gels were stained
with ethidium bromide, and photographic records were made. Gels were
dried as described above and exposed to X-ray film. End fragments were
identified by measuring the distance migrated by the labeled fragments and

comparing the measurements to the photographic record.

EMBL-3 cloning. EMBL-3 DNA was prepared from a phage stock (gift of Paul

 

Bates and Dr. Jerry Dodgson, MSU Dept. of Microbiology) by the methods
described in Maniatis et al. (1982). Sal I cut FHV-l DNA (2ug) was ligated
to Sal I cut EMBL-3 DNA (lug) at 16°C. overnight in 66mM Tris pH 7.2, 10
mM MgCl, 10 mM DTT, 1 mM ATP. Recombinant phage DNA was packaged i9 yiggg
according to the method described by Scalenghe et a1 (1978). Phage were
titered on both 0358 and Q359 strains of E. 921i to determine the number

of recombinant phage. Libraries of recombinant phage were plate amplified

51

on Q359. Libraries were screened by picking individual, well isolated
plaques growing on a lawn of Q359 and placing them into .5 m1 of SM (.lM
NaCl, .2% MgSO4, SOmM Tris-HCl pH 7.5, .01% gelatin). Phage from 1/2 to
1/3 of a resuspended plaque were amplified in 2 ml LB broth cultures and
phage DNA was purified using the small scale procedure described by Leder,
et al (1977).

For 19 gigg plaque hybridizations, plaques were transferred to
nitrocellulose filters (Millipore, HATF, Bedford, MA) and screened with

nick-translated DNA probes as described by Benton and Davis (1977).

Electron Microscopy. Electron microscopic analysis of FHV-l DNA was
performed using the conditions previously described by Ruyechan et a1

(1982).

Results

Restriction enzyme analysis. FHV-l DNA was purified as described above

 

and digested to completion with either Bam HI, Eco RI, Hind III or Sal I.
The end-labeled fragments were separated on agarose gels. After
electrophoresis, gels were dried and exposed to X-ray film. Lambda phage
DNA, digested with either Eco R1, Hind III or Sal I, was run in adjacent
lanes on the gels to provide size markers. Since fragments were labeled
according to their molar concentration in the digest and not their size, a
large number of fragments could be resolved on a single gel. Figure 1
shows that Bam HI, Eco R1, Hind III or Sal I digestion of FHV-l DNA
produced a number of fragments with sizes ranging from approximately 30 kb
to less than .5 kb. The size of each restriction fragment, based on the
average measurements from three different gels, is given in Table 1. Sizes
of smaller fragments were determined on high concentration gels (1.0% and
1.4%), while sizes of larger fragments were based on measurements from
gels of lower concentrations (.5% and .7%).

The molarity of each fragment was determined by scanning
autoradiograms with a spectrodensitometer equipped with an integrator.
Films exposed for varying lengths of time were scanned to ensure linearity
of the film. Figure 2 shows the results of a typical scan of each digest
after electrophoresis on a .8% agarose gel. Molarity of the smaller
fragments was determined by scanning gels of higher concentration (1.0%
and 1.4%). The molarity of each fragment, based on the area under the
peak, is shown in Table 1. Scanning results showed the presence of only

one , two or three molar fragments.

52

53

Figure 1. Restriction endonuclease digestion patterns of FHV-l DNA. FHV-l
DNA was digested with either Bam HI, Hind III, Eco R1 or Sal I and the
fragments were end-labeled and separated by agarose gel electrophoresis.
Hind III digest fragments of lambda DNA with sizes of 23.3, 9.4, 6.6,

4.3, 2.2 and 2.0 kilobase pairs are shown as size markers.

54

H Em
H1 08
a 05:

HI Sum
6356..

 

Figure 1

55

Figure 2. FHV-l DNA restriction fragment molarity determination. Figure
shows typical densitometer scans of autoradiograms of dried .8% agarose
gels conmtaining digested and end-labeled FHV-l DNA. Area under each peak

was used to determine fragment molarity.

56a

Hindiflfl

Ben tiI

 

 

 

 

Figure 2 (continued on next page)

 

 

 

 

56b

0

be
r .’._:“ 3°

.c-o. NA ‘03 a-
o

<

 

Sol I

 

‘90
O
0

' it?

 

 

V"

Figure 2 (continued)

57

Table 1. Restriction enzyme digestion patterns of FHV-l DNA

 

Sal I
mol.
wt.

 

M

mol.

 

Eco R1
mol. M

Hind III
wt.

mol.

Bam HI

(kb) wt. (kb) (kb)

wt. (kb)

 

1.211111111111111

AU.J.U.8.4,o.U.3.8.U.9.4,o.I,o.4
0,5O13nvnvo,o.7.Aussqsoz1i
1.1.1.1111

no

9 fl. *
Annunuwu nuuuriruvaruuununvplnx

11111112111121111121111

.D.U_/.Dnzro.4,o.I.J.U.J.I.J.I,onz.i.i,o.8.5.5
4.9.0.0,nv7.7.avfi.§.h.h.%.1.9.9.9.9.9.11
1.1.1.

* T. n. * V.
AnnununumuP.G.“MIGRATEMANEDInwnacngnunwvAVIqa

12112111111311.1111

052087976526274754

1rO.I4~/.5/.m333332211
311 P

*C G * 0
ABDEFHIJKLMNQRSTUV

211111312111122111121111111

585316285397418766540988754
409876544322221111111

.J

R. T. Mm ewnu Au*

""""

AmCDEFGEKLNOPQR’TVWXYZBCDEFGH

 

133.1

134.4

134.1

133.3

Total:

Average molecular weight: 133.7 :.6

M - fragment molarity

* denotes end fragment,

 

58

To determine the size of the genome, the sizes of restriction
fragments in each digest were totalled and adjustments were made for
fragment molarity. Based on the average of the total fragment size for the
four digestions, the size of the FHV-l genome was determined to be 133.7

kb (Table 1).

Terminal fragment identification. FHV-l DNA was digested with lambda 5'
exonuclease for varying amounts of time and subsequently digested with
either Bam HI, Eco R1, Hind III or Sal I, end-labeled and electrophoresed
with non-exonuclease-treated, digested DNA. Terminal fragments showed
altered mobility compared to untreated controls. Figure 3 shows that Bam
HI fragments K and B', Eco R1 B and Q, and Sal F and L have altered
mobility after exonuclease treatment and presumably are located at the
ends of the viral genome. Only one Hind III terminal fragment, Hind III A,
was observed. Identification of Bam HI, Eco R1 and Sal I termini were
confirmed using kinased synthetic linkers to label the ends of the genome
(data not shown).

To determine which of the terminal fragments were colinear, Sal I
termini F and L were purified from agarose gels and used to probe Eco R1
and Hind III cut FHV-l DNA that had been transferred to nitrocellulose
filters. Figure 4 shows that Sal I L hybridized to Eco RI J, Q and U or V
and to Hind III A. Sal I F hybridized to Eco R1 B and to Hind III E and J.
The faint bands visible when Sal I F was used as a probe were due to
contamination with Sal I E which migrates very close to Sal I F and is
difficult to remove in electrophoretic separations (for the hybridization
patterns of Sal I E to Hind III fragments of FHV-l DNA, see Figure 8).

Hybridization of the Sal I F probe to Sal I digest of FHV-l DNA showed

59

Figure 3. Terminal restriction fragment identification. FHV-l DNA was
treated with lambda 5' exonuclease for 0, 10, 20, 30, or 40 minutes before
being digested with restriction enzymes, end-labeled and subject to
agarose gel electrophoresis on .7% or 1.4% gels. Fragments showing altered

migration patterns after exonuclease treatment are noted.

Bam HI

0 1O 20 30
.mm - o—

‘ '»

---
I4
a»
-e-

f

[IE-1|
!

4K

60

Hind III
A?

O_20>

 

Figure 3

Sal I

MM'
3 .

9 2°49

Eco R1
0 20 4o

61

Figure 4. Blot hybridization patterns of FHV-l Sal I terminal fragments.
Figure shows Southern blots of Hind III and Eco Rl digests of FHV-l DNA

after being hybridized to nick-translated FHV-l Sal I fragments L or F.

Sal I L:

 

Eco R1

Hind Ill

 

62

Figure 4

Sal I F:
Eco R1 Hind Ill

 

63

Table 2. Hybridization and restriction enzyme digestion patterns of FHV-l
Hind III fragment A. "

Hybridization to FHV-l Sal I and Eco R1 restriction digest patterns:

Sal I: B, H, J, L

Eco R1: E, F, J, M, Q, U/V

Fragments (in kb) generated after digestion of FHV-l Hind III A with:
Sal I: 16.0 (B), 6.8 (J), 4.0, 3.9 (L)
Eco R1: 8.2 (E), 7.6 (F), 4.3 (M), 3.2, 2.7 (Q), 2.1 ( U,V)

(corresponding Sal I or Eco R1 fragment given in parenthesis)

64

some hybridization to Sal I E as well as Sal I F (data not shown).
Therefore, Sal I L, Eco R1 Q and Hind III A are located at the opposite
end of the viral genome from Sal I F, Eco Rl B and Hind III E and J.

To confirm the positioning of Sal I L, Eco R1 Q, and Hind III A at
one end of the genome, Hind III A DNA was electrophoretically purified,
digested with both Sal I and Eco R1 and used to probe Sal I and Eco R1
digests of FHV-l DNA. Results of these experiments (Table 2) confirmed
that Hind III A contained both Sal I L and Eco R1 Q. These results also
indicated that the non-terminal end of Hind III A contained only parts of
both Eco RI J and Sal I H. Sal I H and Eco R1 J therefore span the
junction between Hind III A and the remainder of the genome.

Hybridization of Sal I L to the internal fragment Eco R1 J as well
as to terminal fragment Eco R1 Q (Figure 4) suggested that Sal I L and Eco
R1 J and Q shared common sequences. In addition, a bacteriophage clone
(EM-7, see below) containing only Sal I H also hybridized to Sal I L (data
not shown). Sal I F hybridized only to Eco R1 B, located at the opposite
terminus from Sal I L. These results indicated that the FHV-l genome
contains terminal and internal repeat sequences located within
approximately 30 kb of one end of the genome. This type of structure is
consistent with a herpesvirus genome containing a single invertable short

(S) region and suggests that FHV-l has a two isomer genome.

Electron microscopic analysis of FHV-l DNA. To obtain additional proof

 

that FHV-l DNA has two isomeric forms, viral DNA was denatured and allowed
to reanneal and observed by electron microscopy. Figure 5 shows a
representative molecule of reannealed FHV-l DNA showing a single-stranded

loop and double-stranded stem at one end of the viral DNA molecule. This

65

Figure 5. Electron microscopic analysis of FHV-l DNA. Figure shows an
electron micrograph of self-annealed FHV-l DNA molecules. Arrows indicate

the double stranded regions and fd phage single stranded size marker.

66

 

Figure 5

67

Figure 6. Electron microscopic analysis of the S region of FHV-l DNA.
Histogram shows the size distribution of the single-stranded loop region
of self-annealed FHV-l DNA molecules. The sizes (in megadaltons) are the

double stranded molecular weights of the loops.

Number of molecules

68

 

.5
O

.a lo CO I! (h 0’ ‘4 GD 10

 

l

1

5.4 :1: 0.9 x 106.

 

l

2 3 4 5 6 7 e 9 10
Molecular weight x 106

Figure 6

 

69

confirms that inverted repeat sequences are present at one end of the DNA
and suggests that FHV-l has a two isomer genome. Measurement of the
single-stranded loop region of the reannealed DNA indicated that the
average length of the unique DNA of the 8 region was approximately 8 kb
(5.4 Md, Figure 6). Contour length measurements also indicated that the

FHV-l genome is approximately 125 kb in size.

Cloning FHV-l DNA. To facilitate further mapping studies on the genome of

 

FHV-l, viral DNA was inserted into the bacteriophage vector, EMBL-3.
EMBL-3 is a substitution vector capable of accepting fragments from 7 to
22 kb in size. Substitution requires the deletion of the lambda red and
gamma genes. Therefore, recombinant phages will be spi- and can be
selected for by plating on a bacterial host lysogenic for bacteriophage
P2. The spi+ vector phages will not form plaques on Q359, but will grow on
strain Q358 which does not contain a P2 lysogen (Karn et al., 1980, 1983).

Since Sal I digestion produced the greatest proportion of FHV-l DNA
fragments which were capable of being inserted into EMBL-3, Sal I cut
FHV-l DNA was ligated to Sal I cut EMBL-3. The phage library was titered
on both Q358 and Q359. The initial library contained 105 recombinants
and had 64% vector background contamination. Efficiency of spi selection
was tested by 13 gigu plaque hybridization. Filters containing plaque DNA
from platings on both Q358 and Q359 were hybridized to nick-translated
viral DNA. Only 33% of the plaques growing on Q358 hybridized to the FHV-l
probe while nearly 100% of the plaques on Q359 hybridized (data not
shown).

The library was amplified twice by plating on Q359. The amplified

library contained greater than 108 recombinants and less than 10%

70

Figure 7. Analysis of EMBL-3 clones containing FHV-l DNA inserts. Panel A
shows an ethidium bromide stained agarose gel containing Sal I digests of
clones EM-l-9 (lanes 1-9 respectively). Sal I digests of EMBL-3 DNA (B)
and FHV-l (C) and a Hind III digest of lambda DNA (A) are shown as size
markers. Panel B shows a Southern blot of the gel shown in panel A after

being hybridized to nick-translated FHV-l DNA.

71

monomvmu 2.3.".
m

 

mwhomvnww Um<

Figure 7

72

vector background. To screen the library, individual plaques growing on
Q359 were picked and purified. Small scale DNA preparations were made from
each plaque, digested with Sal I, and analyzed by agarose gel
electrophoresis. DNA from over 400 plaques was examined by this method
with the result that nine different clones containing approximately 85% of
the viral genome were isolated. Figure 7A shows that seven clones
containing fragments A, B, C, D, H, I, and J as single inserts (designated
EM-1,2,3,4,7,8 and 9 respectively) were isolated. Two clones contained
double inserts. Clone EM-S contained fragment E and N and another fragment
approximately 4 kb in size that was part of fragment E (data not shown).
EM-6 contained fragments G and N. Figure 7B shows a Southern blot of the
gel shown in 7A after being probed with FHV-l DNA. This experiment
confirmed that all of the clones contained viral inserts. Several clones
contained small Sal I fragments from the internal region of EMBL-3. The
lambda red and gamma genes are not contained on these fragments and give
these phage a spi- phenotype.

Attempts were made to identify clones containing Sal I K and M.
Plaques from both the original and amplified libraries were hybridized to
nick-translated probes prepared from gel purified Sal I K and M DNA. No

positive clones were found.

Genome mapping. To determine linkage relationships between Sal I and Hind
III restriction fragments of FHV-l DNA, Sal I fragments were nick
translated and hybridized to nitrocellulose strips containing Hind III
fragments. Clones containing single inserts were nick translated without
further purification. Sal I fragments E, N, and C were electrophoretically

purified from a digest of phage DNA. Sal I fragments M, and K were

73

Figure 8. Linkage relationships between FHV-l Sal I and Hind III
fragments. FHV-l Sal I restriction fragments were nick translated and
hybridized to filters containing Hind III restriction fragments. Identity

of Hind III fragments is indicated at the left of each lane.

74

. E92

.Eoi

 

8.0
.7152 .I.

.5
9 ..
._
.1
0D.

 

.Gxu .

n . _
t . .05 .03 ‘oa down
0 -< C .<

I mu m n_ U m <

 

"wooed _ .mm

Figure 8

75

Table 3. Hybridization of FHV-l Sal I fragments to FHV-l Hind III
fragments.

 

Sal I Hind III fragments detected:
probe:

 

B/C, D

A

B/C, F/G, H, R, Q,
F/G, I, M, L, s
B/C, N/O/P

E, J

B/C

A, L, T, N/O/P
F/G, R

A

B/C

A

N/O/P

E, N/O/P

zzr‘quznoramunm»

 

76

Table 4. Hind III digestion patterns of FHV-l Sal I fragments.

 

Sal fragment:

ZF‘WQHEQMUOw?

Hind III digestion products (kb):

(D), 3.0, 2.0

O\b

beQJ-‘NO‘O‘OV-
DU!

.7

(H), 3.6 (K), 2.5,
3.2 (M), 2. . 2 5.

A
lot-44>
0V
0"

. .7 (T)

UN
V.
f~h‘
w.
V0

meawasooowbr-JH
100‘
A
v.2
0V

0‘

 

corresponding Hind III fragments are shown in parenthesis

77
electrophorectically purified from a digest of viral DNA. These data are
presented in Figure 8 and summarized in Table 3. DNA from the recombinant
phage clones was digested with both Sal I and Hind III, and gel-purified
Sal I fragments K, L, N were digested with Hind III to determine the
number of internal Hind III sites contained in each Sal I fragment (Table
4). These data were used in combination to determine positions of the Sal
I and Hind III fragments on the FHV-l genome (Figure 10). Fragments in
hypermolar bands were assigned locations in alphabetical order starting at
the S region of the genome.

Sal I L, J, and B hybridized to terminal fragment Hind III A and none
of these fragments contained internal Hind III sites. Sal H hybridized to
to Hind III A, L, N/O/P and T, and contained fragments identical in size
to Hind III N and T along with 4.7 kb of Hind III A and 1.0 kb of Hind III
L. These results indicate that the order of Sal I fragments from the
terminus of the S region must be: L, B or J, and H.

Sal H was linked to Sal I D based on common hybridization to Hind
III L. Sal D was found to contain fragments corresponding to Hind III I,
M, and S along with two 2.5 kb fragments. Since Sal I D hybridized to Hind
III I, M, S, L, and F/G, one of these 2.5 kb fragments must be the part
of Hind III L not contained in Sal I H. The other fragment was therefore
part of Hind III F.

Sal I I contained Hind III R and two fragments 3.4 and 3.2 kb in
size. Sal I I hybridized to Hind III F/G and R only. The 3.4 and 3.2 kb
fragments were part of both Hind III F and G. Sal I I was placed adjacent
to Sal I D since both fragments contained part of Hind III F. Sal I I was
linked to Sal I C based on common hybridization to Hind III G. Sal I C

hybridized to and contained internal Hind III fragments corresponding to

78
Hind III H, K, and Q. The 2.5 kb Hind III digest fragment of Sal I C was

the remainder of Hind III C which was shared with Sal I I. The final Hind
III fragment of Sal C was 4.7 kb in size and was presumably part of Hind
III B.

At this point, examining the terminus of the L region genome and
following the mapping strategy toward the center of the genome is
appropriate. Sal I F was identified as the terminus of the L region of the
genome. Sal I F hybridizes to Hind III J and E. Since Sal I N also
hybridizes to Hind III E and no other Sal I fragments hybridize to Hind
III J, Hind III J must lie cempletely within Sal I F.

Sal I N was cut into two fragments of approximately 1.0 and .6 kb by
Hind III. Hybridization showed that Sal I N overlaped both Hind III E and
N/O/P. Based on the distance from the terminus, Sal I N should share
approximately .6 kb with E and 1.0 kb with Hind III P.

Sal I M was placed next to Sal I N based upon common hybridization to
Hind III N/O/P. Sal I M overlaped the remaining 1.6 kb of Hind III P and
approximately 1.0 kb of Hind III 0.

Sal I E was placed adjacent to Sal M due to common hybridization to
Hind III 0. Sal I E was cut into two fragments of 2.0 kb and 8.6 kb. The
smaller fragment therefore overlaped Hind III 0 leaving 8.6 kb of Sal E
shared with Hind III C.

Sal I A was found to contain all of Hind III D and, in addition, to
hybridize to Hind III B/C. Hind III digestion of Sal I A yielded a
fragment corresponding to Hind III D and two fragments of 3.2 and 2.0 kb
which must overlap both Hind III B and C.

Both Sal I G and K hybridized to Hind III B/C and neither fragment

contained internal Hind III sites. Since it was already determined that

79

Sal I C contained approximately 4.7 kb of Hind III B and that Sal I E
shared 8.6 kb with Hind III C, Sal G was placed adjacent to Sal C and Sal
I K adjacent to Sal I E. This configuration was the only one that also
satisfied the size requirements of the Sal I A overlaps into Hind III B
and C. These assignments completed the Sal I map shown in Figure 10 and
indicated the positions of most of the Hind III fragments.

To confirm the isomeric arrangement of the FHV-l genome and to
determine the order of the Sal I B and J fragments, a more complete map of
the S region was necessary. Sal I fragments L, H, B, and J, which mapped
to that region, were hybridized to Eco R1 fragments of FHV-l DNA which had
been transferred to nitrocellulose strips. Also, clones containing Sal I
B, J, and H were digested simultaneously with Sal I and Eco R1. Results of
these experiments are shown in Figure 9A and B.

Sal I terminal fragment L hybridized to Eco R1 terminal fragment Q
and contained part of Eco R1 U. Sal I L was also contained within Sal I H
and Eco R1 J which contain the internal repeats. Sal I H contained a
fragment equivalent in size to Eco R1 J in addition to two smaller
fragments. One of these smaller fragments contained part of Eco Rl V and
the other Eco R1 T which was located in the L region. Eco R1 fragments U
and V also span the junctions between Sal I L and J and Sal I H and B,
since Sal I H, L, B, and J hybridized to these fragments.

Sal I B contained fragments equivalent to Eco R1 F and M and parts of
Eco R1 E and U/V. Sal I J hybridized to Eco R1 E and F and must contain
most of Eco R1 E since neither Sal I L or H hybridized to Eco R1 E.
Hybridization of Sal I J to Eco R1 F can be accounted for by the presence
of common repeat DNA on both bands. This hypothesis was confirmed by

demonstrating that the electrophoretically purified 5.8 kb Eco Rl/Sal

80

Figure 9. Blot hybridization analysis of the S region of the FHV-l genome.
Panel A shows hybridization of Sal I fragments B, J, H, and L to filters
centaining FHV-l Eco Rl fragments. Panel B shows an ethidium bromide
stained gel of a Sal I/Eco R1 digest of recombinant phage clones containg
FHV-l Sal I fragments H, B, and J. Sizes of fragments (kb) are noted.
Panel C shows a Southern blot of the gel shown in Panel B after being
hybridized with the nick-translated 5.8 kb Sal I/Eco R1 fragment of Sal I

J.

81

«as
'T“?

5m

0|

 

mm:

.v.w

.HN

.Em
Afih

>5. is... >41 >5.

6.! 0..

q...

n.-

I 1 m

”099:. . Em

<

Figure 9

82

Figure 10. Physical map of the FHV-l genome for Sal I, Hind III and Eco
R1. Both isomers of the S region are shown. Vertical dashed line indicates
inversion point. ( Ul-unique long region, Us-unique short region,

TRs-terminal repeat sequences, IRs-internal repeat sequences)

 

 

 

 

83

on. ON. 0: 2.: 0pm 0.0 ON 00 On 0.? 0.» ohm AW. 0 8.2.

- p b

 

 

 

 

 

 

 

oaono=x
III
.5 .mmumammh
“
.: __ :
9.32.23
_ :Jq __ :1 2.03
a Raise:
._— a
.z.. m ._.
:_ Z __ __ .2 4.. Ham
mzzmx <eo.o:_m....
_
.
q... _ a a _=_ _: Bus...
some 0 o a seats: 4
0.x... mignz

Figure 10

84
fragment of Sal I J hybridized to the Eco R1 E fragment generated by Sal
I/Eco R1 digestion of the clone containing Sal I B (Figure 9C). These data
indicates that the inverted repeat sequences extend beyond the Eco R1 U
and V fragments into Eco R1 E and F and therefore must be greater than 4.8
kb in length. The Eco R1 map of the S region of FHV-l DNA is shown in
Figure 10. Both orientations of the 8 region are shown. The orientation of
the Sal I J and B fragments relative to the Eco R1 fragments is the only
configuration that produced the appropriate Eco Rl digestion fragments of
Sal I B and J.

Sizes of the fragments contained within the S region total
approximately 31 kb. Based on the electron microscopic analysis,
approximately 8 kb of the S region is composed of unique DNA. Therefore,
the sizes of the repeats can be calculated to be approximately 11 kb. By
subtracting the size of the S region from the size of the entire genome,

the size of the L region was determined to be approximately 103 kb.

Discussion

 

This paper contains the initial description of the physical structure
of the genome of the C-27 prototype strain of FHV-l. Based on restriction
enzyme digestion analysis, the viral genome is 134 kb in size. This size
estimate is based upon the average of the size total of the restriction
fragments generated by Bam HI, Hind III, Eco R1 and Sal I digestions.
Small variations in the size total calculated for each enzyme is due to
the inherent error in determining the size of individual fragments from
electrophoretic separations. Terminal restriction fragments were
identified by both progressive digestion from the ends of the genome with
lambda 5' exonuclease and by labeling the ends with kinased synthetic
linkers. Two one molar terminal fragments in the Ben HI, Eco RI and Sal I
digests and one terminal fragment in the Hind III digest were identified.
Hybridization patterns of Sal I terminal fragments identified the other
Hind III terminal fragment and indicated that viral DNA contained both
terminal and internal inverted repeat sequences at one end of the genome.
The positions of these repeat sequences was confirmed by electron
microscopic examination of reannealed FHV-l DNA. The size of the genome
based on electron microscopy was shorter than the size determined by
restriction endonuclease digestion. This discrepency was probably caused
by inevitable nicking of very large DNA molecules. Nicking could have
caused an inversion of the L segment of the viral genome to go unnoticed,
however hybridization patterns of the Sal I F terminal fragment indicate
that a L segment inversion is unlikely.

In a previous study (Herrmann et al., 1984), the size of the FHV-l

genome was calculated to be about 10 kb smaller than the size determined

85

86
in this study. However, in that study, fragment molarity was not
determined and some of the smaller restriction fragments might not have
been detected since the fragments were visualized by ethidium bromide
staining.

In this report we also describe the isolation of recombinant phage
clones which contain approximately 85% of the FHV-l genome as Sal I
inserts. Clones containing viral termini were not represented in the
library since they have only one terminus that was capable of ligating to
the Sal I cohesive end on the vector DNA. Two clones were found to contain
the 1.6 kb Sal I fragment N as a multiple insert. It was surprising that
clones containing the smaller fragments Sal I M and K as multiple inserts
were not identified even after screening a large number of plaques with
probes prepared from these fragments. We are currently completing our
clone collection of Sal I fragments by using additional cloning strategies
to isolate clones containing the viral termini and the smaller fragments
that were not represented in the existing clone collection.

The recombinant clones were used both as hybridization probes and as
substrates for double digestion experiments to create a restriction map of
the genome for Sal I and Hind III. The linear order of all Sal I fragments
except the three smallest fragments (less than 1% of the genome) was
determined. The approximate positions of 14 of 22 Hind III fragments were
determined. Based on the data obtained, it was not possible to determine
the order of multiple Hind III fragments lying completely within large Sal
I fragments. .

In addition, an Eco Rl map of the 8 region of the genome was also
created. Lengths of the inverted repeat sequences were determined to be

greater than 4.8 kb, but the exact length could not be determined from

87
the available data. Analysis of the S region with additional restriction
enzymes will be necessary to determine the exact size of the repeats. The
approximate size of the repeats, as determined by electron microscopic
analysis of the S region, was approximately 11 kb.

Previous studies of the structure of herpesviruses genomes have
traditionally involved electron microscopy followed by single and double
restriction enzyme digestion analysis and Southern blot or
cross-hybridization studies (Henry et al., 1981, Spector et al., 1982,
Sullivan et al., 1984). Our strategy in mapping the genome of FHV-l was
basically similar. The availability of cloned DNA fragments for use in
Southern blotting and double digestion experiments was very helpful since
it made it unnecessary to have to electrophoretically purify all of the
restriction fragments in a digest and also allowed us to separate two
molar bands. We observed that individual gel-purified restriction
fragments were frequently contaminated with other parts of the viral
genome. Probes prepared from these fragments had to be tested by
hybridizing them back to the appropriate genomic digest. Often, several
rounds of electrophoretic purification were needed.

Based on the data presented in this report, the genome of FHV-l is
134 kb in size and is composed of two segments. The longer segment (L) is
composed of 103 kb of unique DNA ( U1 ) and is adjacent to a 31 kb short
(S) segment. The short segment contains 8 kb of unique DNA ( Us ) bounded
by 11 kb repeats. The short segment can invert relative to the U1 segment
creating two possibe isomeric forms of the viral genome.

Therefore, FHV-l has a group D genome based on the classification
scheme proposed by Roizman (1980). Other herpesviruses in this group

include pseudorabies virus (PRV) (Ben-Porat et al., 1979),

88

varicella-zoster virus (VZV) (Ecker and Hyman, 1982, Straus et al., 1981),
equine herpesvirus-1 (EHV-l) (Henry et al., 1981), equine herpesvirus-3
(Sullivan et al., 1984) and bovine herpesvirus-1 (Mayfield et al., 1983).
Of these, the size and arrangement of the FHV-l genome is most similar to
that of EHV-l. However, the biological properties of FHV-l are closer to
those of VZV which also has a limited host range. Thus far, no studies
have been conducted to investigate sequence homology or cross reacting
antigens between FHV-l and any other herpesviruses with similar genomic
structure. We have found that FHV-l shares limited sequence homology and
several cross reacting antigens with canine herpesvirus (Rota and Maes,
unpublished observations).

Recent studies have indicated that a small number of VZV DNA
molecules (Kinchington et al., 1985) and DNA from a strain of PRV
(Lomniczi et al., 1984) contain inversions of the L region. It will be
interesting to deterimine, in future studies, if such novel genomic
arrangements exist in FHV-l.

In herpesviruses, the amount of interstrain genetic variability, as
detected by restriction endonuclease analysis, varies considerably. For
example, strains of herpes saimiri (Desrosiers and Falk, 1982) contained
more restriction endonuclease fragment variability than strains of herpes
simplex (Buchman et al., 1980), equine herpesvirus (Studdert et al., 1981)
and PRV (Paul at al., 1982), while strains of VZV (Martin et al., 1982,
Richards et al., 1979) showed far less interstrain variability. Recent
studies have shown that, like VZV, various isolates of FHV-l contain
little restriction fragment size variability. This stability was initially
observed by Herrmann et a1 (1984) in a study of both clinical isolates and

vaccine strains of FHV-l. We have also examined the restriction digestion

89
patterns of a number of case isolates of FHV-l (Rota et al., 1985) and
found that the digestion patterns were very similar. The map that was
constructed in this study for the C-27 prototype strain of FHV-l should be
applicable in the analysis of many FHV-l isolates. Based on this map, most
of the variation between the strains of FHV-l occurred in fragments at the
viral termini and within the internal inverted repeat sequences. This type
of variation has been observed in other herpesvirus genomes (Hayward, et
al., 1975, Paul et al, 1982).

The FHV-l restriction map should prove extremely useful in future
studies aimed at mapping genes on the viral genome and will allow us to
explore herpesvirus infections in the natural host in greater detail.
Also, comparing the relative map positions of important FHV-l genes to the
positions of those genes on other herpesviruses will be interesting
especially in light of recent evidence for colinear genome organization

(Davison and Wilkie, 1983).

References

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Povey, R.C. (1979). A review of feline viral rhinotracheitis (feline
herpesvirus) infection. Comp. Immunol. Microbial. of Infect. Dis. 2,
373-387.

Povey, R.C. and Wilson, M.R. (1978). A comparison of inactivated feline
rhinotracheitis and feline caliciviral vaccines with live-modified viral
vaccines. Feline Pract. 8, 35-42.

Pratt, P.W. (ed.) (1983). Feline Medicine, p.88-97, First Edition,
American Veternary Publication Inc., Santa Barbara CA.

Richards, J.C., Hyman, R.W., and Rapp, F. (1979). Analysis of the DNAs
from seven varicella zoster virus isolates. J. Virol. 32, 812-821.

Roizman B. (1980). The Herpesviruses. vol.l, p.1-23, Plenum Publishing
Corp., New York.

92

Rota, P.A., Maes, R.K., and Evermann, J.F. (1985). Biochemical and
antigenic characterization of feline herpesvirus-l-like isolates from
dogs. Arch. Virol. (in press).

Ruyechan, W.T., Dauenhauer, S.A., and O'Callaghan, D.J. (1982). Electron
microscopic study of equine herpesvirus-l. J. Virol. 42, 297-300.

Scalenghe, F., Buscaglia, M., Steinhal, C., and Crippa, M. (1978). Large
scale isolation of nuclei and nucleoli from vitellogenic oocytes of
Xenopus laevis. Chromsoma 66, 299-319.

Slater, E. and York, C. (1976). Comparative studies on parenteral and
intranasal innoculation of an attenuated feline herpsevirus. Devel. Biol.
Stand. 33, 410-416.

Southern, E. (1975). Detection of specific sequences among DNA fragments
separated by gel electrophoresis. J. Mol. Biol. 98, 503-516.

Spector, D.H., Hock, L., and Tamashiro, J.C. (1982). Cleavage maps for
human cytomegalovirus DNA stain AD169 for restriction endonucleases Eco
Rl, Bgl II, and Hind III. J. Virol. 42, 558-582.

Straus, S.E., Aulakh, H.S., Ruyechan, W.T., Hay, J., Casey, T.A., Vande
Woude, G.F., Owens, J., and Smith, H.A. (1981). Structure of
varicella-zoster virus DNA. J. Virol. 40, 516-525.

Studdert, M., Simpson, T., and Roizman, B. (1981). Differentiation of
respiratory and abortigenic isolates of equine herpesvirus 1 by
restriction endonucleases. Science. 214, 562-564.

Sullivan, D.C., Atherton, S.A., Staczek, J., and O'Callaghan, D.J. (1984).
Structure of the genome of equine herpesvirus type-3. Virology 132,
352-367.

Chapter 3

The Immediate Early Genes and Polypeptides of Feline Herpesvirus-l

Paul A. Rota and Roger K. Maes

93

Abstract

The immediate early (IE) genes and polypeptides of feline
herpesvirus-1, a major upper respiratory tract pathogen of cats, were
studied. RNA purified from cycloheximide-treated, FHV-l infected cells was
radiolabeled and hybridized to restriction fragments of FHV-l virion DNA
and to DNA from recombinant bacteriophage clones containing FHV-l DNA
inserts. The results indicated that IE RNA is transcribed primarily from
the inverted repeat regions which bracket the unique short region of the
viral genome. RNA from mock-infected cells did not hybridize to FHV-l DNA.
In Northern blot experiments, three species of immediate early RNA with
approximate sizes of 5, 1.6 and .8 kb were detected by a FHV-l DNA probe.
The 5 and 1.8 kb RNAs hybridized to cloned FHV-l DNA fragments containing
the S region of the genome. Two polypeptides with approximate molecular
weights of 155 and 120 kilodaltons were present in the i3 yiggg
translation products of IE RNA and were not present in the translation
products of RNA from mock-infected cells. These polypeptides were also
detected in infected cells at four hours post infection, in actinomycin
D-treated, infected cells after reversal of a cycloheximide block, and in

cells infected in the presence of azetidine.

94

Introduction

 

Feline herpesvirus-l is a major viral pathogen of cats and its
distribution is worldwide. Infections in adult cats result primarily in
upper respiratory tract disease, while infections in kittens can cause
generalized disease with high mortality rates (Gaskell and Povey, 1982,
Povey, 1979). Preliminary evidence suggests that FHV-l can establish
latent infections in the trigeminal ganglia and the reactivation of these
latent infections is an important mechanism of virus spread in the cat
population (Gaskell et al., 1985).

Recent studies in our laboratory have focused on the physical
structure of the genome of FHV-l. The viral DNA has two isomeric forms and
is approximately 134 kb in size. Recombinant phage clones containing most
of the genome have been isolated and have been used to construct
restriction endonuclease cleavage maps of the FHV-l genome (Rota et al.,
1985). The goal of this research was to localize the immediate early gene
region and to identify the immediate early polypeptides of FHV-l.

It is well established that during a lytic infection, herpesvirus
gene expression is highly regulated (Feldman et al., 1979, Honess and
Roizman, 1974). Immediate early genes are transcribed from a limited area
of the viral genome immediately after infection. The immediate early gene
products initiate the transcription of early genes. Early gene products
stimulate the transcription of late genes and suppress the further
production of immediate early peptides. The majority of late gene products
are structural components of the virion. Several late genes are
responsible for the suppression of early gene expression. In addition, a

virion component functions to stimulate the expression of immediate early

95

96

genes at the beginning of the replication cycle (O'Hare and Hayward,
1985).

In the presence of cycloheximide, herpesvirus immediate early mRNA
accumulates in infected cells (Ihara et al., 1983). RNA from
cycloheximide-treated, infected cells has been radiolabeled i2 yiggg and
used in blot hybridization studies to identify the regions of the genomes
of cytomegalovirus (Wathen and Stinski, 1982) and pseudorabies virus
(Ihara et al., 1983) containing the immediate early genes. This IE RNA has
been translated ig yiggg to identify the immediate early gene products
(Ihara et al., 1983). When the cycloheximide block is removed from
infected cells immediate early mRNA is rapidly translated into protein.
Addition of actinomycin D to these cells prevents the expression of early
genes and provides another means of identifying immediate early proteins
(Fenwick et al., 1980, Fenwick and Clark, 1983, Fenwick and McMenamin,
1984).

Immediate early peptides of other herpesviruses have also been
identified by treating infected cells with the proline analog, azetidine.
Immediate early peptides accumulated in infected presumably because they
had incorporated the analog and, as a result of this, were defective in
their ability to activate early genes (Fenwick et al., 1980).

In this report, we identify the region of the FHV-l genome containing
the immediate early genes by hybridizing RNA from cycloheximide-treated,
infected feline kidney (CRFK) cells to nitrocellulose filters containing
both virion DNA and cloned viral DNA fragments. The major immediate early
RNA species were identified by Northern blotting. In addition, the major
immediate early peptides of FHV-l were identified by i5 yiggg translation

of infected cell RNA and by SDS-PAGE analysis of polypeptides accumulating

97
in actinomycin D-treated, infected cells after release of a cycloheximide

block or in infected cells treated with azetidine.

Methods and Materials

 

Virus and cells. The C-27 prototype strain of FHV-l was obtained from the
American Type Culture Collection, Rockville, MD (#VR 636). The virus was
propagated in Crandell-Rees feline kidney (CRFK) cells maintained in
Eagle's Minimum Essential Medium (EMEM) (K.C. Biologics, Lenexa, KS.)
supplemented with 10% fetal bovine serum. ( Hyclone defined, Sterile

Systems, Logan, UT).

Immediate early (IE) RNA isolation and labeling. Confluent monolayers of
CRFK cells were preincubated for 1 hour in EMEM with 2% fetal bovine serum
and 100 ug/ml cycloheximide (Sigma, St. Louis, MO) (EMEM-cyc). The
pretreated cells were infected with FHV-l at a multiplicity of infection
of approximately 5 TCIDso/cell. The medium overlaying infected cells

was decanted after a one hour adsorption period and fresh EMEM-eye was
added to the culture flasks. At 4 hours post infection, the infected cells
were scraped into the culture medium and washed twice in ice-cold PBS by
low speed centrifugation (1000 x g, 10 min. 4°C). The washed cell pellet
from approximately 108 cells was resuspended in 8 ml of 5.8 M

guanidinium isothiocyanate, .1 M 2-mercaptoethanol, 1% (wt/vol) Sarkosyl,
50 mM Tris-HCl pH 7.6, and 10 mM EDTA. When the cells were completely
dissolved, the suspension was layered onto a 3 ml cushion of 5.7 M CsCl
and centrifuged for 20 hours at 25,000 rpm in a SW 41 rotor (Beckman
Instruments, Palo Alto, CA) at 20°C. Following centrifugation, the
supernatants were removed by aspiration and the RNA pellets were suspended
in 200 ul of TE (10 mM Tris-HCL pH 7.6, 1 mM EDTA) with .1% SDS. Samples

were extracted twice with phenolzchloroform and precipitated with 2.5 vol

98

99

of ethanol in the presence of .1M NaCl. RNA pellets were washed twice in
95% ethanol before being resuspended in RNase-free water.

Five to ten micrograms of total cellular immediate early or control
RNA were hydrolized in 10 ul of 50 mM glycine pH 9.5, .1 mM EDTA, 1 mM
spermidine at 90°C. for 15 minutes. After hydrolysis, the samples were
chilled immediately on ice and 5 ul of cold kinase buffer (BRL) was added.
250 uCi of gamma 32P-ATP (ICN, Irvine, CA, sp. act. 7,000 Ci/mmole)
and 5 units of T4 polynucleotide kinase (BRL) were added to each sample
and the volume was adjusted to 50 ul with water. Samples were incubated at
37°C. for 30 minutes. Reactions were stopped by addition of 4 ul of .5 M
EDTA and unincorporated label was removed by centrifuging the samples

through .9 ml Sephadex G-50 columns (Maniatis et al., 1982).

Hybridizations. FHV-l virion DNA and cloned FHV-l DNA were purified as

 

previously described (Rota et al., 1985). DNA samples were digested with
restriction enzymes using the reaction conditions recommended by the
manufacturer (BRL) and the fragments were separated by agarose gel
electrophoresis and transferred to nitrocellulose filters using standard
methods (Maniatis et al., 1982). Hybridization and wash conditions were
similar to those described by Wathen and Stinski (1982). Filters were
prehybridized for 24 hours at 68°C. in hybridization buffer (5X
Denhardt's solution, 3X SSC, 150 ug/ml denatured salmon sperm DNA and 50
ug/ml yeast t-RNA. Labeled RNA (108 cpm) was heated to 100°C for 5
minutes before being added to the hybridization solution. Hybridizations
were incubated for 48 hours at 68°C. Filters were washed twice at room
temperature for 30 minutes in 2X SSC, treated with 30 ug/ml DNase-free

(80°C., 10 min.) RNase (bovine pancreas) in 2X SSC for one hour at room

100

temperature, and washed in hybridization buffer at 68°C for one hour.
Filters were given a final wash in 2X SSC before being exposed to Kodak
X-AR film with intensifying screens (Dupont, Wilmington, DE).

Developed films were scanned with an Ortec 4310 densitometer (Oak

Ridge, TN) equipped with an integrator.

Northern blotting. RNA samples from infected or mock-infected CRFK cells

 

were denatured and separated on 1.2% agarose gels containing 7.5%
formaldehyde and transferred to nitrocellulose filters using the methods
described by Maniatis (Maniatis et al., 1982). Total FHV-l virion DNA or
DNA from bacteriophage clones were labeled with 32P by

nick-translation and filters were hybridized and washed as previously

described (Rota et al., 1985).

In vitro translation. IE RNA was translated ip yiggg using a commercially
avialable rabbit reticulocyte lysate kit (BRL) according to the protocol
described by Jackson and Hunt (1983). Labeled translation products were
separated by SDS-PAGE as described by Laemmli (1970). Gels were fixed for
30-60 minutes in 50% ethanol, 10% glacial acetic acid. The fixative was
removed by soaking gels in a large volume of distilled water for 30
minutes. Gels were prepared for flourography by a 30 minute soak in 1M
salicylic acid, dried and exposed to X-ray film. Molecular weight
standards (SDS-6H, Sigma, St. Louis, MO) included myosin (205 kd),
B-galactosidase (116 kd), phosphorylase B (97.4 kd), bovine plasma albumin
(66 kd), egg albumin (45 kd), and carbonic anhydrase (29 kd). Marker lanes

were cut from the gel and stained with Coomassie Brilliant Blue. Molecular

101

weights of the translation products were determined using the method of

Weber and Osborn (1969).

Hybrid selection and hybrid arrest. For the hybrid selection experiments,
the binding of denatured DNA to nitrocellulose filters was performed as
described by Maniatis (1982). Hybridization of IE RNA to filters, washing
of filters, and elution of bound RNA were performed as described by Belle
Isle et al. (l98l).The eluted RNA was precipitated with ethanol, washed
twice with 95% ethanol and translated i3 21252 as described above. For
hybrid arrest of translation experiments, 5 ug of total viral DNA was
hybridized to 1 ug of IE RNA as described by Paterson et a1. (1982) before
being translated igjyiggg. The translation products were analyzed by

SDS-PAGE as described above.

Production of IE pgoteins in infected cells. Two methods were used to
identify IE proteins in infected CRFK cells. The first consisted of
preincubating cells in EMEM-cyc for one hour before infecting them with
FHV-l at an m.o.i. of 5 TCIDso/cell. At 4 hours post-infection,

EMEM-cyc was removed and replaced with EMEM containing 50 ug/ml of
actinomycin D. Cells were harvested 4, 6 and 8 hours after release of the
cycloheximide block. Prior to being harvested, the infected cells were i3
yiyg labeled for 4 hours in labeling medium (EMEM containing 1/10 the
normal concentration of methionine, plus 10 uCi/ml 35S-methionine,
Amersham, Arlington Hgts., I1, sp. act. 800 mCi/mmole) containing 50 ug/ml
of actinomycin D. After the labeling period, the medium was decanted and
the cells were washed three times in cold PBS containing 0.01 mM

phenylmethylsulfonyl fluoride (PMSF) and resuspended in water. The washed

102

cells were lysed by three freeze-thaw cycles. An equal volume of 2X
SDS-PAGE sample buffer (.125 M Tris-HC1 pH 6.8, 20 % glycerol, 4% SDS,
.01% bromphenol blue) was added to each sample and SDS-PAGE was done as
described above.

The second approach consisted of infecting CRFK cells at an m.o.i. of
5 TCIDso/cell in medium containing 4 mM azetidine. Cells were
harvested at 4 and 6 hours post-infection following a 4 hour labeling
period in labeling medium (see above) with 4 mM azetidine. Cells were

harvested and prepared for SDS-PAGE as described above.

Results

Identification of the immediate early gene region of FHV-l DNA. The
region of the FHV-l genome containing the immediate early genes was
initially identified by hybridizing labeled RNA from cycloheximide
treated, infected CRFK cells (IE RNA) to nitrocellulose filters containing
Sal I digested FHV-l DNA. Figure 1A shows that IE RNA hybridized to Sal I
fragments H, B/C, J, and L. The figure also shows that labeled RNA from
mock infected CRFK cells did not hybridize to FHV-l DNA. Films were
scanned with a densitometer and the area under each peak was used to
determine the proportion of labeled RNA binding to each restriction
fragment. Data shown in Figure 1B are based on the average of measurements
obtained by scanning three different gels. Labeled IE RNA hybridized
extensively to Sal I fragments L and H and to a lesser extent to Sal I B/C
and J. Sal I L is a terminal fragment located completely within the
terminal inverted repeat sequences of the viral genome, and Sal I H maps
partially within the internal inverted repeat region. These repeat regions
flank the Us region of the viral genome (Rota et al., 1985) (Figure 4).

To determine which of the fragments in the Sal I B/C band hybridized
to IE RNA, and to confirm the hybridization patterns of the other Sal I
fragments, IE RNA was hybridized to to nitrocellulose filters containing
Sal I digested DNA from a set of bacteriophage clones containing FHV-l DNA
as Sal I inserts (Figure 2). As expected, IE RNA hybridized to cloned Sal
I fragments H and J and did not hybridize to any of the other cloned Sal 1'
fragments which migrate close to Sal I H and J in digests of total viral
DNA. Sal I B, but not C hybridized to IE RNA. It was not possible to

confirm the hybridization of IE RNA to Sal I L since this fragment has not

103

104

Figure l. Hybridization of IE RNA to FHV-l DNA. Panel A autoradiograms of
filters containing Sal I fragments of FHV-l DNA that were hybridized to
labeled viral DNA, IE RNA or RNA from mock-infected controls. A photographi
of an ethidium bromide-stained agarose gel showing sizes (in kilobases) of'
Sal I fragments of FHV-l DNA is shown at the left for reference. Panel B
shows a typical densitometer scan of an autoradiogram of the filter
hybridized to with labeled IE RNA (panel A). The numbers below each band
indicate the percentage of labeled RNA binding to the individual

fragments.

 

105

 

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106

Figure 2. Hybridization of IE RNA to DNA from recombinant clones
containing FHV-l DNA inserts. Sal I digested phage DNA was subject to
agarose gel electrophoresis (a photograph of an ethidium bromide-stained
gel is shown in panel A) and transferred to nitrocellulose filters and
hybridized to labeled IE RNA (panel B). Letters above each lane designate
the FHV-l Sal I fragment contained in each clone. For reference a Sal I

digest of FHV-l DNA was hybridized to nick-translated FHV-l DNA (at left).

 

107

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Figure 2

108

Figure 3. Hybridization of IE RNA to Sal I/Eco Rl digestion fragments of'
bacteriophage clones containing FHV-l IE genes. Panel A shows an ethidiunl
bromide stained stained gel containing Sal I/Eco Rl fragments of clones
containing Sal I fragments H, B, and J which was transferred to
nitrocellulose and hybridized to labeled IE RNA (panel B). The sizes (in

kilobases) of the hybridizing fragments are shown.

 

Had

A

SaH/EcoR1

Sall

San/EcoR1

Sall
A HBJ

 

 

BJ

HBJ

HBJ

109

 

Figure 3

 

110

been cloned. However, Sal I digestion of FHV-l DNA does not produce any
fragments which migrate close to Sal I L in electrophoretic separations.
Therefore, the immediate early genes of FHV-l are contained on Sal I
fragments, H, B, J, and L which are located within the S region of the
viral genome (Figure 4).

Clones containing Sal I fragments H, B, and J were digested with both
Sal I and Eco R1 and the fragments were electrophoresed and transferred to
nitrocellulose filters and hybridized to labeled IE RNA (Figure 3). IE RNA
hybridized to the 5.7 kb Eco R1 J fragment and to the 1.2 kb portion of
Eco R1 V which are contained on Sal I H. Eco R1 J and Sal I H both span
the L-S junction of the viral genome (Figure 4). The 2.0 kb Eco RI
fragment of Sal I H which contains Eco R1 T and is located completely
within the U1 region of the genome did not hybridize. The 7.6 kb Eco R1
fragment of Sal I B which corresponds to Eco R1 F and a small
approximately .5 kb portion of Eco R1 V (barely visible on the 2 week
exposure shown in Figure 3) hybridized to IE RNA. The Eco R1 M fragment
within Sal I B and a 1.9 kb fragment that is part of Eco R1 E did not
hybridize to IE RNA. These two fragments lie completely within the Us
region of the viral genome. Both Eco Rl fragments of Sal I J hybridized to
IE RNA. The .5 kb Eco R1 fargment of Sal I J is barely visible in Figure
3.

The limits of the IE gene region of the FHV-l genome, based on the
data obtained in this study, are shown in Figure 4. All of the fragments
are at least partially contained within the inverted repeat regions of the
viral genome. Some of the hybridizing fragments also contain portions of
both the U1 and Us regions, but none were found that contained only unique

DNA sequences.

111

Figure 4. The immediate early region of FHV-l DNA. Figure shows a Sal I
and Eco R1 map of the S region of the genome generated in a previous.study

(Rota, et al., 1985). The lower solid bar indicates restriction fragments

hybridizing to IE RNA.

112

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.5

m5 8 my:

 

Figure 4

113

Northern blot analysis of IE RNA detected at least three RNA species.
The largest migrated slightly slower than the 288 RNA marker (Figure 5).
and had an approximate size of 5 kb. This RNA hybridized to a total viral
DNA probe as well as to probes prepared from clones containing Sal I
fragments H, B, and J which collectively contain the entire S region of
FHV-l DNA. Sal I L has not been cloned but contains only repeat DNA which
should be present, in inverted orientation, on Sal I H. The hybridization
signal of the Sal I H probe appeared to be more intense than that obtained
with the other cloned fragment probes. Two additional IE RNA species were
recognized by the FHV-l DNA probe. The larger of these two migrated faster
than the 188 RNA marker and its size was estimated to be 1.6 kb. This RNA
was also detected by all of the the cloned fragment probes. A smaller RNA
molecule with an estimated size of .8 kb was detected with the total viral
DNA probe but not with any of the cloned fragments even at exposure times

longer than the one shown in Figure 5.

Identification of FHV-l immediate early peptides. The immediate early
polypeptides induced by FHV-l were identified by analysis of the in giggg
and in yiyg translation products of IE RNA. SDS-PAGE analysis of the in
yitrg translation products of IE RNA and RNA from mock-infected CRFK cells
(Figure 6A) revealed that two polypeptides were present in the translation
products of IE RNA that were not present in those of control RNA. The
largest of these IE-specific polypeptides had an estimated molecular
weight of 155 kilodaltons (Rd). The second had an estimated molecular
weight of 120 kd. Polypeptides which co-migrated with each of the
IE-specific in yiggg translation products were present in in yigg-labeled

extracts of infected CRFK cells which were harvested at 4 hours post

114

Figure 5. Northern blot analysis of FHV-l IE RNA. RNA purified from
infected (lanes B-E) or mock-infected (lane A), cycloheximide-treated CRFK
cells was separated by electrophoresis on a formaldehyde-agarose gel and
transferred to nitrocellulose. Filter strips were hybridized to
nick-translated FHV-l DNA (lanes A, B) or recombinant phage DNA containing
FHV-l Sal I fragments H (C), B (D), or J (E). Positions of ribosomal RNA.

size markers are shown at left.

115

455»-

283»

185»

 

Figure 5

116

Figure 6. SDS-PAGE analysis of FHV-l IE polypeptides. Panel A shows
analysis of in yigrg translation products of IE RNA (I) and RNA from
mock-infected cells (C) compared to radiolabeled extracts from infected
CRFK cells harvested at 4 hours post-infection (i). Panel B shows analysis
of radiolabeled polypeptides accumulating in infected (I) or mock-infected
(C) cells in the presence of actinomycin D and after reversal of a
cycloheximide block. Panel C shows analysis of radiolabeled polypeptides
from infected (I) or mock-infected (I) cells after incubation in 4 mH
azetidine. The positions and sizes of the IE specific polypepties are

marked with arrows.

117

4155
4120

(155

<120
<55

4120

 

Figure 6

118
infection. This suggested that the IE specific polypeptides that were
identified in the analysis of the in yigrg translation products were not
artifactual.

To specifically translate IE RNA in yiyg, infected cells were
released from a cycloheximide block and incubated in labeling medium in
the presence of actinomycin D. The 155 and 120 kd polypeptides were
detected by SDS-PAGE analysis of radiolabeled cell extracts 6 hours after
cycloheximide reversal (Figure 6B). Cell extracts harvested at shorter
and longer times after cycloheximide reversal were also analyzed by
SDS-PAGE (not shown). The samples taken at later times showed essentially
the same SDS-PAGE patterns as the ones shown in Figure 6B. In samples
taken at earlier times, the IE-specific polypeptides were visible but
appeared to be present in smaller amounts. We were not able to detect any
differences in the rate of appearance of the two IE-specific polypeptides
which could indicate a precursor product relationship.

In CRFK cells infected in the presence of azetidine, two IE-specific
polypeptides were identified at 4 hours post infection which co-migrated
with the 155 and 120 kd IE-specific polypeptides identified in the in
yitrg and in yiyg translation studies (Fig. 6C). Additional FHV-l induced
peptides were present in the azetidine-treated cells. Presumably these
represent either breakdown products of immediate early peptides or early
gene products that were expressed in the presence of azetidine.

To demonstrate that the 155 and 120 kd IE-specific polypeptides were
viral gene products, hybrid selection and hybrid arrest experiments were
performed. In gitgg translation of IE RNA hybrid-selected by filters

containing FHV-l DNA produced translation products that corresponded to

119

the 155 and 120 kd polypeptides produced by in ziggg translation of
non-selected IE RNA (data not shown). Control filters were unable to
select for these translation products.

The hybrid arrest experiment provided additional evidence for the
viral origin of the IE-specific polypeptides. Hybridization of FHV-l DNA
to IE RNA prior to in xigrg translation resulted in elimination of only
the 155 and 120 kd IE polypeptides from the translation products (not

shown).

Discussion

This report contains the initial characterization of the immediate
early genes and gene products of FHV-l. Immediate early RNA from FHV-l
infected cells was found to be transcribed from the 11 kb inverted repeat
sequences which flank the unique short region on the viral genome. This
region represents approximately 18% of the viral genome. Most of the IE
RNA hybridized to restriction fragments within the inverted repeats that
are nearest to the viral terminus or the L-S junction. IE RNA did not
hybridize to any fragments which contained only DNA from the Us or U1
regions. Two HSV IE transcripts extend from the repeat regions into the Us
region (Watson et al., 1981). The extent to which FHV-l IE RNA may extend
into the Us region, if at all, cannot be determined until further mapping
studies to delineate the exact boundaries of the FHV-l repeat regions are
completed (Rota et al., 1985).

FHV-l IE RNA was composed of at least three RNA species based on
Northern blot analysis. The largest and apparently most abundant
transcript was approximately 5 kb in size and a smaller less abundant
transcript of 1.6 kb were detected by cloned FHV-l DNA fragments which
collectively contain all of the 8 region sequences of the FHV-l genome. It
is possible that more than one transcript of each size may be present.
Interestingly, a FHV-l DNA probe detected a very small RNA molecule that
was not detected by the cloned fragment probes. This could possibly be a
minor IE transcript that does not map within the 8 region of the genome or
a transcript from an FHV-l cycloheximide-resistant early gene that is
being expressed in the absence of IE mediated activation. This RNA appears

to be in relatively low abundance and it might not have been detected in

120

121

the initial hybridization experiments especially if it is coded for by a
region of the genome that is not represented by our collection of cloned
fragments.

At least two FHV-l encoded IE polypeptides with molecular weights of
155 and 120 kd were identified by both in 3132 and in 21552 translation of
IE RNA. These two polypeptides were also synthesized in infected cells
analyzed at 4 hours post-infection and in cells infected in the presence
of azetidine. Based on observations made for other herpesviruses (Feldman
et al., 1979, Fenwick et al., 1980), it would be expected that FHV-l IE
polypeptides are phosphorylated. we did not observe any major differences
in the SDS-PAGE migration patterns between FHV-l IE peptides synthesized
in gigg and in yitrg. Small variations in molecular weight would not be
detected by our elecrophoretic analysis. Further studies using in yigg
radioactive phosphate incorporation will be needed to detect the presence
and extent of phosphorylation in FHV-l IE proteins. In addition,
monoclonal and monospecific antibodies directed against these IE peptides
would be very useful to study the kinetics of expression, post
translational modification, and extent of interrelatedness between these
proteins.

The organization of the immediate early genes of FHV-l is generally
similar to that of other alpha herpesviruses. IE genes are contained
within the inverted repeat sequences which flank both the Us and U1
regions of HSV DNA (Wagner, 1985) and only the Us region of PRV DNA
(Feldman et al., 1982, Ihara, et al., 1983). In HSV three IE transcripts
with sizes of 4.2, 2.8, and 1.8 kb have been found to code for

phosphoproteins that are 175, 110, 68, 64, and 12 kd in size (Wagner,

122

1985). PRV has a single 6 kb IE transcript which codes for a 180 kd
polypeptide (Ihara et al., 1983). In varicella-zoster virus, a herpesvirus
which is similar to FHV-l in genome stability (Herman et al., 1984, Martin
et al., 1982, Rota et al., 1985) and also in in yiggg and in 3132 growth
characteristics (Roizman, 1980), a 4.3 kb IE transcript mapping within the
inverted repeat regions has recently been identified (Felser et al.,
1985).

The functions of herpesvirus immediate early genes are still poorly
understood. HSV IE proteins have been shown to have DNA binding activity
(Freeman and Powell, 1982) and to interact with host cell heat shock
proteins (Notarianni and Preston, 1982). A recent study has shown that an
HSV immediate early protein functions as a negative regulator of its own
expression and a positive regulator of early gene expression, while other
immediate early proteins were positive regulators of both immediate early
and early gene expression (O'Hare amd Hayward, 1985). The 180 kd PRV IE
polypeptide is a multifunctional molecule which is required for viral
early and late gene expression and appears to be responsible for the
inhibition of host cell protein synthesis (Ihara et al., 1983).

Various attempts have been made to find immediate early expression
during latency. Expression of HSV immediate early genes has been detected
in nervous tissue from latently infected experimental animals (Green et
al., 1981) but not in human tissues (Galloway et al., 1982). The advantage
of identifying the immediate early genes of FHV-l is that this information
can now be used to study the expression of immediate early genes in the

natural host.

References

 

Belle Isle, H., Venkatesan, S., and Moss, B. (1981). Cell-free translation
of early and late mRNAs selected by hybridization to cloned DNA fragments
derived from the left 14 million to 72 million daltons of the vaccinia
virus genome. Virology. 112, 306-317.

Feldman, L., Rixon, F.J., Jean, J.H., Ben-Porat, T., and Kaplan, A.S.
(1979). Transcription_of the genome of pseudorabies virus (a herpesvirus)
is strictly controlled. Virology 97, 316-327.

Feldman, L., DeMarchi, J.M., Ben-Porat T., and Kaplan, A.S. (1982).
Control of abundance of immediate-early mRNA in herpesvirus (pseudorabies)
infected cells. Virology 116, 250-263.

Felser, J.M., Strauss, S.E., Weller, S., and Ostrove, T.M. (1985).
Complementation of an HSV-1 ICP4 temperature-sensitive mutant by VZV.
Abstracts of the Tenth International Herpesvirus WOrkshop, Ann Arbor,
Michigan, p. 8.

Fenwick, M., Walker M., and Marshall, L. (1980). Some characteristics of
an early protein (ICP22) synthesized in cells infected with herpes simplex
virus. J. Gen. Virol. 47, 333-341.

Fenwick, M., and Clark, J. (1983). The effect of cyclohexamide on the
accumulation and stability of functional alpha-mRNA in cells infected with
herpes simplex virus. J. Gen. Virol. 64, 1955-1963.

Fenwick, M., and McMenamin, M. (1984). Synthesis of alpha
(immediate-early) proteins in vero cells infected with pseudorabies virus.
J. Gen. Virol. 65, 1449-1456.

Freeman, M.J. and Powell, K. (1982). DNA-binding properties of a herpes
simplex immediate early protein. J. Virol. 44, 1084-1087.

Galloway, D.A., Fenoglio, C.M., and McDougall, J.K. (1982). Limited
transcription of the herpes simplex virus genome when latent in human
sensory ganglia. J. Virol. 41, 686-691.

Gaskell, R.M. and Povey, R.C. (1982). Transmission of feline viral
rhinotracheitis. Vet. Rec. 111, 359-362.

Gaskell, R.M, Dennis, P.E., Goddard, L.E., Cocker, R.M., and Wells, J.M.
(1985). Isolation of feline herpesvirus from the trigeminal ganglia of
latently infected cats. J. Gen Virol. 66, 391-394.

Green, M.T., Courtney, R.J., and Dunkel, E.C. (1981). Detection of an
immediate early herpes simplex virus type 1 polypeptide in trigeminal
ganglia from latently infected animals. Infect. Immun. 34, 987-992.

Hermann, S.C., Gaskell, R.M., Ehlers, B., and Ludwig, H. (1984).

Characterization of the feline herpesvirus genome and molecular
epidemiology of isolates from outbreaks and latent infections. in: Latent

123

124

Herpesvirus Infections in Veterinary Medicine. p. 321-336. A. Wittman,
R.M. Gaskell and H.J. Rziha (eds), Martinus-Nijhoff Publishing, Boston.

Honess, R.W., and Roizman, B. (1974). Regulation of herpesvirus
marcomolecular sunthesis I. Cascade regulation of the synthesis of three
groups of viral proteins. J. Virol. 14, 8-19.

Ihara, S., Feldman, L., Watanabe, S., and Ben-Porat, T. (1983).
Characterization of the immediate-early functions of pseudorabies virus.
Virology 131, 437-454.

Jackson, R.J., and Hunt, T. (1983). Preparation and use of
nuclease-treated rabbit reticulocyte lysates for the translation of
eukaryotic messenger RNA. Meth. Enzym. 96, 50-74.

Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature (London) 227, 680-685.

Maniatis, T., Fritsch, E., and Sambrook, J. (1982). Molecular cloning: a
laboratory approach. Cold Spring Harbor Laboratories, New York.

Martin, J.H., Dohner, D.E., Wellinghoff, W.J., and Gelb, L.D. (1982).
Restriction endonuclease analysis of varicella-zoster vaccine virus and
wild type DNAs. J. Medical Virol. 9, 69-76.

Notarianni, E.L. and Preston, C. (1982). Activation of cellular stress
protein genes by herpes simplex virus temperature sensitive mutants which
overproduce immediate early polypeptides. Virology 123, 113-122.

O'Hare, P. and Hayward, G.S. (1984). Expression of recombinant genes
containing herpes simplex virus delayed-early and immediate-early
regulatory regions and trans activation by herpes virus infection. J.
Virol. 52, $22-$31.

O'Hare, P. and Hayward, G.S. (1985). Trans-acting regulatory proteins of
herpes simplex virus involved in positive and neagtive regulation of
immediate early gene expression. Abstracts of the Tenth International
Herpesvirus Workshop, Ann Arbor, Michigan, p. 67.

Paterson, B.M., Roberts, B.E., and Ruff, E.L. (1982). Structural gene
identification and mapping by DNA-mRNA hybrid-arrested cell free
translation. Proc. Nat. Acad. Sci. USA 74, 4370-4374.

Povey, R.C. (1979). A review of feline viral rhinotracheitis (feline
herpesvirus) infection. Comp. Immunol. Microbiol. of Infect. Dis. 2,
373-387.

Roizman, B. (1980). The herpesviruses, vol. 1, p 1-23. Plenum Press, New
York.

Rota, P.A., Maes, R.K., and Ruyechan, W.T. (1985). Physical
characterization of the genome of feline herpesvirus-l. (submitted for
publication).

125

Rota, P.A., Maes, R.K., and Evermann, J.F. (1985). Biochemical and
antigenic characterization of feline herpesvirus-l-like viruses from dogs.
Arch. Virol. in press.

Wagner, E.R. (1985). Individual HSV transcripts: characterization of
specific genes. in: The Herpesviruses, vol. 3. p. 45-103. Plenum Press,
New York.

Wathen, M.W., and Stinski, M.F. (1982). Temporal patterns of human
cytomegalovirus transcription: mapping the viral RNAs synthesized at
immediate early, early anfd late times after infection. J. Virol. 41,
462-477.

Watson, R.J., Sullivan, M., and Van Woude, G.F. (1981). Structures of two
spliced herpes simplex virus type 1 immediate early mRNAs which map at the
junctions of the unique and reiterated regions of the virus DNA 8
component. J. Virol. 37, 431-444.

Weber, K. and Osborn, M. (1969). The reliability of molecular weight
determinations by dodecyl-sulfate polyacrylamide gel electrophoresis. J.
Biol. Chem. 244, 4406-4412.

Chapter 4

Mapping the Late Genes of Feline Herpesvirus-l

Paul A. Rota and Roger R. Maes

126

Abstract

In this study, the late genes of feline herpesvirus-1 (FHV-l) were
mapped by hybrid-selection and in yiggg translation of infected cell RNA.
In yitgg translation of non-selected infected cell RNA resulted in the
synthesis of a number of polypeptides which reacted with a goat ant-FHV-l
serum. Several of the translation products comigrated with polypeptides
immunoprecipiatated from FHV-l virions. The SDS-PAGE migration patterns of
two translation products which comigrated with proteins immunoprecipitated
from enzymatically deglycosylated FHV-l virions were altered by the
addition of dog pancreas microsomes to the translation reactions. The
genome locations of the genes coding for 21 of the translation products
was determined by using a set of bacteriophage clones containing 85% of
the viral genome to select infected cell RNA prior to in yiggg
translation. FHV-l late genes map throughout the viral genome and most
are contained within the unique long region. Sal I fragment E selected for
3 translation products which had sizes corresponding to the glycoprotein
precursors identified by the enzymatic deglycosylation of FHV-l virions.
Two of these translation products were apparently converted to a higher
molecular weight form in translation reactions performed in the presence

of the microsome extracts.

127

Introduction

 

Feline herpesvirus-l (FHV-l) is a major cause of upper respiratory
tract disease in young and adult cats. Kittens and debilitated animals may
suffer a more severe generalized infection with high mortality rates (14).
Like other members of the herpesvirus family, FHV-l can establish latent
infections (3). These latent infections can be reactivated by a variety of
stresses, though the biochemical mechanism involved in reactivation is not
understood. The shedding of infectious virus by mostly asymptomatic cats
in which viral reactivation takes place is a significant mechanism of
FHV-l spread in the cat population (2).

FHV-l has been placed in the alpha herpesvirus subfamily (16). The
virus has an extremely narrow in yigg and in yiggg host range and appears
to exist as a single serotype (l4). FHV-l DNA has been found to be less
variable than the DNA of other herpesviruses when analyzed by restriction
endonuclease analysis (5,18). The viral genome is 134 kilobase pairs in
size and has two isomeric forms. The structure of FHV-l DNA is therefore
similar to other alpha herpesviruses such as pseudorabies virus, equine
herpesvirus and varicella-zoster virus (17).

During a lytic infection, herpesvirus true late genes are expressed
after viral DNA replication and most of these genes code for structural
components of the virion (6). The virion of FHV-l contains a minimum of 17
proteins. At least three antigenic glycoproteins with approximate
molecular weights of 105, 68 and 60 kilodaltons have been identified on
the envelope of FHV-l virions by immunoprecipitation analyses. These
glycoproteins are also present on the surfaces of infected cells and the

105 kd glycoprotein has been found in the supernatant medium of infected

128

129
cells. Several abundant non-glycosylated proteins present on the virion
also reacted with the goat antiserum used in this study (10).

We have recently isolated a set of bacteriophage clones containing
most of the FHV-l genome as Sal I inserts (17). Current research is aimed
at using these cloned restriction fragments to determine the map locations
of FHV-l genes which code for important viral antigens.

Herpesvirus genes have been mapped to specific locations on the viral
genome by using cloned or gel-purified restriction fragments of viral DNA
to select RNA from infected cells (4). The in xiggg translation products
of the selected RNA were analyzed either by direct SDS-PAGE or by
SDS-PAGE following immunoprecipitation with monoclonal or monospecific
antibody (9). The addition of dog pancreas microsome extracts to the in
zigrg translation reactions allowed the identification of specific
translation products which required cotranslational processing such as
glycosylation and/or signal peptide cleavage (12,15).

In this study, we have used a set of bacteriophage clones containing
FHV-l DNA inserts to select RNA from infected CRFK cells. RNA was
translated in 21552 in the presence or absence of dog pancreas microsome
extracts and the translation products were compared to intact and
enzymatically deglycosylated FHV-l virions by SDS-PAGE. Translation
products were also analyzed by SDS-PAGE following immunoprecipitation with
the goat anti-FHV-l serum that was used in the initial characterization of
FHV-l antigens (10). The results indicated that, like in other
herpesviruses, FHV-l late genes appear to be distributed throughout the
viral genome. One fragment, Sal I E, selected for four translation
products and at least two of these were identified as glycoprotein

precursors.

Methods and Materials

 

Virus and cells. The C-27 prototype strain of FHV-l was obtained from the

 

American Type Culture Collection, Rockville, MD (#VR 636). The virus was
propagated in Crandell-Rees feline kidney (CRFK) cells maintained in
Eagle's Minimum Essential Medium (EMEM) (K.C. Biologics, Lenexa, KS.)
supplemented with 10% fetal bovine serum. ( Hyclone defined, Sterile
Systems, Logan, UT).

FHV-l virions or infected cell extracts were labeled with
35S-methionine and prepared for subsequent analyses as described
previously (10). Radiolabeled virions and infected cell extracts were
enzymatically deglycosylated using two methods. Samples containing
approximately 105 cpm were incubated for 5 hours at 37’C with either
.5 units of Endo F (NEN, Boston, MA) in 100 mM sodium phosphate pH 6.1, 50

mM EDTA, and 1% NP-40 or with 10 units of Turbo cornutus mixed glycosidase

 

(Miles, Naperville, Ill.) in .01 M phosphate buffered saline (PBS) pH 7.2,
before being immunopecipitated and analyzed by SDS-PAGE as described
below. All immunoprecipitations were performed using a goat anti-FHV-l

serum as previously described (10).

Infected cell RNA isolation. Confluent monolayers of CRFK cells were
infected with FHV-l at a multiplicity of infection of approximately 10
TCIDSO/cell in EMEM with 2% FBS. Infection medium was decanted after a
one hour adsorption period and fresh EMEM with 2% FBS was added to the
culture vessel. After 15-17 hours of incubation, the cells were scraped
from the vessel surface and washed twice in ice-cold PBS by low speed

centrifugation (1000 x g, 10 min., 4°C). The washed cell pellet from
130

131
approximately 108 cells was resuspended in 8 ml of 5.8 M guanidinium

isothiocyanate, .l M 2-mercaptoethanol, 1% (wt/vol) Sarkosyl, 50 mM
Tris-HCl pH 7.6, and 10 mM EDTA. When the cells were completely dissolved,
the suspension was layered onto a 3 ml 5.7 M CsCl cushion and centrifuged
for 20 hours at 25,000 rpm in a SW 41 rotor (Beckman Instruments, Palo
Alto, CA) at 20° C. After centrifugation, the supernatants were removed
by aspiration and the RNA pellets were suspended in 200 ul of TE (10 mM
Tris-HCL pH 7.6, 1 mM EDTA) with .1% SDS. Samples were extracted twice
with phenol:chloroform:isoamyl alcohol (25:24:l) and precipitated with 2.5
vol of ethanol in the presence of .1M NaCl. RNA pellets were washed twice

in 95% ethanol before being resuspended in RNase-free water.

Hybridization selection. Ten micrograms of either FHV-l virion DNA,

 

recombinant phage clone DNA (individual clones contained Sal I fragments:
A, B, C, D, E/N, G/N, H, I, and J), bacteriophage vector (EMBL-3) DNA or
gel-purified restriction fragments prepared as previously described (17),
were denatured and bound to nitrocellulose filters as described by Parnes
(l3). Fifty micrograms of infected cell RNA were hybridized to each filter
in a 100 ul reaction and the subsequent washes and elution of specifically
bound RNA were carried out using the procedure described by Maniatis (ll).
Eluted RNA was precipitated with 2.5 volumes of ethanol in the presence of
15 ug of carrier tRNA (bovine liver, Boehringer MannheiM, Indianapolis,
IN) and washed twice with 95% ethanol before being dried under vacuum and

resuspended in 10-15 ul of water.

In vitro translation. Selected RNA and 3-5 ug of control non-selected RNA

were translated in vitro using a commercially available rabbit

132

reticulocyte lysate system (BRL, Gaithersburg, MD) according to the
protocol described by Jackson and Hunt (7). All in giggg translation
reactions were done in 30 ul volumes containing 10-20 uCi of
35S-methionine (Amersham, Arlington Hgts., IL, sp. act. 800 mCi/mmole)
and l unit/ul RNasin (Promega, Madison, WI) Incubation was for 90 minutes
at 30'C. In some cases, a dog pancreas microsome extract (NEN, Boston
MA) was added to the translation reactions to a final concentration of 3%
prior to the start of incubation. Five ul aliquots of labeled translation
products were diluted with 5 ul of water and incubated in 10 ul of 2x
SDS-PAGE sample buffer (.125 M Tris pH 6.8, 20% glycerol, 4% SDS, .01%
bromphenol blue) for 3 minutes at 100'C before being separated by
SDS-PAGE as described by Laemmli (8). Gels were fixed for 30-60 minutes in
50% ethanol, 10% glacial acetic acid. Fixative was removed by soaking gels
in a large volume of distilled water for 30 minutes. Gels were prepared
for fluorography by a 30 minute soak in 1M salicylic acid, dried and
exposed to X-ray film. Radiolabeled molecular weight markers (BRL) were
run in adjacent lanes of the gel. The molecular weights of proteins were
determined using the method of Weber and Osborn (20).

For immunoprecipitation, 5-10 ul of in giggg translation reactions
were diluted with an equal volume of 2X SA buffer ( .04 mM PBS, 2% NP-40.
.2% SDS, 1% sodium deoxycholate) before being incubated with goat

antiserum as previously described (10).

DNA purificationL7digestion and labeling. For mapping experiments,
bacteriophage clone DNA was digested, separated by agarose gel
electrophoresis and transferred to nitrocellulose filters as previously

described (17). Restriction fragments were isolated from agarose gels by

133

electroelution onto nitrocellulose filters (NA-45, Schleicher and Schuell,
Keene, NH) according to the protocol described by the manufacturer.
Nick-translated probes were prepared as described by Maniatis (11) and

blot hybridzations were carried out as described previously (17).

Northern blotting. RNA samples were denatured and separated on 1.2%
agarose gels containing 7.5% formaldehyde and transferred to
nitrocellulose filters using the methods described by-Maniatis (ll).
Lanes containing RNA from uninfected cells were removed from the gel and
stained with ethidium bromide to visualize 458, 288, and 188 RNA size
markers. Total FHV-l virion DNA or DNA from bacteriophage clones were
labeled by nick-translation (11) and filters were hybridized, and washed

as previously described (17).

Results

FHV-l late RNA was routinely harvested from infected CRFK cells at
l7-18 hours post infection (m.o.i. -10 TCID5o/cell). At this time,
nearly 100% of the cells in the monolayer were showing early signs of
syncytia formation, but extensive degradation of the monolayer or cell
lysis were not apparent. Radiolabeled extracts of whole cells, harvested
at this time, were analyzed by immunoprecipiation analysis with the goat
anti-FHV-l serum and found to contain all of the major FHV-l associated
antigens that were identified in a previous study (10) (data not shown).

RNA from FHV-l infected or mock infected cells was translated in
yigrg and the translation products were analyzed by SDS-PAGE (Figure 1).
Both RNA samples produced a number of translation products with molecular
weights which ranged from 130 kd to less than 25 kd. Most of the
translation products synthesized from infected cell RNA reacted with the
goat anti-FHV-l serum, while none of the translation products from RNA
from mock-infected cells were recognized by the goat antiserum. These
results indicated that at 17-18 hours post-infection, most of the mRNA in
infected cells is of viral origin and presumably most host cell mRNA
production has been inhibited by the infection.

Several of the translation products from infected cell RNA comigrated
with proteins immunoprecipitated from purified, radiolabeled FHV-l virions
(Fig. l). The largest in giggg translation product was approximately 130
kd in size and comigrated with the largest virion-associated protein. This
suggested that complete viral peptides were being synthesized in viggg and

that extensive premature termination of translation was not occurring.

134

135

Figure 1. In yiggg translation products of FHV-l late RNA. RNA purified
from infected (inf.) and mock-infected (con.) CRFK cells was translated in
gitrg in the presence (+) and absence (-) of dog pancreas microsomes.
Translation products were analyzed by direct SDS-PAGE (d.a.) or by
SDS-PAGE after immunoprecipitation with goat anti-FHV-l serum (i.p.).
Lanes at left show SDS-PAGE analysis of proteins immunoprecipitated from
intact 35S-methionine-labeled FHV-l virions (V) or virions treated

with either Endo F (F) or mixed glycosidase (MG). The sizes (in
kilodaltons) of important bands are indicated at either left or right of

the lane.

 

136

INF

d. 3.

WV":
y‘w

rmh

   

 

Figure 1

137
Figure 1 shows SDS-PAGE analysis of FHV-l virions that were

immunoprecipitated with the goat anti-FHV-l serum. Three immunogenic
glycoproteins with molecular weights of 105, 68, and 60 kd that are found
on FHV-l virions are labeled in this figure. Since we were especially
interested in mapping the locations of the glycoprotein genes, it was
necessary to attempt to identify the precursor molecules of these
glycoproteins. The glycoprotein precursors were identified by two methods.
First, to determine the approximate sizes of the glycoprotein precursors,
FHV-l virions were enzymatically deglycosylated with either Endo F or
mixed glycosidases before being immunoprecipitated with the goat
anti-FHV-l serum. Endo F removes N-linked oligosaccharide chains and the
mixed glycosidase preparation contains a number of exoglycosidase
activities. Three possible glycoprotein precursors were identified by Endo
F treatment of FHV-l virions. These molecules had approximate molecular
weights of 82, 58 and 45 kd. Mixed glycosidase treatment produced a
potential glycoprotein precursor with a molecular weight of 74 kd.
Secondly, glycoprotein precursors were identified by comparing the
SDS-PAGE migration patterns of translation products from reactions
performed in the presence or absence of dog pancreas microsomes (Figure
1). Two 12 yiggg translation products with sizes of 74 and 45 kd were
apparently substrates for cotranslational processing since their migration
was altered by the microsomes. Because of the number of bands on the gel
it was difficult to find the higher molecular weight forms of these
translation products, however, no additional bands were visible in the
lower molecular weight ranges which might occur if only signal peptide
cleavage was occurring. These translation products which were altered by

the microsomes were virus specific since both were immunoprecipitated with

138

the goat antiserum and since no translation products were altered by
microsome treatment of the in 21332 translation reactions for
mock-infected cell RNA. The 74 and 45 kd translation products of infected
cell RNA were tentatively identified as glycoprotein precursors since 1)
they comigrated with proteins immunoprecipitated from deglycosylated FHV-l
virions, 2) their migartion was altered by the addition of microsomes to
the translation reaction, and 3) they were synthesized in large amounts
which would be expected since glycoproteins comprise a significant amount
of total virion protein. Though there were translation products which
comigrated with the 82 and 58 kd potential precursors, identified by
deglycosylation of virions, there was no evidence that these proteins were
modified by the addition of microsomes.

To determine the regions of the FHV-l genome which contained the
genes for the various in giggg translation products, infected cell RNA was
hybridized to filters containing total virion DNA or DNA from recombinant
phage clones containing FHV-l DNA restriction fragments. 19 zitgg
translation of the selected RNA revealed that virion DNA selected for a
number of translation products which, with the exception of a 130 kd
protein, were also present in the translation products of non-selected
infected cell RNA (Figure 2). Most of the translation products that were
selected for by virion DNA were also identified in the translation
products selected for by the individual cloned restriction fragments. A
filter control containing only bacteriophage vector DNA (EMBL-B) selected
for products which were 92, 64, and 44 kd in size. These background
contaminants can be seen in each lane of the gel shown in Figure 2.

The locations of the cloned fragments on the FHV-l genome and the

approximate molecular weights of the translation products that were

139

Figure 2. In yigrg translation products selected for by FHV-l Sal I
fragments. Infected cell RNA which was selected for by filters containing
cloned FHV-l restriction fragments (A-J), FHV-l virion DNA (V), or EMBL-3
DNA (fc) was translated in yigrg and analyzed by SDS-PAGE. Sizes (in
kilodaltons) of the translation products are indicated at right. Lanes A-J
are labeled according to the Sal I fragment used in the hybrid selection.

The clones containing Sal I G and E also contain Sal I N.

 

 

140

 

Figure 2

141
selected for are summarized in Figure 3. Immunoprecipitation experiments
revealed that all of the translation products identified in this summary
reacted with the goat anti-FHV-l serum (not shown). It can been seen in
this figure that FHV-l late genes map throughout the genome but are mostly
found in the unique long region. The clone containing Sal I E and N, which
has been previously designated EM 5 (17), selected for six translation
products. Because the molecular weights of some of these proteins
corresponded to the sizes of the putative 74, 58, and 45 kd glycoprotein
precursors identified above, and because several of these translation
products were synthesized in large amounts, this clone was selected for
further study.

Figure 4 shows a map of EM 5 that was generated by restriction digest
and Southern blotting analysis. Bam HI/ Sal I digestion of DNA from this
clone produced three restriction fragments designated E1 to E3. E1 and E2
are unimolar fragments of 4.5 and 2.1 kb in size, respectively. E3
contains three closely migrating bands 1.8 to 1.7 kb in size. El, E2 and
E3 were purified from an agarose gel and were 1) nick-translated and used
as hybridization probes for Northern blot studies, and 2) bound to
nitrocellulose for hybrid-selection studies.

Figure 5 shows the results of Northern blot analysis of FHV-l late
RNA using the Bam HI/Sal I subfragments of clone EM 5. Three major RNA
species with sizes of 2.9, 1.5 and 1.0 kb were detected by total clone
DNA. El showed strong hybridization to the 2.9 kb RNA and weak
hybridization to a series of smaller, minor RNA species. E2.hybridized
intensely to all three major RNA species while E3 showed strong
hybridization to only the two smaller RNAs. The location of these RNA's on

the EM 5 map are shown in Figure 4. These results suggested that these RNA

142

Figure 3. Map locations of FHV-l late gene products. Figure shows location
of FHV-l Sal I restriction fragments on the genome and indicates the sizes
(in kilodaltons) of the in yigrg translation products selected for by each
fragment. Sal I fragments L, K, M, and F have not been cloned and were not

included in this analysis.

 

143

 

e. 9 TI.

 

 

 

 

9.3
3.2.
«0.3. 8.3.230 3.3.2. 2.2. 3.8.
no
.mnmw. s
- . A . ,
:_ _ _ _ _ _ _ . _ _ _
a z... u x 4 o . a z" a .. ..
_
_lll
_
.5 u .5 2.. 3.»

Figure 3

144
molecules may be transcribed from overlapping regions of the genome or
contain common sequences.

Figure 6 shows SDS-PAGE analysis of the translation products selected
for by the subfragments of clone EM 5. In this experiment, only four
translation products were selected for by total clone DNA with sizes of
105, 73, 58, and 45 kd. It is possible that the 82 and 64 kd translation
products identified in the previous hybrid selection experiment were
either premature termination or breakdown products. Both E1 and E2
selected for the 105 and 73 kd peptides and E3 selected for the 58 and 45
kd peptides. It was unclear from these results whether the 58 and 45 kd
translation products were being selected for by the E1 or E2 fragments
since background bands are also visible in this region of the gel. The
Northern blot analysis suggested that E2 should be able to select for the
two smaller RNA species which presumably code for the 58 and 45 kd
proteins. Given this assumption, there is good correlation between the
size of the major RNA's and proteins which map to the individual Bam
HI/Sal I subfragments (Figure 4).

RNA selected by clone EM 5 was also translated in the presence of dog
pancreas microsomes (Figure 6). The migration of all of the translation
products appeared to be altered by the addition of microsomes. Two bands
were visible in the microsome-treated reactions that were slightly larger
than the 45 and 58 kd bands found in the untreated reactions. These could
represent partially glycosylated molecules and their sizes are approaching
those of the 68 and 60 kd glycoproteins found on FHV-l virions. These
results confirmed the initial observation that the 45 kd translation
product is a substrate for cotranslational modification and indicated that

the 58 kd translation product is also processed. Though the 73 and 105 kd

145

Figure 4. Analysis of EM-S. Figure shows locations of Sal I/Bam HI
subfragments E1 to E3. The line under each subfragment indicates sizes (in
kilodaltons) of in giggg translation products selected for by each
subfragment (above of line) and sizes (in kilobases) of late RNA species

which hybridized to each subfragment (below line).

 

 

146

mm

a.
no

mm

mm

a...TIIIl.

Q..= an
meson» 8

«u .u
a

Figure 4

147

Figure 5. Northern blot analysis of FHV-l late RNA. FHV-l late RNA was
subject to electrophoresis through formaldehyde-agarose gels and
hybridized to nick-translated total EM 5 DNA (E) or EM-S subfragments E1

to E3 (lanes 1-3). Sizes of the RNA species (in kb) are indicated at left.

 

148

 

 

 

 

Figure 5

149

Figure 6. In yigrg translation products selected for by subfragments of
EM-S. DNA from EM-S (E), from gel-purified Bam HI/Sal I subfragments El-E3
(1-3), or from bacteriophage vector (EMBL-3) (fc) was denatured, bound to
nitrocellulose and used to select infected cell RNA which was translated
in the presence (E+) or absence (E-, 1-3) of dog pancreas microsomes.
Translation products were analyzed by SDS-PAGE. Sizes (in kd).of the FHV-l

specific translation products are indicated.

 

150

105»

73>

58>

45>

 

 

 

Figure 6

151

translation products were also altered by the addition of microsomes, no
higher molecular weight forms of these molecules were visible on the gel.
A higher molecular weight form of the 73 kd protein which may correspond
to the 74 kd protein immunoprecipitated from deglycosylated virions could
have been obscured by the 92 kd background band.

These results indicate that two and possibly three FHV-l glycoprotein
genes are clustered in a small region of the genome which is located near
the end of the U1 region, and these proteins may be coded for by

overlapping transcripts.

Discussion

This report decribes the initial attempts to map FHV-l late gene
products on the viral genome. In ziggg translation of infected cell RNA
produced a number of translation products which corresponded in size to
proteins found on FHV-l virions. Most of these translation products
reacted with a goat anti-FHV-l serum that was used in the initial
characterization of the FHV-l antigens (10). The migration of two of the
translation products was clearly altered by the addition of dog pancreas
microsome extracts to the translation reaction. These altered translation
products comigrated with bands produced by the enzymatic deglycosylation
of FHV-l virions.

The purpose of deglycosylating virions was to attempt to determine
the sizes of the glycoprotein precursors to provide a reference point for
the analysis of the translation products. Without monoclonal or
monospecific antibody against the individual glycoproteins it is difficult
to study the events involved in the maturation and processing of these
molecules. It is assumed that the 58 and 45 kd precursors are processed
into the 68 and 60 kd glycoproteins found on FHV-l virions.
Deglycosylation of the 105 kd glycoprotein resulted in two different
precursors depending on the enzyme used. Endo F treatment removes only
N-linked carbohyrates while the mixed glycosidase degrades a variety of
oligosaccharide side chains. Therefore, the 82 kd precursor observed in
the Endo F-treated virions could be a partially glycosylated form which is
further degraded to a 74 kd molecule by the mixed glycosidase. The 74 kd
precursor corresponds with an abundant in giggg translation product that

is altered by the addition of microsomes to the translation reaction. Of

152

153
course, it is possible that there may be more than one 105 kd glycoprotein
and more than one precursor. In fact, other researchers (1) using gradient
SDS-PAGE analysis have identified three glycoprotein bands with
approximate sizes of 120-112 kd on FHV-l virions. It is not clear if these
three bands are glycosylation intermediates of the same glycoprotein or
represent more than one glycoprotein.

Using recombinant clones to hybrid select RNA it was possible to
determine the map locations of 21 of the in yitrg translation products.
FHV-l late genes were found to map throughout the genome with the majority
of them concentrated in the unique long region. Few late genes mapped in
the 8 region. It is interesting that several neighboring fragments
selected for proteins of the same approximate molecular weight suggesting
that the genes which code for these proteins are contained on both
fragments. This analysis is incomplete since several fragments of the
genome were not represented in the clone collection. The results are
sufficiently complete to show that the distribution of FHV-l late genes is
similar to the late gene arrangement of other herpesviruses (4,19).

An abundant protein of 130 kd was present in the in yiggg translation
products of infected cell RNA and comigrated with an immunoprecipitatble
non-glycosylated protein on FHV-l virions. This protein may be the major
viral capsid protein because of its size, immunogenicity, relative
abundance, and because it is not present in detergent extracts of FHV-l
virions (Rota and Maes, unpublished observations). Unfortunately, neither
total virion DNA nor DNA from any of the cloned fragments was able to
select for this protein under the conditions used in this study. Is is
possible that RNA coding for larger proteins is less resistent to the

manipulation required for hybrid selection experiments than RNA which

154

codes for smaller proteins. Other researchers have been able to hybrid
select RNAs which code for large molecular weight proteins using
conditions that were similar to those used in this study. However, in
these studies the amount of high molecular weight translation products was
reduced compared to the smaller translation products (4).

A region of the genome containing Sal I fragment E hybridized to
three late RNA species which coded for four polypeptides. Further studies
will be needed to determine to what extent these transcripts may overlap
or whether they contain common sequences. Clusters of late genes which
produce several overlapping transcripts have been identified on the genome
of HSV (19).

Two of the polypeptides selected for by the Sal I E region of the
FHV-l genome were cotranslationally processed to higher molecular weight
forms by the microsome extracts. Complete glycosylation usually does not
occur in zitgg (12) so it would be difficult, without specific antibody,
to determine if these higher molecular weight forms of the translation
products correspond to any of the glycoproteins found on intact virions.
Rea et a1 (15) have used a similar approach to identify a restriction
fragment of pseudorabies virus which selected for a translation product
that was modified by the addition of microsomes. This protein reacted with
monoclonal antibody directed against the viral glycoprotein that is found
in the medium of infected cells.

The migration of the other two peptides selected for by Sal I E was
altered by the microsomes. Since no higher molecular weight forms were
apparent, the possibility that the translation of these proteins was
inhibited by the microsomes cannot be ruled out. Therefore, while there is

good evidence that the 58 and 45 kd translation products are glycoprotein

155

precursors, the identity of the 73 kd translation product is unclear.
These initial studies indicate that further analysis of this region of the

genome are justified.

References

1) Fargeaud, D.C., C. Benoit-Jeannin, F. Kato, and G. Chappuis. 1984.
Biochemical study of the feline herpesvirus-l: identification of
glycoproteins by affinity. Arch. Virol. 80:69-82.

2) Gaskell, R.M. and R.C. Povey. 1982. Transmission of feline viral
rhinotracheitis. Vet. Rec. 111:359-362.

3) Gaskell, R.M, P.E. Dennis, L.E. Goddard, R.M. Cooker and J.M. Wells.
1985. Isolation of feline herpesvirus from the trigeminal ganglia of
latently infected cats. J. Gen Virol. 66:391-394.

4) Hell, W., S. Modrow, and H. Wolf. 1985. Mapping of herpes saimiri
proteins on the viral genome: proteins dependent and not dependent on
viral DNA synthesis. J. Virol. 56:414-418.

5) Hermann, S.C., R.M. Gaskell, B. Ehlers and H. Ludwig. 1984.
Characterization of the feline herpesvirus genome and molecular
epidemiology of isolates from outbreaks and latent infections. in: Latent
Herpesvirus Infections in Veterinary Medicine. p. 321-336. A. Wittman,
R.M. Gaskell and H.J. Rziha (eds), Martinus-Nijhoff Publishing, Boston.

6) Honess, R.W., and B. Roizman. 1974. Regulation of herpesvirus
marcomolecular sunthesis I. Cascade regulation of the synthesis of three
groups of viral proteins. J. Virol. 14:8-19.

7) Jackson, R.J. and T. Hunt. 1983. Preparation and use of
nuclease-treated rabbit reticulocyte lysates for the translation of
eukaryotic messenger RNA. Meth. Enzym. 96:50-74.

8) Laemmli, U.K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature (London) 227:680-685.

9) Lee, Y.T., M. Para, and P.G. Spear. 1982. Location of the structural
genes for glycoproteins gD and gE and for other polypeptides on the S
component of herpes simplex type 1 DNA. J. Virol. 43:41-49.

10) Maes, R.K., S. Fritsch, L. Herr, and P. Rota. 1984. Immunogenic
proteins of feline rhinotracheitis virus. J. Virol. 51:259-262.

11) Maniatis, T., E. Fritsch, and J. Sambrook. 1982. Molecular cloning: a
laboratory approach. Cold Spring Harbor Laboratories, New York.

12) Matthews, J., G. Cohen, and R.J. Eisenberg. 1983. Synthesis and
processing of glycoprotein D of herpes simplex virus types l_and 2 in an
in vitro system. J. Virol. 48:521-533.

13) Parnes, J.R., B. Velen, A. Felsenfeld, L. Ramanathan, V. Ferrini, E.
Apella, and J.C. Seidman. 1981. Mouse B2 microglobulin cDNA clones: a
screening procedure from cDNA clones corresponding to rare mRNAs. Proc.
Nat. Acad. Sci. USA. 78:2253-2257.

156

157

14) Povey, R.C. 1979. A review of feline viral rhinotracheitis (feline

herpesvirus) infection. Comp. Immunol. Microbiol. of Infect. Dis.
2:373-387.

15) Rea, T.J., J. Timmins, G. Long, and L.E. Post. 1985. Mapping and
sequence of the gene for the pseudorabies virus glycoprotein which
accumulates in the medium of infected cells. J. Virol. 54:21-29.

16) Roizman, B. 1980. The herpesviruses, vol. 1, p 1-23. Plenum Press, New
York.

17) Rota, P.A., R.K. Maes and W.T. Ruyechan. 1985. Physical
characterization of the genome of feline herpesvirus-l. (submitted for
publication).

18) Rota, P.A., R.K. Maes and J.F. Evermann. 1985. Biochemical and
antigenic characterization of feline herpesvirus-l-like viruses from dogs.
Arch. Virol. in press.

19) Wagner, E.R., 1985. Individual HSV transcripts: characterization of
specific genes. in: The Herpesviruses, vol. 3. p. 45-103. Plenum Press,
New'York.

20) Weber, K. and M. Osborn. 1969. The reliability of molecular weight
determinations by dodecyl-sulfate polyacrylamide gel electrophoresis. J.
Biol. Chem. 244:4406-4412.

Chapter 5

Biochemical and Antigenic Characterization of Feline Herpesvirus-l-Like

Isolates from Dogs.

Paul A. Rota and Roger K. Maes

158

Summagy

The DNA and polypeptide patterns of feline herpesvirus-1 (FHV-l), a
virus usually associated with feline respiratory infections, were compared
to those of five herpesvirus isolates from dogs. These canine isolates had
been shown to be antigenically similar to FHV-l by cross neutralization
tests. DNA from FHV-l (C-27 strain) and each canine isolate was digested
with either Bam HI, Eco R1 or Sal I and analyzed on .8% agarose gels. The
restriction digest patterns of the canine isolates were nearly identical
to.C-27 for all three restriction enzymes. Interestingly, all of the
canine isolates showed a small extension of the largest Bam HI fragment
(14.5 kb) that was not present in the C-27 strain. Bam HI digested FHV-l
DNA from clinical cases in cats had digest patterns that were very similar
to the canine isolates and also showed an extension of the 14.5 kb
fragment. Southern blotting experiments revealed that DNA from the canine
isolates has extensive homology to C-27 DNA. SDS-PAGE analysis of
radiolabeled polypeptides from C-27 and the canine isolates showed
identical virion-associated polypeptide profiles. In addition, a goat
anti-FHV-l antiserum precipitated three glycoprotein antigens from the
canine isolates with migration patterns that were identical to the three
major antigenic glycoproteins found on C-27. Hind III and Eco R1 digestion
patterns of canine herpesvirus DNA showed no similarity to C-27 DNA. In
addition, canine herpesvirus DNA had no homology to C-27 DNA under the

stringent conditions used.

159

Introduction

 

Feline herpesvirus-1 (FHV-l) is a major upper respiratory tract
pathogen in cats. The virus can also cause more severe generalized disease
in kittens, abortions in pregnant cats as well as latent infections (17).
The virus has been classified as a member of the alphaherpesvirinae and
has been found to have a limited in yigg and in 21539 host range infecting
only Felidae or cells of feline origin (18).

Herpesviruses which were shown to be antigenically similar to feline
herpesvirus-l (FHV-l) have recently been isolated from dogs. The primary
clinical sign in each of the cases examined was diarrhea. The age,
vaccination history and antibody status of the dogs at the time of virus
isolation were not consistent from case to case. Virus isolates from these
dogs grew only in Crandell-Rees feline kidney (CRFK) cells and not in
monkey kidney (Vero) cells or Madin Darby canine kidney (MDCK) cells. The
isolates were chloroform sensitive, showed basophilic intranuclear
inclusion bodies and were neutralized by antiserum to FHV-l but not by
antiserum to canine herpesvirus (CHV) (4). It was concluded that these
infections were caused by a virus that was similar to FHV-l.

Thus far, CHV and pseudorabies virus have been the only recognized
herpesvirus infections in dogs (1, 5, 9, 16), but they have not been
associated with enteric infections in dogs over two weeks old. Also,
previous studies have indicated that FHV-l was incapable of infecting dogs
(7). Current research in our laboratory has focused on the physical and
antigenic characterization of FHV-l. The report on the canine isolates was

therefore very interesting to us. We obtained five canine isolates in

160

161
order to do a more detailed biochemical and antigenic comparision of these
canine herpesvirus isolates to the C-27 prototype strain of FHV-l.

We report here that the five canine isolates have genomes which show
restriction enzyme digestion patterns that are nearly identical to those
of C-27 and to those of FHV-l isolates of feline origin. Also, these
canine isolates appear to contain all of the major antigens and
virion-associated polypeptides of C-27 including the three major

glycoproteins.

Methods and Materials

 

Virus and Cells. Five previously described (4) canine FHV-l-like isolates
and five FHV-l strains of feline origin, isolated at the Washington Animal
Disease Laboratory, Pullman WA, were used in this study. The C-27
prototype strain of FHV-l was obtained from the American Type Culture
Collection, Rockville, MD (#VR 636). The viruses were propagated in
Crandell-Rees Feline Kidney (CRFK) cells which were maintained in Eagle’s
Minimum Essential Medium (EMEM) (K.C. Biologics, Lenexa, KS.) with 10%
fetal bovine serum (Hyclone defined, Sterile Systems, Logan, UT). Canine
herpes virus was obtained from the A.T.C.C. (#VR-552) and grown in Madin

Darby canine kidney (MDCK) cells using the same medium.

Plaque Assay Procedure. Confluent monolayers of CRFK cells growing in
tissue culture plates (60 mm, Corning) were infected with serial dilutions
of the virus sample in EMEM. After a one hour adsorbtion period, the
inoculation medium was removed and EMEM containing 1% agarose was layered
over the cells. Plaques were visualized 48 hours later by staining the
viable cells with 2% neutral red in Hank's Balanced Salt Solution. The
plaques were picked with a sterile Pasteur pipette, diluted into EMEM, and
allowed to incubate at 4°C. for 4 hours before being used to infect CRFK

cells.

Isolation of Viral DNA. Confluent monolayers of CRFK cells were infected

 

with virus at an multiplicity of infection of .01. When the CPE was
advanced, the infected cells were shaken from the culture vessel surface

and pelleted by low speed centrifugation (500 rpm, 10 min). The washed

162

163
cells were resuspended in 10 ml of TE (lOmM Tris-HCl pH 7.5, 1 mM EDTA)
and left on ice for 15 minutes. NP-40 (Sigma, St. Louis, MO.) was added to
a final concentration of .5% and the cells were incubated for another 15
minutes on ice. Cell nuclei were pelleted by centrifugation at 2,000 x g
for 10 minutes and the supernatant containing the viral nucleocapsids was
adjusted to 1% SDS and 25mM EDTA. Pre-digested (80°C., 15 min.) pronase
was added to a final concentration of 1 mg/ml and the supernatant was
incubated overnight at 37°C. Saturated sodium iodide (1.5 volumes)
containing 1 ug/ml ethidium bromide was added to the supernatant. The
mixture was centrifuged for 48 hours at 44,000 rpm at 20°C. in an SW
50.1 Rotor (Beckman Instruments, Palo Alto, CA.). The viral DNA bands
were visualized by u.v. illumination and harvested by side-puncturing the
tube with a 20 ga. needle. The DNA-containing samples were extracted
three times with isoamyl alcohol (5 volumes) before being dialyzed

extensively against TE.

Restriction Enzyme Analysis. One microgram amounts of viral DNA were
incubated with 10 units of either Bam HI, Eco R1, Sal I or Hind III
(Bethesda Research Laboratories, Gaithersburg, MD.) using the reaction
conditions recommended by the manufacturer. When the digestion was
complete, 1 uCi of the appropriate a32P-labeled trinucleotide (Bam

HI: dGTP, Eco R1 and Hind III: dATP, Sal I: dTTP, Amersham, Arlington
Heights, 111., sp. act. 800 Ci/mmole) was added to the reaction along with
1 unit of the Klenow fragment of DNA polymerase (BRL). The samples were
incubated at room temperature for 45 minutes, precipitated twice with

ethanol and loaded onto .8% agarose gels and electrophoresed (20 V, 20

164
hrs). When electrophoresis was complete, the gel was dried on a gel dryer

without heat and exposed to Kodak X-AR film.

Southern Blotting. After electrophoresis, restriction fragments were

 

transferred to nitrocellulose filters by the method of Southern (21).
32P-labeled DNA probes were prepared by nick translation using

standard methods (13). Filters were prehybridized for 4 hrs at 42°C. in
20% formamide, .6M NaCl, .06M sodium citrate, .01M EDTA, .1% SDS, 5X
Denhardt's solution (.1% ficoll, .1% polyvinylpryollidone, .1% bovine
serum albumin), and 50 ug/ml denatured salmon sperm DNA. Hybridizations
were performed at 42°C. in 50% formamide, 1X Denhardt's, 4X SSC (1X SSC:
.15M NaCl. .015M sodium citrate, pH 7.0), .1M EDTA, .1% SDS, 50 ug/ml
denatured salmon sperm DNA. After Hybridization, the filters were washed
twice for 15 min with 2X SSC at room temperature followed by two 1 hr
washes with .1X SSC at 68°C. The filters were allowed to dry and exposed

to X-ray film.

Immunoprecipitation Analysis. 14C-glucosamine or 35S-methionine
labeled virions were prepared as previuosly described (14) The virions
were partially purified from the supernatant medium by centrifugation
through cushions of 25% potassium tartarate in TE at 100,000 x g for 1
hour at 4°C. in a Beckman SW-27 rotor.

Following preincubation with normal goat serum, the samples were
incubated overnight with goat anti-FHV-l serum. The complexes were
pelleted by addition of 10 volumes of a protein-A producing strain of

Staphylococcus aureus prepared by the method of Kessler (10). The

165

immunoprecipates were then analyzed by SDS-PAGE (11). The gels were fixed

and dried (12) and exposed to X-ray film.

Results

Figure 1 shows a comparison of the Bam HI, Eco R1 and Sal I digestion
patterns of DNA from the C-27 strain of FHV-l and DNA from five
plaque-purified canine isolates. The sizes of the Bam HI, Eco R1 and Sal I
restriction fragments of C-27 DNA and the locations of some of these
fragments on the viral genome have been determined in a concurrent study
(19). In the Bam HI digest of C-27 DNA, the largest two fragments migrate
as a single two molar band of 14.5 kb. In all of the canine isolates, one
of these fragments is slightly larger and two one molar bands are
observed. The canine isolates also show some variation in the Ben HI 4.8,
2.7 and 1.4 kb fragments. However, these variations are not consistent
among the individual isolates. The 4.8 and 1.4 kb Bam HI fragments have
been identified as terminal fragments (19) and the size differences may
be due to variations in the size of the terminal repeat DNA.

Consistent differences were not observed between the canine isolates
and C-27 in both the Eco R1 and Sal I digests (Fig.1). The Eco R1 12.0,
5.7, 2.7 and 2.1 kb fragments and Sal I 10.1, 6.8 and 3.9 kb fragments
show size variation from isolate to isolate. The Eco R1 12.0 and 2.7 kb
and Sal I 10.1 and 3.9 kb fragments are located at the viral termini. The
Eco R1 5.7 and 2.1 kb and Sal I 6.8 kb fragments are located within the
internal inverted repeat regions of the viral genome (19). Size variation
was apparent in other fragments whose locations have not yet been
determined.

For each canine isolate, DNA was purified from a number of individual
plaque isolates and compared by restriction endonuclease digestion (data

not shown). The restriction digest patterns were consistent for the

166

167

Figure 1. Restriction enzyme digest patterns of C-27 DNA (lane 1) and DNA
from five canine isolates (lanes 2-6). Restriction fragments were
end-labeled with 32P and separated on .8% agarose gels. Figure shows

autoradiogram of dried gels. The molecular weights (kb) of certain bands

are shown on the left.

 

 

 

 

 

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169
individual isolates suggesting that the variations observed in Figure l
are the result of interstrain and not intrastrain variation.

To determine if there were any consistent differences between the
canine isolates and field strains of FHV-l, DNA was also purified from
five plaque-purified FHV-l clinical isolates of feline origin. Figure 2
shows a comparison of the Bam HI digestion patterns of these isolates to
C-27. All of the feline isolates showed the same extension of one of the
largest Bam HI fragments that was observed in the digests of DNA from the
canine isolates. The feline isolates also showed the same general
variation in the size of Bam HI terminal fragments.

Southern blotting experiments were done to examine the extent of
similarity between the genomes of the canine isolates and C-27. DNA from
the canine isolates was digested with Dan HI, electrophoresed, transferred
to nitrocellulose, and probed with nick-translated C-27 DNA (Figure 3).
All of the Bam HI fragments of the canine isolates were detected by the
C-27 probe indicating that the canine isolates share extensive sequence
homology to C-27 throughout their entire genomes.

Purified virus stocks of the C-27 strain and the canine isolates,
labeled with 1“C-glucosamine or 35S-methionine, were used to
compare the patterns of the virion-associated and major antigenic
structural polypeptides. Figure 4A shows a direct SDS-PAGE comparison of
one of the canine isolates with C-27. The polypeptide patterns based upon
one dimensional SDS-PAGE appear to be identical suggesting that the canine
isolates do not differ significantly from C-27 with respect to the
structural components of the virion.

Figure 4 also shows a comparison of the immunoprecipitation analysis

of a canine isolate and C-27 obtained with a goat anti-FHV-l antiserum

170

Figure 2. Bam HI digestion patterns of C-27 DNA (lane 1) and five isolates
of FHV-l of feline origin (lanes 2-6). Restriction fragments were
end-labeled with 32P and separated on .8% agarose gels. Figure shows

autoradiogram of a dried gel.

171

 

0“.

 

Figure 2

172

Figure 3. Southern blot analysis of C-27 DNA ( lane 1) and DNA from five
canine isolates (lanes 2-6). DNA samples were digested with Dan HI,
separated on .8% agarose gels and transferred to a nitrocellulose filter.
The filter was probed with nick-translated C-27 DNA and washed as

described in Methods and Materials.

173

 

 

Figure 3

174

Figure 4. SDS-PAGE and immunoprecipitation analysis of C-27 and canine
isolate purified virions. Panel A shows direct SDS-PAGE analysis of
35S-methionine-labeled C-27 virions (lane 1) and canine isolate

virions (lane 2). 1“C-glucosamine-labeled C-27 virions are shown (lane

3) as size markers. Panels B and C show SDS-PAGE analysis of
immunoprecipitates formed after the incubation of radiolabeled C-27
virions (lanes 82, Cl) and canine isolate virions (lanes B3, CZ) with goat
anti-FHV-l antiserum. Panel B shows 35S-methionine labeled virions,

panel C shows 1l‘C-glucosamine labeled virions. 14C-glucosamine

labeled virions are shown in panel B (lane 1) as size markers.

3:1de

ysis of

1mm (1‘

(:2) with!

310851

175

 

 

Figure 4

176
(14). SDS-PAGE analysis of the immunoprecipitates reveals that both
viruses appear to be antigenically equivalent (Fig. 4B). Three of the
common antigens also labeled with 14C-glucosamine indicating that both
viruses contain the same major antigenic glycoproteins (Fig. 4C).
Radiolabeled virion preparations from the other four canine isolates
reacted with the goat serum in the same manner as the isolate shown in
Figure 4 (data not shown).

Figure 5 shows a comparison of the Hind III and Eco R1 digestion
patterns of CHV and FHV-l DNA. It is obvious from the data that the
digestion patterns are completely different . Also, in Southern blotting
experiments C-27 DNA did not hybridize to CHV DNA under the conditions
used in Figure 3 (data not shown). There appears to be no homology between
the genomes of feline and canine herpesviruses using the hybridization and

wash conditions described above.

177

Figure 5. Restriction enzyme digest patterns of C-27 DNA (lanes 1,3) and
canine herpesvirus DNA (lanes 2,4). Lanes 1 and 2 show an Eco R1 digest,
lanes 3 and 4 a Hind III digest. DNA fragments were end-labeled with
32P and separated on .8% agarose gels. Figure shows an autoradiogram

of a dried gel.

178

 

Figure 5

Discussion

 

Five herpesvirus isolates from dogs were compared to the C-27
prototype strain.of feline herpesvirus-1 by restriction endonuclease
digestion, Southern blotting, SDS-PAGE and immunoprecipitation. DNA from
the five canine isolates showed Bam HI, Eco R1 and Sal I digestion
patterns that were nearly identical to those of the prototype strain of
FHV-l and to DNA purified from clinical isolates of FHV-l. The majority of
the variation observed in the digest patterns between individual virus
isolates occurred in fragments located at the viral termini or within the
inverted repeat regions of the viral genome. Therefore, some of the
heterogeneity presumably is due to variations in the DNA of terminal or
internal repeat sequences. The feline and canine case isolates all had an
extension of one of the two 14.5 kb Bam HI fragments observed in C-27 DNA.
It is of molecular epidemiological importance that the restriction
patterns generated by a number of clinical isolates from both dogs and
cats were very similar. Generally, only minor variations are observable in
restriction digestion patterns of a number of case isolates. This
stability of the genome of FHV-l has been noted in a previous study which
examined the restriction endonuclease digestion patterns of DNA from a
number of FHV-l isolates from Europe, the United Kingdom and the United .
States (6). In this study, some variation was found in the sizes of
terminal restriction fragments. Another interesting aspect is the
extension of the 14.5 kb Bam HI fragment in all of the clinical isolates
but not in the C-27 strain. The role of this extension is unclear and the
location of this fragment on the viral genome has not yet been determined.

It may code for viral functions that are lost on repeated cell culture.

179

180

Both the canine isolates and the clinical isolates of FHV-l were studied
after a low number of passages in cell culture, while the C-27 strain had
been passed repeatedly in culture in our lab before this study. We did not
notice any difference in plaque morphology between C-27 and the canine
isolates, however.

Southern blotting experiments confirmed and extended the restriction
analysis data in the sense that a C-27 DNA probe was able to detect all of
the Bam HI fragments of the canine isolates.

The canine isolates and the C-27 strain of FHV-l showed identical
virion-associated polypeptide patterns by SDS-PAGE analysis. Also, a goat
anti-FHV-l antiserum recognized the same major virion-associated antigens
from purified virions of both the canine isolates and the C-27 strain. In
addition, both the canine isolates and the C-27 strain appear to share at
least three glycoprotein antigens. These three glycoproteins have been
previously identified as the major glycoprotein antigens of FHV-l (14).

It is theorectically possible that the canine isolates contained unique
antigens that were not recognized by the goat antiserum. These antigens
would most likely be minor since they could not be visualized in the
direct SDS-PAGE analysis of the viral structural proteins. It would be
necessary to use more rigorous methods of analysis to determine if such
minor differences exist between the protein antigens of these virus
isolates. It will be especially important to further characterize the
common glycoproteins because herpesvirus glycoproteins are extremely
important in viral infectivity and in host range specificity (2,15). These
molecules are also important targets of the host's immune response (20).

We conclude from the results of the restriction digestion, Southern

181

blotting, SDS-PAGE and immunoprecipitation that the canine isolates we
examined were very similar to the C-27 strain of FHV-l.

The isolation of feline-related viruses from dogs is not without
precedent. Previous studies have reported the isolation of feline
calicivirus (3) and a parvovirus related to feline panleucpaenia virus (8)
from dogs.

The disease potential of the canine FHV-l-like isolates remains
unclear. The initial isolations were made from dogs showing readily
observable clinical signs. Since these isolates had no HA activity and
grew only in CRFK cells, is seems unlikely that the reported clinical
signs were due to simultaneous infection with other viruses, especially-
canine parvovirus. In a small scale experiment in our laboratory, dogs
orally and intranasally exposed to the canine isolates developed mild
clinical signs. These included a rise in temperature, mild leucopenia and
in some dogs, soft stools. Postinfection, but not preinfection, sera from
these dogs were able to immunoprecipitate several antigens from FHV-l
virions (Maes and Rota, unpublished observations).

Data from this study show that the canine isolates are clearly
different from CHV, a recognized herpesvirus pathogen of dogs. CHV DNA
gave restriction digest patterns that were markedly different from FHV-l
and did not hybridize to a FHV-l probe under the stringent hybridization
and wash conditions used. Southern blotting studies using varying
stringency conditions are currently underway. This will allow us to obtain
a more precise definition of the extent of homology between the DNA's of
CHV and FHV-l. Furthermore, antiserum against both CHV and FHV-l is being
used in immunoprecipitation studies to determine if there are common

antigens shared by these two viruses.

References

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response. J. Amer. Vet. Med. Assoc. 555, 1714-1721 (1970).

2. Eberle, R., Courtney, R.: Preparation and characterization of specific
antisera to individual glycoprotein antigens comprising the major
glycoprotein region of herpes simplex type 1. J. Virol. 55, 982-987
(1980).

3. Evermann, J.F., Bryan, G.M., McKeirnan, A.J.: Isolation of a
calicivirus from a case of canine glossitis. Canine Pract. 5, 36-39
(1981).

4. Evermann, J.F., McKeirnan, A.J., Ott, R.L., Reed, L.A.: Diarrheal
condition in dogs associated with viruses antigenically related to feline
herpesvirus. Cornell Vet. 12, 285-291 (1982).

5. Gustafson, D.P.: Pseudorabies. In: Leman, A.D., Glock, R.D.,
Mengeling, W.L., Penny, R.H.C., Scholl, E., and B. Straw (eds.), Diseases
of Swine, 209-223. Ames, Iowa: Iowa State University Press. 1981.

6. Hermann, S.C., Gaskell, R.M., Ehlers, B., Ludwig, H.: Characterization
of the feline herpesvirus genome and molecular epidemiology of isolates
from natural outbreaks and latent infections. In: Wittman, G., Gaskell,
R.M., and H.J. Rziha (eds.), Latent Virus Infections in Veterinary
Medicine, 321-336. Boston, Massachusetts: Nijhoff Publishers. 1984.

7. Horvath, Z.A., Bartha, L., Papp L., Juhasz, M.: On feline
rhinotracheitis. Acta. Vet. 55, 415-420 (1965).

8. Johnson, R.H., Spadbrow, P.B.,: Isolation from dogs with severe
enteritis of a parvovirus related to feline panleucopaenia virus, letter.
Aust. Vet. J. 55, 151 (1979).

9. Karpas, A., King, N.W., Garcia, F.G., Calva, F., Cross, R.E.: Canine
tracheobronchitis: Isolation and characterization of the agent with
experimental productiuon of the disease. Proc. Soc. Expt. Biol. Med. 121,
45-52 (1982).

10. Kessler, S.W.: Cell membrane antigen isolation with the staphylococcal
protein-A antibody adsorbent. J. Immunol. 511, 1482-1490 (1978).

11. Laemmli, U.K.: Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature (London) ggz, 680-685 (1970).

12. Laskey, R.A., Mills, A.D.: Quantitative film detection of 3H and
14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 55,
335-341 (1975).

13. Maniatis, T., Fritsch, E., Sambrook, J.: Molecular Cloning: A

Laboratory Manual, 109-112. Cold Spring Harbor, New York: Cold Spring
Harbor Laboratory. 1982.

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14. Maes, R.K., Fritsch, S.L., Herr, L.L, Rota, P.A.: Immunogenic
proteins of feline rhinotracheitis virus. J. Virol. 51, 259-262 (1984).

15. Pereira, L., Cassai, R., Honess, R., Roizmam, B., Temi, M., Nahmias,
A.: Variability in the structural polypeptides of hepres simplex virus I
strains: Potential application in molecular epidemiology. Infect. Immun.
55, 271-275 (1976).

16. Poste, G., King, N.: Isolation of a herpesvirus from the canine
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19. Rota, P., Maes, R.K., Ruyechan, W.: Physical characterization of the
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SUMMARY

While the description of the FHV-l genome provided by this report is
far from complete, it is sufficient to indicate that the structure and
general organization of the FHV-l genome is similar to the structure and
organization of other alpha herpesviruses. These experiments provide the
first step toward understanding the molecular biology of FHV-l and should
provide the basis for future studies in several areas.

The cloned FHV-l DNA fragments should be useful in the study of FHV-l
latency. These clones could initially be used to construct hybridization
probes of high specific activity for use in Southern blotting and 55 5555
hybridization studies. Once the tissue or tissues of latency have been
identified, a rapid and sensitive method could be developed to determine
if cats are FHV-l carriers. This assay would be needed to fully evaluate
the efficacy of current and proposed FHV-l vaccines. Also, these clones
could be used as probes in Northern blotting experiments to measure gene
expression from various regions of the FHV-l genome during latency in the
natural host.

When monoclonal and monospecific antibodies to FHV-l antigens become
available, the gene mapping studies described in Chapter 4 could be
continued to isolate genes for individual FHV-l antigens. These antigens
could be expressed via recombinant DNA techniques and assessed for their
ability to prevent clinical disease and the establishment of latent

infections in the natural host.

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185

Finally as more information on the biochemical structure of FHV-l is
obtained, a more complete comparison could be made between FHV-l and other
herpesviruses. It will be interesting to compare the genome arrangements

of different herpesviruses with similar biological properties.