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Membrane proteins of alcelaphine herpesvirus 1

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Membrane proteins of alcelaphine herpesvirus 1
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Adams, Steven Wade
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Antibodies ( jstor )
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Cattle ( jstor )
DNA ( jstor )
Fever ( jstor )
Infections ( jstor )
Molecular weight ( jstor )
Molecules ( jstor )
Monoclonal antibodies ( jstor )
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Cattle ( mesh )
Herpesviridae ( mesh )
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Thesis (Ph. D.)--University of Florida, 1989.
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Includes bibliographical references (leaves 93-98).
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Typescript.
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Vita.
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by Steven Wade Adams.

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MEMBRANE PROTEINS OF ALCELAPHINE HERPESVIRUS 1


By

STEVEN WADE ADAMS
















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1989




MEMBRANE PROTEINS OF ALCELAPHINE HERPESVIRUS 1
By
STEVEN WADE ADAMS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1989


ACKNOWLEDGEMENTS
A summary of work such as that presented in a dissertation cannot
be complete without the acknowledgement of the many individuals that
have played much more than minor roles in the journey to completion.
Members of my committee, Drs. Howard Johnson, Alvin Moreland and Sue
Moyer must be mentioned for their help and encouragement. The great
collage of personalities that shared laboratory space during my tenure
as a graduate student are now too numerous to name individually, but
their influencessocial, philosophical and scientificshould not go
overlooked. Thanks are extended to Dr. Ed Stephens for the use of his
laboratory and large amounts of x-ray film. Dr. N. Balachandran was
extremely helpful in my early days in the lab, and he continues to be
a welcome source of support. Susan Turk has been invaluable in
operating a reagent-locating service and providing hearty laughter.
Last, but certainly not least, I cannot say enough to adequately
acknowledge the long hours of support and assistance provided by my
mentor and major professor, Dr. Lindsey Hutt-Fletcher. Her
indefatigable dedication to graduate student education goes far beyond
the call of duty and must be highly commended.
ii


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS
ABSTRACT V
CHAPTERS
1 INTRODUCTION
Historical Perspective 1
Clinical and Pathological Manifestations 3
Transmission 5
Virus Characteristics 6
Diagnosis 9
Importance of Membrane Proteins 12
2 IDENTIFICATION OF VIRUS-INDUCED PROTEINS
Introduction 15
Materials and Methods 16
Results 21
Discussion 30
3 MONOCLONAL ANTIBODIES
Introduction 32
Materials and Methods 32
Results 36
Discussion 40
4 RELATIONSHIPS OF AHV-1 WITH OTHER BOVID HERPESVIRUSES
Introduction 51
Materials and Methods 52
Results 55
Discussion 63
5 PROTOTYPE DIAGNOSTIC ELISA
Introduction 65
Materials and Methods 65
Results 67
Discussion 71
iii


6 THE gp115 SURFACE PROTEIN
Introduction 75
Materials and Methods 75
Results 77
Discussion 87
7 SUMMARY 90
REFERENCES 93
BIOGRAPHICAL SKETCH 99
iv


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MEMBRANE PROTEINS OF ALCELAPHINE HERPESVIRUS 1
By
Steven Wade Adams
May 1989
Chairman: Lindsey M. Hutt-Fletcher
Major Department: Pathology and Laboratory Medicine
Alcelaphine herpesvirus 1 (AHV-1) is carried as a latent
inapparent infection in Alcelaphine bovid species. In contrast, when
cattle and several species of exotic bovids are infected, mortality
approaches 100%. Although AHV-1 is endemic to and is largely confined
to Africa, a very similar disease thought to be carried by sheep is
found in the United States. Both diseases are known as malignant
catarrhal fever (MCF). Our overall goal was to study the membrane
proteins of AHV-1 and identify those molecules that play a role in the
initiation of virus infection. We intended to apply this information
to the development of novel methods of diagnosis of latently infected
carrier animals. The antigenic relationships of AHV-1 and other
herpesviruses infecting cattle were also examined, in the hopes of
identifying a protein entirely unique to AHV-1 that might be used as a
reagent in the development of a diagnostic test. We were able to
identify a minimum of 6 putative virus-induced membrane proteins in
AHV-1 infected cells, and produce monoclonal antibodies reactive with
4 of them. We were unable to identify a single protein as unique to
v


AHV-1, but determined that two monoclonal antibodies, 12B5 and 31B3,
reacted with epitopes unique to AHV-1. Monoclonal antibody 12B5
exhibited neutralizing activity and was used to assemble a competitive
enzyme-linked immunosorbant assay. The assay tested the ability of
serum known to contain antibodies against AHV-1 to compete with the
monoclonal antibody 12B5 for binding to AHV-1 infected cell antigen.
Preliminary results indicated that an assay of this type represents a
feasible method of diagnosis. The test also detected antibodies in
sheep that were capable of competing with the monoclonal antibody,
supporting the concept that the African form and the sheep associated
form of MCF cure caused by related viruses. Since the monoclonal
antibody 12B5 possessed neutralizing activity, the proteins bound by
this protein were examined more closely. The antibody immuno-
precipitated a complex of 5 proteins whose precursor/product
relationships were elucidated. The mature form of the protein found
in the virion was identified as a disulfide-linked dimer.
vi


CHAPTER 1
INTRODUCTION
Historical Perspective
Alcelaphine herpesvirus 1 (AHV-1) is carried as a latent
infection by African antelope of the family Bovidae, subfamily
Alcelaphine. Alcelaphine species include wildebeest (Connochaetes)
hartebeest (Alcelaphus), and topi (Damaliscus), and are not clinically
affected by infection. In contrast, infection in cattle, exotic
bovids, and several species of deer results in a generalized
lymphopro1iferative disease with a mortality approaching 100%
(18,46,48).
The disease in susceptible species has been known as malignant
catarrh, malignant head catarrh, snotsiekte, and malignant catarrhal
fever (MCF). Malignant catarrhal fever due to Alcelaphine herpesvirus
1 is an exotic disease but is important to the United States for
several reasons. First, MCF has been diagnosed on several occasions
in the United States since the 1970s, and many speculate that the
incidence of MCF is presently increasing (20,35). Sporadic outbreaks
pose an expanding threat to the domestic cattle population as well as
susceptible wildlife species. Second, increased popularity of exotic
game ranching in areas such as Texas has provided increased
opportunities for the dissemination of the disease in nearby cattle
operations. Third, zoological collections have suffered substantial
1


2
mortality in endangered species as a result of transmission of AHV-1
(9,16).
Epidemiologic data suggest that sheep may also serve as
reservoirs for MCF. The sheep-associated form of MCF was first
described in Europe in 1798 and has been called the European or
American form, although its distribution is worldwide (21). This
endemic disease is clinically and pathologically indistinguishable
from the disease caused by AHV-1. Serological surveys have indicated
that approximately 27% of sheep and goats possess antibodies reactive
with AHV-1 by virus neutralization, immunofluorescence, and ELISA
(22). As early as 1929 investigators were successful in transmitting
the sheep-associated form by inoculation of infected blood, although
no virus has yet been isolated from sheep or clinically infected
animals associated with sheep (53,54).
The wildebeest-associated or African form of MCF has been known
to the Masai tribesmen of eastern Africa for centuries, and it has
been estimated that 7% of the annual mortality in cattle in eastern
Africa is due to this disease (40). Masai pastoralists have long
realized the importance of preventing the exposure of their cattle to
the wildebeest, especially during the wildebeest calving season. This
association of wildebeest and MCF was first investigated in 1923 when
Mettan raised a wildebeest calf on a Friesen cow, which resulted in
the death of all exposed cattle. The etiological agent, AHV-1, was
first isolated in 1960 from a blue wildebeest (46).


3
Clinical and Pathological Manifestations
MCF is an acute, generalized disease with mortality near 100%.
It is characterized by fever, depression, profuse nasal and ocular
discharge and encrustation, drooling, photophobia, keratitis, erosion
and diptheresis of mucous membranes, generalized lymphadenopathy, skin
lesions, and, occasionally, cystitis and central nervous involvement
(20). Gross post mortem lesions vary considerably depending on the
course of the disease. Animals that die very acutely may show fewer
lesions other than hemorrhagic enterocolitis. In more protracted
cases, lesions are more severe. The muzzle is often encrusted and
raw, and cutaneous lesions may be hyperemic and occur as a generalized
exanthema with exudation of lymph. These lesions are frequently seen
on the ventral thorax and abdomen, inguinal region, perineum and loin.
Lesions in the respiratory system may vary from mild serous nasal
discharge and hyperemia of the mucous membranes to severe ulceration
of epithelia and formation of prominent pseudomembranes. Epithelia of
the nasal passages and sinuses may be affected as well as pharyngeal,
and laryngeal epithelium. Lesions may also extend to the tracheal and
bronchial mucosa. The mucosa of the alimentary tract may vary from
mild mucosal hyperemia and edema to severe erosions and ulceration on
the dental pad and gingival surfaces, tongue and buccal papilla.
Mucosal inflammation, hemorrhage and ulceration may also be found in
the rest of the gastrointestinal tract including the esophagus,
abomasum, small intestine, colon and rectum. If lesions are present
in the urinary tract, they usually include hyperemia of the bladder


4
mucosa and, occasionally, marked dilatation of bladder mucosal
vessels. Petechiae and ecchymoses may also occur in the renal pelvis
and ureters. The liver is usually slightly enlarged with a prominent
reticular pattern. There may be hemorrhages and erosions in the gall
bladder. A generalized lymphadenopathy is characteristic of MCF and
is most consistently prominent near the head and neck. Fibrinous
polyarthritis has been seen in a number of cases of MCF. Corneal
edema, with ulceration and/or vascularization is often seen.
Malignant catarrhal fever can be confused with other viral diseases,
namely bovine viral diarrhea-mucosal disease, rinderpest, bluetongue,
foot and mouth disease, and vesicular stomatitis. These diseases may
all result in similar gross lesions which necessitate histopathologic
and serologic examination for diagnosis (20).
Lesions that provide a consistent basis for diagnosis of MCF are
found in the epithelia of the gastrointestinal and urinary tracts, the
lymphoid tissue and the adventitia and walls of small blood vessels in
any organ. Vascular lesions consist of fibrinoid necrotizing
vasculitis with a predominantly mononuclear cell infiltrate of the
adventitia. Lymphoid tissue shows destruction and loss of lymphocytes
and an increase in macrophages and lymphoblast proliferation.
Several investigators have pointed out the similarity of lesions
of lymphoproliferation with those of malignant lymphoma and infectious
mononucleosis, raising speculation as to the oncogenic potential of
AHV-1 (23,26). In addition, recent work in which AHV-1 was
inoculated into rats describes neoplastic-like lymphoproliferation


5
in gut-associated lymphoid tissue. This lesion may represent a
potential model for herpes virus-induced oncogenesis (24).
Transmission
Alcelaphine species harbor the virus as a latent infection and
are apparently unaffected clinically. The virus is thought to be
highly cell-associated in the host and is rarely transmissible between
adult wildebeest, although virus has been isolated iron nasal
secretions of captive adult wildebeest after stress or administration
of corticosteroids (56).
Neonatal wildebeest have been shown to shed cell-free AHV-1 in
nasal and ocular secretions and in feces (38). Virus can be isolated
from infected wildebeest calves up to 3 months of age, which coincides
with the appearance of neutralizing antibodies in the nasal secretions
(41). Transmission to cattle or other susceptible species is thought
to occur by inhalation of cell free virus from aerosol droplets or
ingestion of food or water contaminated with infectious secretions or
feces (40). The Masai of eastern Africa have firmly believed for
centuries that transmission of MCF occurs from contact of cattle with
fetal membranes and birth fluids from calving wildebeest. However,
attempts to isolate virus from fresh placentas from calving wildebeest
have been unsuccessful (55).
Virus is also apparently highly cell-associated in susceptible
species, and cattle-to-cattle transmission has not been demonstrated
to be significant (47). The mode of transmission of the sheep
associated form of MCF is unknown, but is believed to require close


6
contact with carrier animals and does occur with greater incidence
near the lambing season. Both wildebeest-associated and sheep-
associated forms of malignant catarrhal fever have been experimentally-
transmitted to a variety of susceptible bovids and cervids, as well as
rabbits, guinea pigs, rats and hamsters, by inoculation of infected
blood and/or other tissues (8,25,27,31,42,45,63).
Two genetically distinct groups of AHV-1 have been identified,
and are designated AHV-1 and AHV-2. AHV-1 refers to virus isolated
from wildebeest, and AHV-2 refers to virus isolated from topi and
hartebeest (30). Epidemiological data suggests AHV-2 has not been
associated with MCF in cattle (50). AHV-1 and AHV-2 have been
demonstrated to be in two distinct groups by restriction endonuclease
patterns and DNA cross-hybridization (57).
Virus Characteristics
Alcelaphine herpesvirus 1 has been examined by electron
microscopy of infected cell monolayers. The virions have typical
herpesvirus morphology and have icosohedral nucleocapsids of 94-194
nm. They apparently develop in the nucleus and pass through the
nuclear membrane, acquiring an envelope (7).
The best source of virus for isolation is buffy coat cells which
are then cocultivated with fibroblasts. Although less consistent,
virus isolation can also be made from spleen, lung, lymph nodes,
adrenals and thyroids. Virus is quickly inactivated in the carcass,
and tissues must be collected within one hour post-mortem for virus
isolation to be successful.


7
Cytopathic effect (CPE) may not be evident before several
subculture passages of cells. Early CPE consists of formation of
syncytia followed by contracting and rounding of cells. As the virus
becomes more adapted to cell culture, there is formation of focal
clusters of shrunken rounded cells, and a decrease in the formation of
syncytia. Cowdry type A inclusion bodies are formed in infected cell
cultures, but have not been identified in tissues of AHV-1-infected
animals (20).
The virus apparently infects lymphocytes, and cocultivation of
lymphocytes with fibroblast feeder layers results in infection of the
fibroblast cell lines. Because of this ability to infect lymphocytes,
AHV-1 has been classified as a gamma herpesvirus, the same designation
given to Epstein-Barr virus, Marek's disease virus, Herpesvirus
saimiri and Herpesvirus teles. The first wildebeest virus, isolated
by Plowright in east Africa in 1960, is designated WC11 and has become
the prototype laboratory strain. This virus has been adapted to grow
in variety of cells including bovine, ovine, rabbit and African Green
Monkey kidney (Vero) cells. At 37C AHV-1 forms syncytia in
susceptible cell lines, which slowly round up and detach (15). If
grown at lower temperatures of 32-34C, development of CPE is slightly
accelerated and larger amounts of cell-free virus are produced (15).
The replicative cycle of WC11 is quite long, and foci of CPE are not
visible until 72-96 hours post infection. A new AHV-1 isolate,
designated 1982, was recently obtained from the Zoological Society of
San Diego from a diseased Formosan sika deer (Cervus nippon) and has


8
been used in our laboratory for a portion of these studies. This
isolate has proven very useful, as its replication cycle is
considerably shorter than that of WC11. Extensive CPE resulting in
complete destruction of the monolayer occurs in less than 72 hours
with 1982, as opposed to 2-3 weeks with WC11. Virus titers are 2 to
3 logs higher with 1982 than with WC11.
Alcelaphine herpesvirus 1 has also been successfully isolated
from hartebeest, topi, and the scimitar-horned oryx (Oryx gazella
dammah), in addition to the species that are susceptible to induction
of clinical disease by the virus. Analysis of DNA iron these various
isolates has demonstrated that all isolates except the hartebeest and
topi isolates have a high degree of homology with the prototype WC11
strain of AHV-1. The DNA from the hartebeest and topi isolates
hybridize with each other, but not with the isolates from other
species (57).
In addition to Alcelaphine species, a high prevalence of
neutralizing antibody to AHV-1 has been found in the subfamily
Hippotraginae (addax and various oryx species), but no virus has yet
been isolated from these species (37).
The DNA structure of AHV-1 appears very similar to that of
H.saimiri and H.teles, which are both gammaherpesviruses. DNA from
purified virions sediments into two components in cesium chloride
gradients which appear equivalent to the M and H components of saimiri
and teles. The M component is thought to consist of unique coding
regions surrounded by flanking repeated sequences of varying length.


9
These are extremely high in guanine plus cytosine content relative to
the non-reiterated DNA. The H component is thought to be a
heterogeneous mixture of similar reiterated sequences (6).
The pathogenesis of the disease is poorly understood. Viral
antigens have been demonstrated in mature lymphocytes by immuno
fluorescence, although only a small number of infected cells were
detectable by this technique (44). Antigen has only rarely
demonstrated in cells other than lymphocytes (52).
Diagnosis
The similarity of the clinical and gross pathological signs
associated with MCF and those of other viral diseases infecting cattle
necessitates the use of serological means for definitive diagnosis.
Serological identification of infected animals is, however,
complicated by immunological cross-reactivity that exists among bovid
herpesviruses. The assay most commonly used to test animals for
latent herpes infections is an indirect immunofluorescence assay using
virus-infected target cells. Unfortunately, this assay fails to
discriminate animals carrying AHV-1 from those previously infected
with other more common or less potentially devastating herpesviruses
(19).
There are at least five herpesviruses infecting cattle, bovine
herpesvirus 1 (BHV-1), bovine herpesvirus 2 (BHV 2), bovine
herpesvirus 4 (BHV 4) pseudorabies virus (PRV or Suid herpesvirus-1),
and alcelaphine herpesvirus 1.


10
Bovine herpesvirus 1 (BHV-1), also known as infectious bovine
rhinotracheitis virus, is a very common disease of cattle which is
associated with fever and mild to severe respiratory tract infections.
These infections may be complicated by secondary infections with a
variety of other viruses and bacteria. Encephalitis, abortions and
occasional enteritis can result from BHV-1 infections. Because BHV-1
is so commonly encountered in cattle populations, the possibility of
diagnostic cross-reactivity is a valid concern.
Bovine herpesvirus-2 (BHV-2), which is also known as bovine
herpes mammillitis virus, is far less significant clinically, but may
be responsible for occasional outbreaks of mammillitis and secondary
bacterial mastitis. Epidemiological surveys estimate that 20% of the
U.S. cattle population may be seropositive for BHV-1 (58). Previous
work in our laboratory has demonstrated significant cross-reactivity
between BHV-2, BHV-1, PRV and Herpes simplex virus type 1 (HSV-1). It
therefore seems probable that there will be some cross-reactivity with
this rather common virus and AHV-1.
Bovine herpesvirus 4 (BHV-4) encompasses a group of viruses of
minor clinical significance which have been isolated from animals
showing variable clinical signs. This group of viruses has also been
referred to as bovine herpesvirus 3, a designation previously reserved
for AHV-1. This group includes Movar virus, which we are using as a
group prototype, as well as DN599, 66-P-347, and UT. Viruses in this
group are closely related serologically, and restriction endonuclease
patterns of DNA from various isolates appear quite similar (43).
There is evidence that, of all the bovid herpesviruses, those in the


BHV-4 group are the most similar to AHV-1; they have the highest level
of serological cross-reactivity and similar DNA restriction
endonuclease patterns (33). Pseudorabies, also known as Aujesky's
disease, infects a variety of species, but domestic swine are thought
to be the principal reservoir (14). As cattle may be infected with
pseudorabies virus, we must also evaluate its serological relatedness
to AHV-1.
Virus neutralization is the only accepted asay for providing a
definitive diagnosis of AHV-1 infection (19,49). This a time-
consuming test that can only be done at only a few laboratories
certified by the United States Department of Agriculture to work with
the virus under Biohazard level 3 conditions. The assay is currently
performed by mixing serial dilutions of test sera with approximately
100 tissue cultures infectious doses (TCID) of WC11 in each test well
containing confluent Vero cell monolayers. The test is then read at
10 days and the endpoint (titer) is defined as the dilution of sera
that results in prevention of cytopathic effect in 50% of the test
wells. An alternate method for quantifying virus is the use of plaque
assays. Although undoubtedly more accurate than a TCID50 assay, a
plaque assay with AHV-1 is more difficult to read and its use has not
been readily adopted (17). An ELISA assay would provide a rapid,
simple, and sensitive diagnostic test for AHV-1. This assay would not
require the use of live virus and could be routinely used by
diagnostic laboratories.
An ELISA has been tested by one group for its ability to detect
antibodies to AHV-1. Polyclonal serum from gnotobiotic cattle


12
infected with BHV-1, BHV-2, Movar or SHV-1 did not cross-react in this
assay, but the specificity of the ELISA was only 83.9% when tested
with bovid sera that may have possessed antibodies to related viral
antigens (60,61). An ELISA that utilizes monoclonal antibodies to
compete for serum antibodies from infected animals might have a higher
degree of specificity, thereby resulting in a lower percentage of
false positives.
Importance of Herpesvirus Membrane Proteins
In order to begin critical study of AHV-1, its pathogenesis,
diagnosis and prophylaxis, examination of the virus membrane proteins
is pertinent.
Herpesvirus membrane proteins are found on the surface of
infected cells and the envelope of the mature virion, and these
proteins are important for several reasons. Specific interactions of
membrane proteins and host cell receptors are at least one important
aspect of cell tropism and host range. It has been demonstrated in
Epstein-Barr virus that the large membrane glycoproteins gp350/220
bind to the cellular receptor 012 (12). Once attachment occurs, the
virus nucleocapsid must penetrate the host cell by a fusion event
which is also thought to be a function of the envelope proteins (36).
The fusion event may occur at the cell surface, which is the case with
paramyxoviruses (62), or may occur only after endocytosis of the
virion and subsequent fusion of the endocytic vesicle and the virus
envelope. This latter mechanism has been shown to occur in vesicular
stomatitis virus, Semliki Forest virus, and influenza virus
(10,11,62,64). The fusion event in this case is apparently dependent


13
upon a decrease in pH within the endosme that results in a
conformational change of the proteins responsible for the fusion event
(59). Once the nucleocapsid enters the cell it is probably
transported to the nuclear membrane where DNA is released into the
nucleus (4).
Virus membrane proteins are also involved in virus assembly and
egress from the host cell (60). High mannose glycoprotein precursors
of HSV-1 membrane proteins accumulate at the inner surface of the
nuclear membrane, the site of herpesvirus envelopment. It has been
suggested that interaction of the glycoproteins and the nucleocapsid
or tegument proteins culminates in their aggregation during the
process of envelopment (60). Virus is transported through
intracytoplasmic membranes to the cell surface. Further processing of
the glycoproteins occurs within the Golgi, and interactions between
the glycoproteins and intracytoplasmic membranes are thought to direct
egress of the virus from the cell, although the exact mechanism is not
known (56). Although these processes have not yet been studied in
AHV-1, similar mechanisms of virus/host cell interactions probably
exist.
Animals that are naturally infected with herpesviruses tend to
have high antibody titers to various membrane proteins, and these
antibodies often posses virus-neutralizing activity in vitro. It has
also been demonstrated that passive transfer of antibody to the
glycoproteins of HSV confers protection to mice subsequently
challenged with an otherwise lethal dose of virus (2).


Virus proteins may thus represent potential reagents for the
detection of antibodies in infected animals, as well as potential
14
immunogens for prophylaxis. Further, more detailed characterization
of these proteins will provide information useful to better understand
the pathogenesis of infection.


CHAPTER 2
IDENTIFICATION OF VIRUS-INDUCED PROTEINS
Introduction
Knowledge and understanding of viral proteins provide a basis for
study of infection and pathogenesis, as well as for the development of
novel diagnostic reagents and protective immunogens. At the outset of
this study little was known and nothing reported concerning the
identification and characterization of AHV-1 proteins. It was
essential to complete a preliminary analysis of the major virus-
induced proteins prior to more detailed characterization of individual
virus components.
In order to obtain a profile of the major viral induced proteins
and an indication as to when after infection these various proteins
were produced in the largest amounts, a series of time course studies
of AHV-1 infected cells was done. Not only did these experiments
allow us to gain an appreciation of relative molecular weights of
virus induced proteins that are metabolically labeled with 353-
methionine, but they also provided two additional pieces of
information. We determined the point during the virus-replication
cycle individual polypeptides were produced in the highest
concentrations, and indentified the times we might maximize
radiolabeling of particular proteins of interest. Since glycoproteins
present in cell and virus membranes are highly immunogenic and
15


16
responsible for initiating a virus neutralizing antibody response,
later experiments focused on their identification (1,59).
Materials and Methods
Cells and Virus
The WC11 strain of AHV-1 originally isolated in 1960 by Plowright
and coworkers (44) was grown in Vero (African green monkey kidney)
cells. Cells were infected at multiplicities of infection (MOI) of 1
plaque-forming unit (PFU) to 100-1000 cells. Higher multiplicities of
infection were difficult to obtain, as yields of infectious virus were
low. Virus was allowed to absorb to the cells for 2 hours, and then
was overlaid with Dulbecco's minimal essential medium (DMEM,Gibco)
supplemented with 100 IU/ml penicillin and 100 ug/ml streptomycin and
2% heat-inactivated fetal bovine serum. Infection was allowed to
proceed at 34C until cytopathic effect was extensive and the cell
monolayer no longer adhered to the plate. Cells were pelleted at 1500
rpm for 10 minutes, and the supernatant removed. The supernatant
provided stock virus for further experiments. The cells were stored
at -70 C for later use as immunogen, or as antigen in ELISA assays.
Strain 1982 of AHV-1 was obtained from the San Diego Zoo and
grown in primary bovine embryonic lung (BEL) cells. Infection and
harvest of virus were done as for WC11. Multiplicities of infection
of 5-10 were possible as yields of infectious virus were approximately
1000 fold greater with the 1982 strain than with the WC11 isolate.
Virus titers were determined by counting the number of plaques formed


17
in Vero cell monolayers at 10 days post infection. Titers of WC11
ranged from 1.5 x 102 to 1 x 10 PFU per ml. Titers of 1982 pools
ranged from 1 x 10 to 1 x 107 PFU per ml.
Radiolabeling
Vero cells were infected with WC11 virus at an MOI of 1 PFU to
100-1000 cells, and labeled with 25s-methionine or ^H-glucosamine in
minimal amounts of media containing 1/10 the normal concentration of
methionine or glucose. Replicate cultures of infected cells were
labeled at 24-hour intervals from days 1 through 10 postinfection. At
the end of each labeling period cells were scraped off with a rubber
policeman into ice-cold, phosphate-buffered saline (PBS), pelleted by
centrifugation, washed two times in PBS, and the pellets stored
at -20C. Bovine embryonic lung cells were infected with strain 1982
virus at an MOI of 5-10 PFU/cell and labeled with -^S-methionine
between 24 and 48 hours postinfection. Cells were harvested as
described above for WC11-infected samples above.
Virus that was to be extrinsically labeled with 12^I was
concentrated from spent culture media by centrifugation at 100,000 g.
Virus harvested from 50 ml of spent culture media was suspended in PBS
and labeled with 0.5-1.0 mCi of 125j by -(-he use of Iodogen
(tetrachlorodiphenylglycouril; Pierce Chemical Co.) (13). Free iodine
was removed by sieving on a 5 ml desalting column (P6-DG;Biorad).
Efficacy of labeling was determined by comparing the radioactivity
present in trichloroacetic acid insoluble material with total counts
in 10 Ml samples.


18
Labeled samples were either suspended immediately in 100 il
sample buffer containing 0.65 M Tris, 1% sodium dodecyl sulfate (SDS),
10% glycerol and 1% 2-mercaptoethanol, or prepared for immuno-
precipitation.
Antibody Production
Polyclonal antibody to AHV-1 was prepared by repeated
immunization of a rabbit. For the first injection, approximately 108
infected cells were suspended in 2.0 ml of PBS, emulsified with an
equal volume of complete Freund's adjuvant, and injected at multiple
subcutaneous sites of a young adult female rabbit. Freund's
incomplete adjuvant was used for all subsequent immunizations. The
rabbit was injected three times at biweekly intervals with infected
cell lysate, and 3 times at biweekly intervals with virus concentrated
by centrifugation from 100 ml of spent culture media. After the
immunization schedule was completed, the rabbit was sedated with 0.1
ml/kg of Innovar-vet (fentanyl 0.04% and droperidol 2%) and bled iron
the central auricular artery.
Antibody was purified from serum by affinity chromatography on
protein A-agarose. Purified antibody was then repeatedly absorbed at
4"C for 24 hours with approximately 5 x 108 Vero cells and repeatedly
passed over two sets of affinity columns, the first composed of Vero
cell proteins coupled to cyanogen bromide activated Sepharose, and the
second comprised of bovine testicle cell proteins coupled to the same
matrix. The rabbit antibody obtained by these procedures was
initially highly reactive with bovine serum albumin in an ELISA assay
and efficiently immunoprecipitated a very heavy band of an 128I


19
labeled protein of approximately 70 kDa. Further absorption over an
affinity column made of BSA coupled to Sepharose beads reduced the
ability to immunoprecipitate the 70 kDa protein. The antibody was
chromatographed on protein-A agarose and the protein concentration
determined with BCA Protein Assay (Pierce Chemical Co.)
Immunoprecipitation
Pellets of radiolabeled cells or viruses that were to be
immunoprecipitated were solubilized by suspension in radio-
immunoprecipitation buffer (RIPA), containing 0.05 M Tris
hydrochloride pH 7.2. 0.15 M NaCl, 0.1% deoxycholate, 1.0% Triton X-
100, 0.1% SDS, 100 U aprotinin per ml and 0.1 M phenylmethyl-
sulfonylflouride (PMSF), and sonicated for 30 seconds. Sonicated
material was then centrifuged at 100,000 g for 1 hour to remove the
non-soluble portion of the lysate. Four hundred microliter samples of
the lysate were added to 125 /I of protein A-agarose beads (Genzyme
Corp., Boston, Massachusetts) and 100 /ug of purified R21 rabbit
antibody and rocked on a platform at 4 C for 3 hours. The agarose
beads were then pelleted by centrifuging in a Eppendorf centrifuge for
4 min, washed 4 times with RIPA buffer and boiled for 3 min in 105 ¡xl
of sample buffer to dissociate immunoprecipitated proteins. The
amount of radioactivity in a 5 a1 sample was determined and the
proteins were either immediately analyzed or stored at -20 C for
analysis at a later time.


20
Electrophoresis
Immunoprecipitated samples were analyzed by sodium dodecyl
sulfate-polyacrylamide electrophoresis (SDS-PAGE) (30). Gels
consisting of 0.65 M Tris pH 8.8, 9% acrylamide, 0.1% SDS, 0.1%
glycerol, 0.83% ammonium persulfate, and 0.28% N,N'diallyl-
tartardiamide (DATD) were poured, overlaid with 1-butanol and allowed
to polymerize for at least 2 hours. After polymerization, the gels
were washed with 0.65 M Tris pH 6.8 and stacking gels consisting of
0.65 M Tris pH 6.8, 4% acrylamide, 0.1% SDS, 0.05% glycerol, 0.83%
ammonium persulfate, and 0.28 % DATD were poured and allowed to
polymerize for 45 minutes. Immunoprecipitated samples and molecular
weight standards were loaded into individual wells in the stacking
gels and submerged in reservoir buffer of 25 mM Tris and 200 mM
glycine with 1% SDS. A constant voltage of 50 V was applied for
electrophoretic separation of proteins. Gels were stained with 0.1%
Coomassie blue stain for 5 minutes and destained with several changes
of destain (7% methanol and 7% acetic acid) over a 12- to 24-hour
period to visualize the molecular weight standards. Gels containing
proteins labeled with ^5j were dried on filter paper and placed in
contact with XAR film (Eastman Kodak Co., Rochester, N.Y.) at -70 C
for fluorography. Gels containing 35g or 3^ labeled proteins were
infused with 2,5-diphenyloxazole (PPO) by treating the gel in two 30
min changes of dimethyl sulfoxide (DMSO) followed by 22% PPO in DMSO
for 3 hours. The PPO was precipitated in the gel by soaking in IH2O
for at least 1 hour and dried on filter paper. The gels were then
placed in contact with XAR film at -70 C for fluorography (5).


21
Results
Time course experiments
Replicate cultures of infected cells were labeled at 24-hour
intervals from days 1 through 10 postinfection. By 10 days post
infection 100% of the cell monolayer was damaged by virus infection
and by day 11 postinfection, the majority of cells were sloughed from
the surfaces of the tissue culture flasks. At the end of each
labeling period, cells were harvested as described and prepared for
electrophoresis. Identification of virus induced proteins was
complicated by continuation of host cell protein synthesis (Fig 2.1);
host cell proteins continued to be made in large amounts even in the
face of 100% cytopathic effect (CPE) at 10 days postinfection.
However, upon close examination at least 4 virus-induced proteins
could be distinguished. These four proteins had molecular weights of
145,000, 135,000, 125,000, and 110,000. It was also apparent that
each of these proteins increased over time, being first readily
visible at day 6 and reaching maximum amounts at day 10 postinfection
(Figure 2.1).
In order to visualize virus induced proteins more clearly, a
portion of the 3^S-methionine labeled AHV-1 infected cell lysate from
each 24-hour postinfection time interval was immunoprecipitated with
rabbit anti-AHV-1 antibody (Figure 2.2). Although host cell proteins
continued to complicate interpretation, it was now possible to discern
a minimum of 15 virus induced proteins, ranging in molecular weight
from 32,000 to 180,000. In an effort to determine the relationships
of molecular weights of proteins specified by strain 1982 and strain


22
Figure 2.1 SDS-PAGE analysis of -^S-methionine labeled proteins in
mock infected Vero cells harvested after labeling between 0 and 24
hours post infection (V) and AHV-1 infected Vero cells harvested at
the end of each of 10 consecutive 24-hour labeling periods between 0
and 10 days post infection (1-10). Numbers to the right indicate
virus induced proteins and their apparent molecular weights (xIOOO).


23
Figure 2.2 SDS-PAGE analysis of -^S-methionine labeled proteins
immunoprecipitated by rabbit anti-AHV-l antibodies from mock infected
Vero cells harvested after labeling between 0 and 24 hours post
infection (V), and AHV-1 infected Vero cells labeled for 24-hour
intervals for 10 days after infection (1-10); mock infected and
infected cell lysate used in this experiment were also used in the
experiment depicted in Figure 2.1. Numbers to the right indicate
virus induced proteins and their apparent molecular weights (xIOOO).


24
WC11, cells were infected with each strain, labeled with
35s-methionine, harvested and immunoprecipitated with polyclonal
rabbit antibodies as described above. The -^^S-methionine labeled
protein profiles of WC11 and 1982 are virtually identical with one
exception. A 37 kDa protein is immunoprecipitated from WC11-infected
Vero cell lysate, but not from lysate of 1982-infected BEIL cells. All
other viral induced proteins identified in WC11 appear to have
counterparts in the 1982 strain with similar molecular weights (Figure
2.3).
Identification of Membrane Proteins
Our primary interest was in the membrane proteins of AHV-1, and
to begin to identify these, WC11 virions were extrinsically labeled
with 1iySed, immunoprecipitated with polyclonal immune serum and
analyzed by SDS-PAGE. Six virus-specific proteins were identified
that were not visible in labeled lysate of mock infected Vero cells.
These proteins ranged in molecular weight from 48,000 to 145,000
(Figure 2.4).
Strain 1982 was also extrinsically labeled with 125j iySed in
RIPA and the lysate immunoprecipitated with rabbit polyclonal
antibodies to AHV-1 (R21). A pattern of bands representing proteins
of molecular weights similar to those of WC11 was evident (Figure
2.4). These virus proteins ranged in molecular weights from 32,000 to
130,000. One notable difference between the 2 strains in 125j
labeling experiments was the lack of reactivity of R21 antibodies with
1982 surface protein of 95 kDa, and the reactivity of R21 with a
diffuse band of 32 kDa in strain 1982 that was not seen in WC11


25
WC11
1982
200K-
200K-
--175
Figure 2.3 SDS-PAGE analysis of 3%-methionine labeled proteins
immunoprecipitated by rabbit anti-AHV 1 antibody from AHV-1 strain
WC11 (WC11) and strain 1982 (1982) infected cells. Numbers to the
right indicate virus induced proteins and their apparent molecular
weights (x1000).


26
WC11
1982
**pv
45 k-
^48
42k-
^32
Figure 2.4 SDS-PAGE analysis of labeled proteins
immunoprecipitated from extrinsically labeled AHV-1 strain WC11 (WC11)
and strain 1982 (1982) labeled virions. Numbers to the right indicate
virus proteins and their apparent molecular weights (xIOOO).


27
(Figure 2.4). Proteins of 130 kDa, 110 kDa, 105 kDa, and 48 kDa were
readily identified when either strains WC11 and 1982 were labeled with
12^I (Figure 2.4). Also seen in both strains was a protein with a
molecular weight of 175,000. This protein is probably cellular in
origin, as monoclonal antibodies were produced that immunoprecipitated
an abundant protein of similar molecular weight from both uninfected
BEL and Vero cells (see below).
Many membrane proteins of herpesviruses are glycosylated. It was
therefore of interest to determine which of the molecules that labeled
with 125j also contained sugars. Vero cells were infected with WC11
and labeled with 3H-glucosamine for 24 hours at day 10 post infection
Immunoprecipitation with rabbit anti-AHV-1 antibody and analysis by
SDS-PAGE (Figure 2.5) allowed identification of ten virus induced
glycoproteins with molecular weights of 145,000, 130,000, 115,000,
110,000, 105,000, 95,000, 78,000, 72,000 and 48,000 and 32,000.
Polyclonal rabbit antibody immunoprecipitated a nearly identical
pattern of proteins from 1982 infected cells labeled with 3H-
glucosamine. One minor difference was the presence of a 62 kDa
glycoprotein in cells infected with 1982 and the lack of a 72 kDa
protein identified in Vero cells infected with WC11. A comparison of
35S-methionine labeled proteins from WC11 and 1982 infected cells was
also made. An almost identical pattern of proteins was seen with each
strain (Figure 2.3).
A summary of WC11 and 1982 induced proteins thus far identified by
labeling with various radioisotopes is given in Table 2.1.


28
200K
CN
200K
Figure 2.5 SDS-PAGE analysis of %-glucosamine labeled proteins
immunoprecipitated from uninfected (Vero, BEIL) and AHV-l strain WC11
(WC11) and AHV-1 strain 1982 (1982) cells. Numbers to the right
indicate virus induced proteins and their apparent molecular weights
(xIOOO).


Table 2.1- Proteins Identified by Iimiunoprecipitation with
Polyclonal Antibodies to AHV-1.
29
a- Molecular weight (x1000).
t,- A glycoprotein of 44 Kd was not precipitated with polyclonal
antibody, but was identified with monoclonal antibody 11C3
(see Chapter 4).


30
Discussion
It was readily apparent that host protein synthesis continues
relatively unabated throughout infection with AHV-1. A total of only
14 virus induced proteins could be identified by metabolically
labeling cells with 35g_methionine. The genome of AHV-1, however, is
approximately 8 x 106 daltons, and it is probable that its total
coding capacity is far greater than this experiment would suggest
(31). We assume, therefore, that some virus transcripts are either
translated in amounts too small to be detected by protocols used, that
they have mobilities greater or smaller than the ranges analyzed on
gels, or that the translation products are poorly labeled with 3%
methionine. The experiment did, however, allow us to conclude that
metabolic labeling of WC11 induced proteins for further analysis was
optimally done at 10 days post infection.
Our long-term objective was to characterize the envelope proteins
of AHV-1. Six virus proteins were immunoprecipitated with rabbit
polyclonal antibodies from virions extrinsically labeled with 12^i.
All six of these proteins were also glycosylated and therefore were
presumed to be surface glycoproteins. Two interesting differences
were found between WC11 and 1982 extrinsically labeled with 12^I. A
surface-labeled protein of 95 kDa was identified in WC11 but lacking
in the strain 1982, whereas a protein of 32 kDa was exclusive to
strain 1982. In contrast, proteins of identical size were visible,
although not prominent, in analyses of material from cells infected
with both strains of virus if they had been labeled with 2H-
glucosamine or 2^S-methionine. If we assume that the proteins


31
labeled by these techniques are identical, then we suggest that they
may in fact be molecules that are not actually found on the virus
surface. Their apparent presence on the surface of the virion may
then be a result of damage to the virions, allowing access to normally
internal proteins and subsequent labeling with 12^I. It is also
conceivable that the proteins are actually found on the surface of one
strain but not the other, or that the portions of each molecule
possessing tyrosines reactive in the labeling process are
differentially accessible.
More extensive characterization of the envelope proteins required
monoclonal rather than polyclonal reagents. The next step was to
prepare such reagents.


CHAPTER 3
MONOCLONAL ANTIBODIES
Introduction
Monoclonal antibody technology, as originally described by Kohler
and Milstein (29), has provided invaluable tools for the study of
proteins. Monoclonal reagents represent a distinct advantage over
polyclonal reagents for a variety of reasons. Of particular relevance
to our work was the fact that since monoclonal antibodies are specific
for a single epitope, or at least a very limited number of cross
reactive epitopes, they can be used to isolate and analyze individual
proteins within a mixture. Our goal was to prepare monoclonal
reagents that would allow us to identify and characterize individual
envelope proteins of AHV-1. In addition, we were able to use the
antibodies we produced to identify at least one protein that can
induce a virus-neutralizing response. These antibodies also provided
the basis of a diagnostic assay for AHV-1 infection that is described
in Chapter 5.
Materials and Methods
Mouse Immunizations
Virus was concentrated by ultracentrifugation from 50 ml of spent
culture media and used for subcutaneous immunization of 3 Balb/c mice.
The virus was emulsified in an equal volume of complete Freund's
adjuvant to give a final volume of 0.5 ml per mouse. Mice were
32


33
subsequently immunized at 1-2 week intervals by subcutaneous injection
with a similar amount of virus emulsified in incomplete Freund's
adjuvant. After three injections a small amount of blood was taken
from the tail veins of the mice and sera separated from the blood were
tested in surface immunofluorescence assays for the presence of
antibody to AHV-1 induced surface proteins. When mice tested positive
by this assay, they were boosted by intraperitoneal injection with 0.5
ml of antigen in PBS and sacrificed 3 days later for preparation of
hybridomas.
Fusion
Hybridomas were made by fusing mouse spleen cells with the
myeloma cell line SP2/0 Ag 14. Mice were anesthetized with
methoxyflurane and exsanguinated by cardiac puncture using a 23-gauge
needle. The spleen was then removed and aseptically placed in sterile
DMEM supplemented with non-essential amino acids, sodium pyruvate, 0.1
M Hepes and 20% fetal bovine serum (SDMEM). The spleen cells were
released from the spleen capsule by grinding the spleen between two
frosted-end microscope slides. Red blood cells were lysed with
ammonium chloride and spleen cells washed twice with SDMEM.
Hyperimmune spleens yield more than 108 white blood cells. The spleen
cells were combined with the SP2/0 myeloma cells at a ratio of five
spleen cells to one SP2/0 cell, washed in serum-free SDMEM and fused
together with 50% polyethylene glycol 1500 for one minute. Serum-free
media was slowly added and the fused cells were washed to remove PEG
and plated into 96 tissue culture plates at 1 x 105 cells in 100 /I


34
SDMEM with 20% fetal bovine serum (FBS) per well. After 24 hours, 100
Hi of SDMEM containing two times the normal concentration of
hypoxanthine, thymidine and aminopterin was added to each well. Cells
were fed when media was depleted. Outgrowths were screened for the
production of antibody and transferred first into 24 well tissue
culture plates, and then into petri dishes for further growth. When
cultures had expanded to approximately 5 million cells they were
frozen in SDMEM containing 20% FBS and 10% dimethyl sulfoxide.
ELISA
Culture media from hybridomas were initially screened by an
enzyme linked immunosorbent assay that used WC11-infected Vero cells
as antigen. Virus-infected cells were collected when virus CPE was
approaching 100%. Infected cells and uninfected control cells were
pelleted by centrifugation, suspended in carbonate/bicarbonate buffer
pH 9.6 and lysed by freeze thawing three times. These antigen
preparations were then tested at various dilutions to determine the
concentrations that produced the maximum differential signal between
infected and non-infected cells when polyclonal mouse anti-AHV-1
antibody was reacted with each. Ninety-six well flat bottomed plates
were incubated overnight with varying amounts of antigen in
carbonate/bicarbonate buffer 9.6. Plates were then incubated
sequentially with 5% skimmed milk in PBS-Tween 20, dilutions of mouse
antibody, goat anti-rabbit or anti-mouse antibody conjugated to horse
radish peroxidase (Organon Teknika Corp., Durham, N.C.) and the
substrate orthophenylenediamine (Sigma). Plates were washed between


35
each incubation with 0.05% Tween 20 in 0.85% NaCl; the final enzyme
reaction was stopped with 2 N H2SO4 and analyzed for color change at
492 nm.
Surface Fluorescence
Vero cells that had been infected with WC11 10 days previously,
were placed at 4C for 30 minutes and removed from tissue culture
flasks by vigorous pipetting. Cells were pelleted by centrifugation,
fixed with 0.1% paraformaldehyde, washed three times with PBS, and
incubated with culture media that had been concentrated to one-tenth
their original volume by dialysis against polyethylene glycol. Cells
were then washed three times in PBS, incubated for 30 minutes with
fluorescein conjugated rabbit anti-mouse antibody, washed with PBS and
examined with a fluorescence microscope.
Virus Neutralization
Hybridomas were screened for their ability to neutralize virus in
a standard plaque reduction assay. A known number of PFU were
incubated for one hour at 37"C with hybridoma culture media that had
been concentrated 10 fold by dialysis against polyethylene glycol.
The mixture was titrated in confluent Vero cell monolayers in 24 well
plates. Virus was allowed to absorb for 2 hours, at which time each
well was overlaid with 1 ml of SDMEM containing 2% fetal bovine serum.
Infection was allowed to proceed for 10 days, at which time the number
of plaques in the cell monolayer was counted. Hybridoma culture media
that reduced the number of plaques by 50% were determined to possess


36
neutralizing activity. Subclones of hybridoma 12B5 were further
screened with this technique to confirm neutralizing activity.
Results
ELISA
The difference in reactivity of mouse anti-AHV-1 sera with
infected and uninfected Vero cells was greatest if ELISA plates were
coated with 30 /g of cell protein. The difference in reactivity
between infected and uninfected cell proteins did not begin to
diminish appreciably until a protein concentration of less than 3 /g
per well was used (Figure 3.1) Three micrograms of protein per well
were used in all subsequent ELISA assays. Non-immune mouse serum
reacted at equally low levels with infected and non-infected Vero
cells. This indicated the specificity of this ELISA as a test for
anti-AHV-1 antibodies, especially at the higher concentrations of
antigen.
Hybridoma screening
After several unsuccessful fusions, one fusion resulted in
outgrowth in a total of 165 wells of five 96 well culture dishes.
Culture supernatant was harvested from the 96 well plates, and tested
in the ELISA assay. Of the 165 hybridomas tested, a total of 97
tested positive by ELISA. A positive reaction was determined to be
one in which the optical density in wells coated with infected cell
antigen was at least 0.300 and approximately 2 times higher than the
optical density in wells coated with uninfected cell lysate. The 97
hybridomas positive by at least two of these criteria were then
screened for their ability to bind surface proteins of infected but


37
Antigen concentration (/ug/ml)
Figure 3.1 Titration curve of AHV-1 infected and uninfected Vero
cell ELISA antigen. Polyclonal mouse anti AHV-1 serum reacted
with AHV-1 infected cell lysate. O-Polyclonal mouse anti-AHV-1 serum
reacted with uninfected cell lysate. Non-immune mouse serum
reacted with AHV-1 infected cell lysate. -Non-immune serum reacted
with uninfected cell lysate.


38
not uninfected cells in an immunofluorescence assay. A total of 56
hybridomas tested positive by these criteria.
Immunoprecipitations with Monoclonal Antibodies
Monoclonal antibodies that were positive by surface fluorescence
were screened for their ability to immunoprecipitate virus induced
proteins that had been radiolabeled with 12^i or 35g_me-t:hionine.
Hybridomas immunoprecipitated 8 different virus-induced proteins
(Table 3.1). Despite previous screening, hybridomas appeared to
recognize cell proteins. These antibodies immunoprecipitated
molecules of 220 kDa and 180 kDa from both infected and uninfected
cells. Hybridomas capable of immunoprecipitating putative surface
glycoproteins from both WC11-and 1982-infected cells are described in
more detail.
The 145 kDa protein
Five hybridomas were identified that immunoprecipitated a 145 kDa
protein. Two of these hybridomas, 14B6 and 41B1 were cloned and
culture media containing antibody was concentrated by dialysis against
polyethylene glycol. Results of immunoprecipitation with both
monoclonal antibodies were identical; therefore only results for 14B6
are shown. The 145 kDa protein was immunoprecipitated with monoclonal
14B1 from virus infected cells labeled with 35S-methionine (Figure 3.2
and 3.3) or 3H-glucosamine (Figure 3.6). Antibodies to this protein
did not iirmunoprecipitate a labeled protein from the lysate of 125I
labeled 1982 or WC11 virions (Figure 3.4 and 3.5). The protein was
also not immunoprecipitated by rabbit polyclonal antibodies from 125I
labeled 1982 virions. It is therefore probable that this protein is
not present on the surface of the 1982 virion.


39
The 95 kDa protein
Eighteen hybridomas were identified that reacted very strongly
with a 95 kDa protein induced by strain WC11. Five of these were
cloned and concentrated from culture media. Neither the polyclonal
rabbit antibody R21 nor monoclonal antibodies 21B1 and 24C1 could
immunoprecipitate this 95 kDa molecule from 12^i labeled strain 1982
(Figure 3.4 and 3.5). All antibodies had similar patterns of
reactivity and the results of immunoprecipitating virus are shown only
for 21B1. As noted in Chapter 2, a protein of 95 kDa was identified
in 25S-methionine nd 2H-glucosamine labeled virus-infected cells that
had been immunoprecipitated with polyclonal rabbit antibodies to AHV-
1. Monoclonal antibodies 24C1 and 21B1 did not immunoprecipitate this
protein from 3H-glucosamine labeled 1982-infected cells (Figure 3.6),
but did immunoprecipitate a 95 kDa protein from 2H-glucosamine labeled
WC11- infected Vero cells (data not shown). Monoclonal antibody 21B1
also reacted weakly with a protein of 95 kDa from WC11- or 1982-
inf ected cells metabolically labeled with 25S-methionine (Figure 3.2
and 3.3)
The 44 kDa protein
Three hybridomas were identified as reactive with a protein of an
apparent molecular weight of 44,000. One of these hybridomas, 11C3
was cloned and antibody was concentrated from culture media. The 44
kDa protein was labeled with 35S-methionine and %-glucosamine. It
was immunoprecipitated weakly by polyclonal serum, but was readily
identifiable after immunoprecipitation with monoclonal 11C3. It was


40
particularly prominent in infected cells that had been labeled with
3^S-methionine, but could not be identified by labeling of virions
with 1. The protein was present in both strains of AHV-1 with
identical apparent molecular weights.
The 115 kDa protein
Seven hybridomas were found to immunoprecipitate a group of 5
proteins from lysates of cells infected with WC11 or 1982 which had
been labeled with ^H-glucosamine and 2 ^S-methionine and
electrophoresed under reducing conditions. The proteins had molecular
weights of 115,000, 110,000, 105,000, 78,000, and 48,000. Two
hybridomas reactive with the group, 12B5 and 31B3 were monocloned and
used to make ascites in mice. Antibody was purified from ascites by
affinity chromatography on protein A-agarose. The patterns of
proteins immunoprecipitated by either of these two antibodies were
identical, so only analyses of proteins immunoprecipitated with 12B5
are given (Figure 3.2 and 3.3). Monoclonal antibody 12B5 irrmuno-
precipitated only 2 proteins of the group from lysate of 12^I
labeled virus, namely those of 78 kDa and 48 kDa. The monoclonal
antibody 12B5 had virus neutralizing activity when assessed by a
standard plaque reduction assay (Table 3.2). This interesting
complex of proteins was therefore selected for closer examination
(see Chapter 6).
Discussion
Production of monoclonal antibodies allows analysis of individual
AHV-1 proteins. A summary of the data presented in this chapter


41
Figure 3.2 SDS-PAGE Analysis of -^^S-methionine labeled proteins
immunoprecipitated from cells infected with WCl1 by rabbit polyclonal
anti-AHV 1 antibodies (R-AHV), and monoclonal antibodies 14B6, 12B5,
21B1 and 11C3. Arrows to the right indicate proteins immuno
precipitated from infected cells and their apparent molecular weights
(x1000).


42
Figure 3.3 SDS-PAGE analysis of 35s_methionine labeled 1982 proteins
immunoprecipitated by rabbit polyclonal anti-AHV 1 antibodies (R-AHV),
non-immune rabbit sera (PB) and monoclonal antibodies 14B6, 12B5, 21B1
and 11C3. Arrows to the right indicate proteins immunoprecipitated
from infected cells and their apparent molecular weights (x1000).


43
>
Figure 3.4 SDS-PAGE analysis of 1^5j labeled proteins of WC11
virions, immunoprecipitated by polyclonal rabbit anti-AHV 1 antibodies
(R-AHV), non-immune rabbit sera (PB), and monoclonal antibodies 14B6,
12B5, 21B1 and 11C3. Arrows to the right indicate proteins
immunoprecipitated from virion lysate and their apparent molecular
weights (x1000).


44
Figure 3.5 SDS-PAGE analysis of extrinsically labeled proteins
of 1982 virions, iircnunoprecipitated by polyclonal rabbit anti-AHV 1
antibodies (R-AHV), non-immune rabbit sera (PB), and monoclonal
antibodies 14B5, 12B5, 21B1 and 11C3, Arrows to the right indicate
proteins immunoprecipitated from virion lysate and their apparent
molecular weights (xl000)-


45
Figure 3.6 SDS-PAGE Analysis of ^H-glucosamine labeled 1982 proteins
iinmunoprecipitated by rabbit polyclonal anti-AHV 1 antibodies (R-AHV),
non-immune rabbit sera (PB) and monoclonal antibodies 14B6, 12B5, 21B1
and 11C3. Arrows to the right indicate proteins immunoprecipitated
from infected cells and their apparent molecular weights (x1000).


46
Table 3.1 Summary of Characteristics of Hybridoma Antibodies
Reactive with Virus Induced Proteins.
MWa
Hybridoma
Antibodies
ELISA Reactivity
Surface
Floresc.
Irnmunoppt. 5
Neut.c
Infected
Antigen
Uninfected
Antigen
220
12C2
.761
.208
+
+
_
21B2
.986
.556
+
+
+
22D6
.490
.239
+
+
-
31D1 m
.456
.160
+
+
-
180
23C3 m
32D2
.863
.247
+
+

42C5
.621
.253
ND
+

44A5
.522
.218
+
+
-
145
11A1
.390
.192
+
+
+/-
11D3
1.559
.890
+
+
14B6 m
.675
.227
+
+
-
21A2 m
.562
.151
+
+

41B1 m
.667
.354
+
+
-
115
12B5 m
.711
.116
+
+
+
13A6
1.486
.264
+
+

23A1
1.185
.172
+/-
+
-
31B5 m
.828
.199
+
+

32A4
1 .002
.252
+
+
-
22A2
.599
.389
+
+
-
32D2
.863
.247
+
+
-


Table 3.1 continued
47
MWa
Hybridoma
Antibodies
ELISA reavtivity
Surface
Flouresc.
Immunoppt.b
Virus
Neut.c
Infected
Antigen
Uninfected
Antigen
95
11A6 m
1.452
.521
+
+

14D4
1.505
.680
+
+
-
21B1
.448
.241
+
+
ND
21C5
1.125
.267
+/-
+
-
21D3 m
.665
.288
+
+
ND
22A3
.481
.228
+
+
ND
22C6
.809
.413
+
+
ND
24C1
.391
.243
+
+
-
24C3
.429
.318
+
+
-
31A5
.329
.174
+
+
-
31C3
1.931
1.062
+
+
-
32B3
.411
.232
+
+
ND
41B1
.667
.354
+
+
-
41D2
.456
.129
+
+
-
44D2
1 .954
.614
+
+
-
22A6
.379
.190
+
+
-
22B5 m
.555
.283
+
+
ND
31C5 m
.553
.233
+
+
-
44
11C3 m
1.532
.514
+
+

21B2
.986
.556
+
+
+
44D2
1.954
.614
+
+
-
a molecular weight of proteins immunoprecipitated by antibodies,
b positive indicates hybridomas that were capable of iirmunprecipitating
proteins radiolabeled with 12^I and/or 3^S-methionine.
c virus neutralizing ability as determined by plaque reduction assay,
m Indicates hybridomas that have been monocloned.
ND Indicates not done.


Table 3.2 Comparison of the Neutralizing Ability
of Hybridoma Supernatants, and Known Positive and
Negative Sera.
Experiment 1
Experiment 2
Antibody
#Plaques
Antibody
#Plaques
None
25
None
36
12C2
22
Goat (-)
32
13A6
24
Calf (-)
34
21B2
11
31B1
34
24C3
26
24B6
31
14C4
26
21B2
23
44A5
21
33B2
31
32B2
26
14E4
26
33A4
22
33A2
34
44B1
20
21B6
35
31D1
22
31C4
30
24C1
21
R21 (+)
11
11A1
17
21A5
30
14B6
23
11C4
24
11C3
22
31A4
36
22D6
24
11A1
32
31C5
21
12B5
9
21C5
21
24A4
26
41D2
20
12A4
25
21A5
20
24A2
33
23C3
25
23B3
41
44D2
20
42A4
36
12B5
11
41B3
32
31C3
28
33C4
35
11A6
23
42D5
38
42C5
20
23A1
21
22A6
25
32D2
22
(-) Known negative sera
(+) Known positive sera


49
appears in Table 3.1. Ninety-five hybridomas were produced to AHV-1
virus induced proteins. Thirteen of these were cloned and the eight
proteins of 145, 95, 44, 115, 110, 105, 78, and 48 kDa were more
extensively described.
The 145 kDa protein was a glycosylated molecule, but was very
poorly labeled with 125i. Since antibodies to the protein reacted
with the surface of infected cells, it is presumably a membrane
protein that either merely labeled poorly with the 125I or failed to
be incorporated into the virion envelope.
The 95 kDa protein was present in virus infected cells although
it labeled poorly with 2^S-methionine or 2H-glucosamine and/or was
weakly immunoprecipitated with available hybridoma antibodies.
Monoclonal and polyclonal antibodies immunoprecipitated the iodinated
95 kDa protein from strain WC11, but not from strain 1982. This
difference could reflect damage to the virion envelope of WC11 and
exposure to iodine of proteins that are not normally seen on the
surface of the virion or could mean that the protein is differently
distributed in the two strains.
The 44 kDa protein was immunoprecipitated from both 35g_
methionine, and 3H-glucosamine labeled infected cells. This protein
was not appreciably labeled by 125i.
The remaining 5 proteins of 115, 110, 105, 78 and 48 kDa were all
precipitated as a group by several monoclonal antibodies. One of the
monoclonal antibodies was also capable of neutralizing virus
implicating the complex in initiation of infection. It was also noted
that not only were the number of plaques reduced by the antibody, but


50
the plaques appeared to be smaller in diameter. The 5 proteins
labeled very well with 35S-methionine and -^-glucosamine in cells
infected with either strain WC11 or 1982. They were immuno-
precipitated with polyclonal antibodies to AHV-1 and monoclonals 12B5
and 31B3. Monoclonals 12B5 and 31B3 also precipitated at least 2 of
the 5 proteins from 1^5j labeled virions. The evaluation of the
relationships between the proteins of the group is the subject of
Chapter 6.


CHAPTER 4
RELATIONSHIPS OF AHV-1 WITH OTHER BOVID HERPESVIRUSES
Introduction
Herpesviruses of man and animals vary widely in their DNA
structures, host ranges and growth characteristics. These differences
provide the basis for classification into the subfamilies a., £, and r
(51). The herpesviruses do however share common morphology, mode of
entry into host cells (fusion with cell membranes), nuclear location
of viral DNA replication and virus assembly, and acquisition of an
envelope from the inner nuclear membrane (51 ). In all herpesviruses
examined, virus DNA transcription and protein synthesis cure regulated
in a cascade fashion during the lytic cycle (51 ). This would imply
that in addition to unique proteins responsible for differential cell
tropism, they probably possess some conserved structural and non-
structural proteins responsible for common functions. A great deal of
DNA homology has been observed between various herpesviruses, and a
number of common antigenic determinants have been reported among human
and animal herpesviruses (3,28,32). Based on these previous
observations, ENA homology as well as antigenic relationships cure also
likely to be present among herpesviruses infecting bovid species.
Moreover, it has been observed that diagnosis of AHV-1 has been often
complicated by the presence of antibodies to other herpesviruses (19).
This has prevented adoption of alternate methods of serologic
51


52
diagnosis to replace the cumbersome and time consuming virus
neutralization assay. In this chapter the degree of antigenic cross
reactivity between various herpesviruses is examined. It was the
intent of these studies to attempt identification of a protein or
proteins that are entirely unique to AHV-1, and not recognized by
antisera to any of the other bovid herpesviruses. The identification
of such a protein, and monoclonal antibodies against it, would provide
the basis for a diagnostic competitive ELISA assay.
Materials and Methods
Cells and virus
WC11 strain of AHV-1 obtained from the San Diego Zoo was grown in
Vero cells. Bovine herpesvirus 1 (BHV-1;Colorado strain 1 ATCC VR
864) and bovine herpesvirus 2 (BHV-2;NY-1 strain, ATCC VR 845) were
grown in primary bovine testicle (BT) cells. Movar 33/63 was grown
in Maden-Darby bovine kidney (MDBK) cells, and Suid herpesvirus 1
(SHV-1) was grown in baby hamster kidney (BHK) cells. Cells were
infected with BHV-1, BHV-2 and SHV-1 and were labeled with 35g_
methionine (40 /Ci/ml) by addition of radioisotope at 5 hours post
infection in methionine free media and harvested 24 hours later. Cells
infected with Movar 33/63 were labeled between 24 and 48 hours post
infection.
Polyclonal antibodies
Antisera was made in rabbits to each of the four herpesviruses.
Monolayers of susceptible cells were infected with virus at a
multiplicity of infection of 5-10 plaque-forming units (PFU) and
incubated at 37C until CPE was extensive. Cells were harvested and


53
subjected to two cycles of freeze/thaw to produce a cell lysate.
Adult New Zealand White rabbits were initially immunized with 2 ml of
cell lysate emulsified in Freund's complete adjuvant. Three
subsequent inoculations were given at weekly intervals with 2 ml of
the cell lysate emulsified with incomplete Freund's adjuvant, and
blood was collected 2 weeks later. Immune rabbit sera (2ml) were
absorbed 4 times with 5x10^ cells of the type in which the virus was
propagated, and 4 times with packed Vero cells and were passed 6 times
over a column of Sepharose 4B coupled to a frozen and thawed lysate of
noninfected cells of the type in which the virus was propagated.
After these absorptions, antisera no longer reacted with the
noninfected cells in indirect immunofluorescence assays. Antibody was
then purified from sera by chromatography on protein A-agarose,
concentrated by dialysis against polyethylene glycol, and protein
concentrations determined.
Virus and DNA purification
Virus was pelleted from spent culture media from AHV-1 infected
Vero cell cultures. Virus was suspended in TE buffer (1OmM Tris
chloride, ImM EDTA) and layered onto a 20-60% continuous sucrose
gradient and centrifuged in a SW 28.1 rotor at 15,000 rpm for 50
minutes. The virus band was harvested by penetrating the poly
carbonate tube with a needle and withdrawing the band with a syringe.
The virus was then diluted in TE buffer and pelleted by centrifugation
for 30 minutes at 150,000 g.


54
The purified virus pellet was suspended in 0.5 ml of TE buffer
and protein was digested by the addition of Proteinase K (1 OO/ug/ml)
and SDS (0.1%). Digestion was allowed to proceed for 2 hours and the
preparation was extracted 2 times alternating with phenol and
chloroform. Virus DNA was then precipitated with 100% ethanol and 3M
sodium acetate, then pelleted by centrifugation at 15,600 g in an
Eppendorf centrifuge. The supernatant was discarded and the pellet
was dried under vacuum. The DNA was suspended in TE buffer and stored
at 4C.
DNA Digestion and Southern Blotting
A sample of DNA was added to equal volumes of tracking dye (10%
glycerol and bromophenol blue in TE buffer) and electrophoresed in a
1% agarose gel with 0.5% ethidium bromide. The amount of viral DNA
was estimated by comparison of intensity of the viral DNA to Hind III
digested fragments of lambda DNA. One microgram of DNA was then
digested overnight with 2 1 of restriction endonuclease Pst I, or
Hind III. The digested DNA was loaded and electrophoresed on a 1%
agarose gel. The gel was denatured in 1.5 M NaCl and 0.5 M NaOH, then
neutralized in 1M Tris (pH 8.0) and 1.5 M NaCl. The DNA was then
transferred to nitrocellulose and immobilized by baking the
nitrocellulose for 2 hours at 80C under vacuum (34).
Hybridization of Southern Filters
The nitrocellulose containing DNA was wetted with 6X SSC (34) and
prehybridized in 6X SSC, 0.5% SDS, 5X Denhardt's solution (34) and 100
tg/ml of denatured herring sperm DNA in a sealed bag at 56C for 3


55
hours. The ^2P labeled AHV-1 DNA with total counts per minute (CPM)
of 2.2 x106 was added and allowed to hybridize for 12 hours at 56C.
The nitrocellulose was then washed 3 times, for 30 minutes at 56C, in
2X SSC and 0.1% SDS, dried on Whatman 3MM paper and applied to X-ray
film to obtain an autoradiographic image.
DNA Probe
Radiolabeled AHV-1 DNA was made by random primer extension using
the Boehringer Manheim Random Primed DNA Labeling Kit. In brief, 1 /-g
of AHV-1 DNA was denatured at 95C for 10 minutes, then cooled on ice.
Unlabeled nucleotides dGTP, dTTP, and 32P labeled dCTP and dATP, and
Klenow enzyme were added to the denatured mixture, which was allowed
to incubate for 1 hour. The reaction was then stopped with 2 /I of
.25 M EDTA and non-incorporated nucleotides were removed with an 8 ml
desalting column. Fractions were collected from the column, counted
on a scintillation counter and the fractions with peak activity were
pooled. The radiolabeled probe was added to hybridization solution
(5ml formamide, 0.1 ml 20% SDS, 2 ml 20X SSC, 0.5 ml Denhardt's
solution, with 100 /g/ml of denatured herring sperm DNA) and brought
to a total volume of 10 ml with sterile autoclaved double deionized
water. The probe was denatured by boiling for 5 minutes before
hybridization with nitrocellulose.
Results
Cross-reactivity of AHV-1 Proteins
Virus-induced proteins (strain WC11 ) radiolabeled with 35g_
methionine were immunoprecipitated with equal concentrations (35 /tg)


56
^^^nfecte 205k-
Figure 4.1 SDS-PAGE analysis of ^S-methionine labeled proteins
immunoprecipitated from uninfected (Vero) and AHV-1 infected (AHV
Infected) Vero cells with equal concentrations of rabbit polyclonal
antibodies to AHV-1, Movar, BHV1, SHV1 and BHV2. Arrows to the right
indicate proteins immunoprecipitated from infected cells and their
apparent molecular weights (x1000).


57
AHV1 Infected
Vero
>
x
w
ro
>
r-
CN
T-
r-
w
TO
CNJ
>
>
>
>
>
>
>
X
X
X
X
o
X
I
CO
CQ
<
c/)
5
CQ
CQ
Figure 4.2 SDS-PAGE analysis of ^^S-methionine labeled proteins
immunoprecipitated from uninfected (Vero), and AHV-1 infected (AHV
Infected) Vero cells with unequal concentrations of rabbit polyclonal
antibodies to SHV1, Movar, BHV1, BHV2 and AHV1. Numbers to the right
indicate position of molecular weight standards and their apparent
molecular weights (x1000).


58
of purified rabbit polyclonal antibody against AHV-1, BHV-1, BHV-2,
Movar, and SHV-1. Some antigenic cross reactivity was seen in this
analysis with most AHV-1 proteins (Figure 4.1). This cross-reactivity
was especially notable for proteins of 52 kDa and 54 kDa. There was
however, also a notable lack of reactivity of a 110 kDa protein with
any antibodies other than those made to AHV-1 itself. In fact this
protein appeared completely unique to AHV-1.
If this experiment was repeated using equal volumes of antibody,
a rather different pattern was seen (Figure 4.2). The total amounts
of each polyclonal antibody used was 35 /-g anti-AHV-1, 48 /g anti-SHV-
1, 55 yug anti-BHV-1, 115 /g anti-BHV-2, and 175 /g anti-Movar. Cross
reactivity now appeared extensive. The 110 kDa protein that appeared
unique when equalized concentrations of antibody are used, was
precipitated by the antibodies raised to Movar when these were present
in higher concentrations.
DNA Hybridization
DNA was isolated from AHV-1, BHV-2 and Movar. This DNA was
digested by restriction endonucleases Eco RI and Hind III, and run on
a 1% agarose gel. The DNA was then transferred to nitrocellulose and
blotted with the ^2p labeled AHV-1 DNA. Hybridization was extensive
in lanes containing AHV-1 DNA (Figure 4.3). Hybridization was also
seen with one major band and several (at least six) minor bands
ranging in size from approximately 4 to 9 kilobases in both the Eco RI
and Hind III digests of Movar 33/63. No hybridization was noted
between the AHV-1 probe and digested BHV-2 DNA even after long


59
Figure 4.3 Southern blot analysis of DNA isolated from WC11 strain
of AHV-1, BHV-2, and Movar digested with restriction endonucleases Pst
I and Hind III and hybridized with WC11 AHV-1 labeled with ^P, by
random primer extension. Numbers to the right indicate positions of
lambda-Hind III fragment standards, and their sizes in kilobases.


60
exposure of the probed nitrocellulose to radiographic film (Figure
4.3).
Unique epitopes
Cross immunoprecipitation experiments did not identify a single
protein that could be assumed to be unique to AHV-1. We therefore
sought to identify at least individual epitopes unique to AHV-1. In
order to do this, susceptible cell lines were infected with BHV-1,
BHV-2, SHV-1 and Movar and labeled with 35g_methionine as described
above. Lysates were made of the infected cells, and samples were
immunoprecipitated with polyclonal rabbit antibodies to the respective
viruses, as well as to AHV-1. Samples of lysate were also irrcnuno-
precipitated with 2 monoclonal antibodies. Since a neutralization
assay is currently the only method capable of distinguishing animals
infected with AHV-1 from those carrying other herpesviruses, we
reasoned that antibodies capable of neutralizing infectivity might be
the most likely to recognize unique AHV-1 epitopes. One antibody,
12B5, was selected because of its ability to neutralize virus in
vitro. This antibody recognizes the AHV-1 115 kDa and associated
proteins. The second antibody selected was 31B3. This antibody does
not have neutralizing activity at similar antibody concentrations, but
immunoprecipitates the same protein complex as monoclonal 12B5. It
was found that monoclonal antibodies 12B5 and 31B3 did not
specifically react with 35S-methionine labeled proteins of BHV-1, BHV-
2, Movar or SHV-1 (Figure 4.4, 4.5). These data suggested that the 2
epitopes recognized by the monoclonal antibodies were likely to be
unique to AHV-1.


61
Figure 4.4 SDS-PAGE analysis of -^^S-methionine labeled BHV-1
proteins (left 4 lanes), and BHV-2 proteins (right 4 lanes)
immunoprecipitated with their respective polyclonal antibodies (anti-
BHV-1 or anti-BHV-2), monoclonal antibodies 12B5 and 31B3 and non-
immune antibodies (PB). Numbers to the left indicate positions of
molecular weight standards and their apparent molecular weights
(xlOOO).


62
m
a.
200K-
116K
97K
66K- *
20 OK-
Figure 4.5 SDS-PAGE analysis of 35S-methionine labeled Movar
proteins (left 4 lanes), and SHV-1 proteins (right 4 lanes)
immunoprecipitated with their respective polyclonal antibodies (anti-
Movar or anti-SHV-1), monoclonal antibodies 12B5 and 31B3 and non-
immune antibodies (PB). Numbers to the left indicate positions of
molecular weight standards and their apparent molecular weights
(xlOOO).


63
Discussion
Antigenic cross-reactivity between AHV-1 and the other
herpesviruses tested were expected because of earlier work of others
(3,28). Immunoprecipitation of AHV-1 proteins with polyclonal
antibodies to other bovid herpesviruses suggested that AHV-1 was more
closely related to Movar than it was to BHV-1, BHV-2 or SHV-1. To
further test the degree of relationship between these two viruses, the
degree of DNA homology as evaluated by cross-hybridization of DNA from
AHV-1 and Movar was determined. Although not extensive, a moderate
degree of homology was shown to exist at the DNA level. This finding
further substantiated a closer relationship between these two viruses
and was consistent with data generated by ourselves and others that
indicated antigenic cross reactivity between AHV-1 and Movar (43,57).
Although the various herpesviruses are undoubtedly antigenically
related to some degree, the fact that the virus neutralization assay
was discriminating suggested that proteins with neutralizing epitopes
might be wholly or in part unique to AHV-1. It was our intention to
identify a protein that was entirely unique to AHV-1, that might
provide the basis of a diagnostic assay. Monoclonal antibodies or
polyclonal monospecific antibodies to this protein might then be used
in the development of a competitive ELISA. Alternatively, cloning of
the gene encoding this protein, and its subsequent expression, might
allow its purification in relatively large amounts. The purified
protein could then be used as antigen in a direct or indirect ELISA.
This approach could provide a very specific and rapid means of
diagnosis.


64
An entirely unique protein could not be definitively identified
by immunoprecipitation of AHV-1 proteins with polyclonal antibodies
to other bovid herpesviruses. An alternate approach was thus pursued.
Identification of monoclonal antibodies that reacted with individual
unique epitopes on AHV-1 that were not shared with the other viruses
provided reagents that could be used in the development of a
prototype diagnostic assay for AHV-1 antibodies. These monoclonal
reagents provide the basis for a prototype of an improved diagnostic
assay described in Chapter 5.


CHAPTER 5
PROTOTYPE DIAGNOSTIC ELISA
Introduction
The following chapter describes a preliminary assessment of a
prototype diagnostic assay for infection with AHV-1. This assay will
be based on a competitive ELISA, which tests the ability of sera from
animals exposed to AHV-1 to compete for the binding of a monoclonal
antibody to AHV-1 antigen. If an assay of this type proves to be
sensitive and specific, it will provide a useful tool for rapid, safe
and relatively inexpensive diagnosis of AHV-1 infection.
Materials and Methods
ELISA Reagents
The antigen in this assay was composed of protein from WC11
infected Vero cells, and was used at concentrations equal to that used
in the ELISA described previously for the screening of hybridomas.
Plates were coated with antigen in bicarbonate/carbonate buffer pH 9.6
and washed 3 times in wash buffer containing 0.05% Tween 20, and 0.85%
NaCl. Samples being tested for the presence of antibodies against
AHV-1 were added to plates for one hour at 37"C, and followed by
addition of monoclonal antibody 12B5 at a concentration of 0.1 /ig per
well. The mixture was allowed to incubate for an additional 2 hours,
washed 3 times with wash buffer and incubated for 1 hour at 37"C with
goat anti-mouse antibody conjugated with horse radish peroxidase
65


66
and washed again. The amount of anti-mouse antibody bound was
visualized by a change in the color of the substrate ortho-
phenylenediamine (Sigma). The reaction was stopped with 2M H2SO4,
and the absorbance at 492 nm measured with an ELISA reader. The
ability of the serum samples to compete with the monoclonal antibody
for binding to the antigen was expressed as percent of control values
(see below).
The optimal concentration of monoclonal antibody to be used in
the assay was determined in tests using a serum sample from a bison
with clinical disease and known to contain antibodies to AHV-1, and a
known negative serum sample. Concentrations of monoclonal antibody
ranging from 0.1 yug to 100 Ag per well were examined. Results were
expressed as a percentage of the optical density of a well that had
been reacted with the monoclonal antibody, but no competing serum
sample. That is to say that the value is expressed as a percentage of
the maximum possible optical density in the absence of competing serum
antibodies.
Serum samples
Polyclonal rabbit sera made against AHV-1, BHV-1, BHV-2, Movar,
and SHV-1, were tested for their ability to compete with the
monoclonal antibody 12B5 for binding with AHV-1 antigen. Also tested
were 3 bovid serum samples (2 wildebeest and 1 bison) known to be
positive and 4 bovid sera (1 wildebeest, 1 domestic cow, 1 moose and 1
domestic goat) known to be negative by virus neutralization. In
addition a total of 8 serum samples from sheep associated with a


67
recent outbreak out malignant catarrhal fever in Alaskan moose were
tested. Five samples were known to be positive and two samples were
known to be negative by virus neutralization. One sheep sample was
considered a borderline positive by the neutralization assay.
Thirteen serum samples obtained from a sentinel cattle herd belonging
to the University of Florida, and presumed to be negative, were also
assayed.
Results
Titration of 12B5
The optimum concentration of monoclonal antibody 12B5 for use in
the competitive ELISA was found to be 0.1 /g per well. This
concentration produced the maximum difference in reactivity between
sera with and without antibodies to AHV-1 (Figure 5.1).
Rabbit antibodies
Equal concentrations of purified polyclonal antibodies against
AHV-1, BHV-1, BHV-2, Movar and SHV-1 were tested in the ELISA to
determine their ability to compete with the monoclonal antibody 12B5
for binding to AHV-1 antigen. In this experiment results were
expressed as a percentage of the optical density in a well containing
equal concentrations of non-immune rabbit antibody. At 20 tg of
antibody there was a clear distinction between the ability of AHV-1
antibody to compete for antigen versus that of the other polyclonal
antibodies. Figure 5.2 illustrates that when between 0.5 /tg and 20 ag
of each polyclonal antibody was used in this assay, the AHV-1 antibody
was better able to crpete with binding of antibody than any of the


68
Figure 5.1 Titration of monoclonal antibody 12B5 for use in a
competitive ELISA. Percent control is calculated as the optical
density of wells containing serum and monoclonal antibody 12B5 divided
by the optical density of wells containing monoclonal antibody only
x100. - Positive bison serum. O-Negative goat serum. Monoclonal
antibody 12B5 used at concentrations of a) 100 /tg/ml b) 10 /*g/ml
c) 1 /tg/ml d) 0.1 /tg/ml.


69
o
H
-p
c
8
Q

<#>
Figure 5.2 Analysis of the ability of rabbit polyclonal antibodies
raised against various bovine herpesviruses to reduce binding of
monoclonal antibody 12B5 to AHV-1 infected cell lysate in a
competitive ELISA. Percent control is calculated as the optical
density of wells containing the polyclonal antibody sample divided by
the optical density of wells containing equal concentrations of non-
immune rabbit antibodies x 100. o- rabbit anti-AHV-1, - rabbit
anti-Movar, n rabbit anti-BHV-1, O- rabbit anti-BHV-2, -
rabbit anti-SHV-1.


70
other polyclonal antibodies. When the anti AHV-1 antibody concen
tration dropped below 0.5 /tg, it no longer competed with the mono
clonal antibodies for binding to antigen.
Bovid sera
The bovid serum samples were diluted 1:10 and tested for their
ability to compete with monoclonal antibody for binding to antigen.
The data illustrating the ability of the serum samples to compete are
expressed as percent inhibition of binding. This was calculated by
the formula: % inhibition of binding = a-b/a, where a = the optical
density in wells containing negative sera and monoclonal antibody
12B5, and b = the optical density in wells containing test sera and
monoclonal antibody 12B5. The 3 known AHV-1 positive serum samples at
dilutions of 1:10 were able to each compete with monoclonal antibody
for binding to the antigen. When these 3 samples were compared with
known negative serum samples, they were able to reduce the binding of
monoclonal antibody from 35 to 50% (Figure 5.3). The 3 known negative
serum samples reduced the ability of the monoclonal antibody to bind
AHV-1 antigen by less than 1% (Figure 5.3). The serum samples from
the UF sentinel herd reduced the binding from 0 to a maximum of 25%
(Figure 5.3). Four of the known positive sheep samples reduced
binding from 22 to 30%, and all of the known negative sheep samples
reduced binding less than 6%. One sheep sample that was determined to
be borderline-positive by a virus neutralization conducted by the San
Diego Zoo, was the median value of the sheep samples in its ability to


71
reduce binding to AHV-1 antigen. One known positive sheep serum
sample reduced binding only 8% (Figure 5.4).
Discussion
Although very preliminary, the results thus far generated with
the prototype competitive ELISA strongly suggested that it has premise
for identification of animals infected with AHV-1. The assay was
clearly capable as differentiating rabbit polyclonal antibodies raised
against AHV-1 and the other bovid herpesviruses. In addition, serum
from bovids testing positive with the virus neutralization assay were
better able to compete with monoclonal antibody 12B5 for binding with
antigen than were 4 serum samples shown to be negative by the same
test. When the 13 cattle from the UF sentinel herd were tested, the
ability to crpete for binding with monoclonal 12B5 was more variable,
but no samples were able to conpete more than 25%.
It has been demonstrated that serum from sheep associated with
outbreaks of malignant catarrhal fever in cattle are capable of
neutralizing AHV-1 virus in plaque reduction protocols (22). The
interpretation of these findings was that the sheep associated form of
malignant catarrhal fever is caused by an as yet unidentified virus
which is very similar to AHV-1. Our data support this interpretation.
The total number of animals tested in this assay is to date very
small, and must be expanded before statistical analysis of its
predictive value can be determined. If after further analysis, the
monoclonal 12B5 and the infected cell lysate as antigen does not allow
specific diagnosis, several alternative approaches might be pursued.


72
Bovine & Sentinel
Alcelaphine Bovine
Figure 5.3 Ability of serum to inhibit binding of monoclonal
antibody 12B5 to AHV-1 infected cell antigen. Percent inhibition of
binding = a-b/a; where a = Optical density in wells containing
monoclonal antibody and known negative serum, b = Optical density in
wells containing monoclonal antibody and test sera. - Samples known
to be positive by virus neutralization assay. O- Bovine and
Alcelaphine serum samples known to be negative by virus neutralization
and Bovine samples from sentinel herds- presumed to be negative.


73
Bovine & Ovine
Alcelaphine
Figure 5.4 Ability of Alcelaphine, Bovine, and Ovine serum to
inhibit binding of monoclonal antibody 12B5 to AHV-1 infected cell
antigen. Percent inhibition of binding = a-b/a; where a = Optical
density in wells containing monoclonal antibody and known negative
serum, b = Optical density in wells containing monoclonal antibody and
test sera. - Samples known to be positive by virus neutralization.
O- Samples known to be negative by neutralization. - Borderline
result in virus neutralization assay.


74
An alternate monoclonal antibody to the 115 kDa or some other protein
may yield better results. The fact that antibody 12B5 does neutralize
in vitro implies that the epitope recognized by the antibody is unique
to AHV-1. If an alternate antibody were to be tested in this assay,
it would seem logical to select another capable of neutralizing AHV-1
in vitro. If the assay does prove to have a good predictive value,
production of antigen on a large scale might be attempted by the
cloning of the gene gp115. Cloning of the gene into a plasmid
expression vector might suffice to produce large amounts of a fusion
protein for use as an antigen. One potential problem with this
technique is that the bacterially expressed protein will not be
glycosylated and therefore may not be recognized by the monoclonal
antibodies that we have available. This would necessitate one of two
additional approaches. We could use a mammalian vector system such as
vaccinia virus to produce a protein that was processed more like the
native molecule, but a better approach would probably be to use our
fusion protein to immunize mice to produce additional monoclonal
antibodies. Sera would then need to be tested for their ability to
compete these new monoclonal antibodies iron the purified fusion
protein. One or more of these approaches should lead to the
refinement of this assay as a tool for the identification of carrier
animals infected with AHV-1, and possibly the sheep-associated virus.


CHAPTER 6
THE gp115 SURFACE PROTEIN
Introduction
Several hybridoma antibodies made against AHV-1 were found to
react with a virus induced complex that consisted of 5 proteins that
could be labeled with 35S-methionine. One of the antibodies was also
capable of neutralizing virus in plaque reduction assays. The
neutralizing antibody recognized the same epitope, or one very close
to it, as sera taken from naturally infected animals inhibited binding
of the monoclonal antibody to AHV-1 antigen (see Chapter 4). The
complex thus may include a molecule!s) of significance to both
diagnosis and prevention of the disease. Two hybridoma antibodies
that recognized the complex, 12B5 which neutralized infectivity and
31B3 which did not, were repeatedly cloned and used to investigate the
nature and origin of the protein complex.
Materials and methods
Gel electrophoresis
SDS-PAGE electrophoresis was performed as described previously,
except that 2-mercaptoethanol was omitted from the sample buffer when
gels were to be run under non-reducing conditions.
75


76
Pulse-chase experiments
Monolayers of BEL cells were infected with approximately 5 PFU
per cell of strain 1982 of AHV-1 and infection was allowed to proceed
for 36 hours. At 36 hours post infection the cells were pulsed with
100 MCi/ml of 35g_methionine in methionine free media for 15 minutes
and chased with cold methionine for periods of 15, 30, 45, 60, 120
minutes or 24 hours. To achieve chase of radiolabel the media
containing the ^^s-methionine was removed and replaced with fresh
media containing 100 Mg of cycloheximide and 10OX the normal
concentration of methionine. At the end of each chase period cells
were scraped from the flask with a rubber policeman into ice cold PBS,
pelleted and stored at -20C.
N-glycanase digestion
Strain 1982 infected BEL cells were labeled with 3^S-methionine,
and lysed as described previously. Two samples were immuno-
precipitated with monoclonal antibody 12B5 and protein A-agarose. The
protein A-agarose beads with bound 12B5 antibody and radiolabeled
protein were washed 3 times with RIPA buffer and suspended in 33 Ml of
.2M phosphate buffer pH 8.6 and 0.5% SDS and 0.7% 2-mercaptoethanol.
The samples were then boiled for 3 minutes, and to each was added 20
Ml 0.67 M phosphate buffer pH 8.6, 10 Ml 0.1 M phenanthroline, 16.7 Ml
7.5% NP-40 and 10 Ml of distilled water. The mixture was vortexed.
To one sample was added 2.5 U of N-glycanase in 10 Ml glycerol, and to
the other an equivalent volume of glycerol without enzyme. The
mixtures were mixed with an equal volume of sample buffer and analyzed
by SDS-PAGE.


77
Results
Reducing vs. Non-Reducing Conditions
When the complex immunoprecipitated by antibody 12B5 was analyzed
by SDS-PAGE under reducing conditions 5 proteins were seen. The
molecular weights of these proteins were 115,000, 110,000, 105,000,
78,000, and 48,000. When the same complex was analyzed under non
reducing conditions, only 3 proteins were seen. The proteins of 78
kDa and 48 kDa were not detected, and the 115 kDa protein was present
in very large amounts (Figure 6.1).
If the monoclonal antibody 12B5 was used to immunoprecipitate a
lysate of 12^i labeled 1982 virions, only one protein of 115 kDa was
identified under non-reducing conditions. Under reducing conditions
it was replaced by two smaller proteins of 78 kDa and 48kDa (Figure
6.2). This suggested to us that the mature protein found on the
virion was a heterodimer composed of 2 fragments of 78 kDa and 48 kDa
linked by disulfide bonds. We supposed that the molecules of 110 kDa
and 115 kDa seen in cells labeled with 2^S-methionine were processing
intermediates. To elucidate the relationships of the proteins further
we carried out pulse chase experiments as described above. Proteins
were immunoprecipitated from each pulse and chase period and analyzed
under reducing or non-reducing conditions. Irrespective of the
presence or absence of 2-mercaptoethanol, only one protein of 110 kDa
was specifically immunoprecipitated by 12B5 from the 15-minute pulse
period (Figure 6.3 and 6.4). We presumed this to be the primary
translation product. During the 15-minute chase period two proteins
were visible under both reducing and non-reducing conditions, the


78
R
NR
Figure 6.1 SDS-PAGE analysis of ^^S-methionine labeled proteins
immunoprecipitated from cells infected with monoclonal antibody 12B5,
and electrophoresed under reducing (R) and non-reducing (NR)
conditions. Arrows to the right indicate proteins immunoprecipitated
from infected cells and their apparent molecular weights (x1000).


79
NR
R
200k-
42k-
48
Figure 6.2 SDS-PAGE analysis of proteins immunoprecipitated from
labeled 1982 virions immunoprecipitated with monoclonal antibody
12B5 and electrophoresed under reducing (R), and non-reducing (NR)
conditions. Arrows to the right indicate proteins immunoprecipitated
from virion lysate and their apparent molecular weights (x1000).


80
heavily labeled 110 kDa protein and a lighter band representing a
protein at 105 kDa. After a 30-minute chase period an additional
molecule of 115 kDa could be seen. The 45-minute chase period was the
first time interval at which the protein patterns were clearly
different under reducing and non-reducing conditions. During this
chase period, the 115 kDa protein seen under non-reducing conditions
was a rather heavy band.In contrast the 115 kDa protein was barely
noticeable when run under reducing conditions. Instead, the 115 kDa
protein appeared to be replaced by 2 proteins, one at 78 kDa and one
at 48 kDa. This pattern remained essentially unchanged throughout the
remainder of the chase periods examined, except that the 115 kDa
protein gradually became more prominent with time under non-reducing
conditions (Figure 6.3) and the 78 kDa and 48 kDa proteins likewise
increased in intensity under reducing conditions.
Identity of 115 kDa protein
The -^S-methionine labeled proteins immunoprecipitated by 12B5
from infected cells migrated slightly faster under non-reducing
conditions than under reducing conditions. Molecules of 115, 110, and
105 kDa were seen in reducing gels. These we assumed to be the same
proteins as those that migrated with apparent molecular weights of
slightly less than 115, 110 and 105 kDa. This made it difficult to be
absolutely certain of the identity of the 2 groups. We were most
interested in substantiating what our earlier data had suggested;
namely that the 115 kDa molecule was actually a heterodimer composed
of the 78 kDa and 48 kDa molecules. In order to test this hypothesis,
the following experiment was performed. Bovine embryonic lung cells


81
were infected and labeled with 35S-methionine and immunoprecipitated
with 12B5. The immunoprecipitate was then analyzed by SDS-PAGE. The
gel was fixed for 15 minutes with a solution of 7% acetic acid and 7%
methanol. A 3 mm punch was then used to cut out a series of
acrylamide plugs starting from the top of the gel to below the 58 kDa
molecular weight marker. All plugs were then placed in scintillation
fluid and counted in a scintillation counter. Two major peaks of
radioactivity were seen in regions of the gel to which the proteins of
115 kDa and 110 kDa were expected to migrate (Figure 6.5). The
regions of the gel corresponding with location of these peaks were
excised and protein electroeluted from them at 100 V overnight at 4'C.
A sample containing a total of 8700 CPM of radioactivity from each
protein fraction was loaded onto a 9% gel in an equal volume of
reducing sample buffer and electrophoresed. Exposure of the original
gel confirmed that the 115 kDa and the 110 kDa bands had been excised.
Exposure of the second gel indicated that 2 proteins of 78 kDa and 48
kDa originated from the 115 kDa protein (Figure 6.6). The 110 kDa
protein apparently remained intact except that a new molecule was seen
under reducing conditions with molecular weight of approximately 160
kDa and on overexposure of the gel a faint band of 48 kDa was seen.
The 115 kDa protein was almost entirely reduced to 48 kDa and 78 kDa
molecules, although a small amount of protein could be seen as a light
band running with a mobility of 155 kDa (Figure 6.6).


82
66k-
42k-
Figure 6.3 SDS-PAGE analysis of proteins immunoprecipitated by
monoclonal antibody 12B5, and electrophoresed under non-reducing
conditions. Lanes show infected cells (P) pulsed with 35g_met-hionine
for 15 minutes at 24 hours post infection, and chased for 15 minutes
at 24 hours post infection followed by chase for 15 minutes, 30
minutes, 45 minutes, 60 minutes, 120 minutes, and 24 hours,
respectively, in media containing cycloheximide and 100X cold
methionine. Arrows indicate proteins immunoprecipitated with
monoclonal antibody 12B5.


83
O.
o
n
'in
o
lO
'o
CN
-C
200k-
66 k-
<4i78
42 k-
Figure 6.4 SDS-PAGE analysis of proteins iimmunoprecipitated by
monoclonal antibody 12B5, and electrophoresed under reducing
conditions. Lanes show infected cells (P) pulsed with 35g_methionine
for 15 minutes at 24 hours post infection, and chased for 15 minutes
at 24 hours post infection followed by chase for 15 minutes, 30
minutes, 45 minutes, 60 minutes, 120 minutes, and 24 hours,
respectively, in media containing cycloheximide and 100X cold
methionine. Arrows indicate the positions of the proteins specifically
immunoprecipitated with 12B5.


Punch Sample
84
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
221
y////A
V////////////A
y//////////////////////////^^^^
y///////////,v//////////////////^^^^
y////////////z
'///////A
100
200
cpm
300
400
Figure 6.5 Histogram indicating counts per minutes in 3 rnm punch
samples that were used to identify the location of ^^S-methionine
labeled proteins of AHV-l immunoprecipitated by monoclonal antibody
12B5 and electrophoresed in a 9% polyacrylamide gel under non-reducing
conditions.


35
1 2
200K-
42 K-
Figure 6.6 SDS-PAGE analysis of ^%-methionine labeled AHV-1 110 kDa
protein (lane 1) and 115 kDa protein (lane 2) excised from a non
reducing gel, electroeluted and subsequently run on a 9%
polyacrylamide gel under reducing conditions. Numbers to right
indicate molecular weights of proteins (x1000).


86
Figure 6.7 SDS-PAGE analysis of 35S-methionine labeled AHV-1
proteins immunoprecipitated by monoclonal antibody 12B5 mock digested
(lane 1) or digested (lane 2) with N-glycanase. Numbers to left
indicate proteins immunoprecipitated from AHV-1-infected cells and
their apparent molecular weights (x1000).


87
N-Glycanase digestion
Digestion of the immunoprecipitated 115 kDa complex with N-
glycanase under reducing conditions resulted in a loss of the 115 kDa,
110 kDa and 105 kDa proteins, and the appearance of a new protein at
approximately 100 kDa. The 78 kDa and 48 kDa proteins were not seen
in the digested sample, but new proteins of 46 kDa and 60 kDa became
apparent (Figure 6.7).
Discussion
Pulse-chase experiments indicated that the first protein in the
115 kDa complex to be made was the 110 kDa molecule. We also knew
from previous experiments that this molecule was glycosylated (Figure
2.5). This was consistent with the 110 kDa protein being the primary
translation product to which N-linked sugars were added. The next
protein to appear in the pulse-chase experiments was the 105 kDa
protein. This protein was seen in small amounts under both reducing
and non-reducing conditions, and is considered to be an intermediate
form that possibly has undergone trimming of sugars. The 115 kDa
molecule first appeared in the 30 minute chase period, and label
continued to accumulate in this protein for the next 24 hours if
samples were analyzed under non-reducing conditions. When the pulse-
chase samples were run under reducing conditions, most of the 115 kDa
molecule was replaced with a 78 kDa and a 48 kDa molecule. This
provided evidence to suggest that the 115 kDa molecule was a
heterodimer comprised of a 78 kDa and a 48 kDa molecule held together
by disulfide bonds. Since the 115 kDa molecule appeared last in the
pulse-chase experiment, we hypothesized that the 115 kDa molecule was


88
the mature form of the protein. Further support for this assumption
was provided by the fact that when lysate of 125I labeled virions were
immunoprecipitated with monoclonal antibody 12B5 and run under non
reducing conditions, only one protein of 115 kDa was seen. When this
same precipitated molecule was analyzed under reducing conditions, it
was replaced with two proteins, one at 78 kDa and one at 48 kDa. A
very small amount of the 115 kDa molecule was visible in auto
radiographs of gels run under reducing conditions. We hypothesized
that this may represent a transient uncleaved form of gp115.
Since relative mobilities of proteins were slightly different
when they were electrophoresed under reducing rather than non-reducing
conditions, an experiment was undertaken to be certain that the 115kDa
molecule represented the heterodimer. The 115 kDa and the 110 kDa
proteins were excised from preparative gels run under non-reducing
conditions, and subjected to reducing conditions before being
separated by SDS-PAGE. The 115 kDa molecule was almost completely
degraded into a 48 kDa and a 78 kDa protein. The 110 kDa molecule
remained largely intact with a small amount of protein visible at 48
kDa. A new species, heavily labeled appeared at approximately 160
kDa. This probably represents aggregation of protein as a result of
oxidation of cysteine residues during electroelution, as only a very
small region corresponding to a molecular weight of 110 kDa was
excised from the preparative gel for electroelution. The small amount
of proteins at 48 kDa that could be seen when the 110 kDa molecule was


89
reduced with 2-mercaptoethanol we believe might result from breakdown
of the 110 kDa protein at the cleavage site.
When the 115 kDa protein complex was immunoprecipitated with
monoclonal antibody 12B5 and digested with N-glycanase under reducing
conditions, the 115 kDa, 110 kDa and the 105 kDa molecules were
replaced by a molecule of approximately 100 kDa. The 78 kDa and 48
kDa molecules were also replaced by molecules of approximately 60 kDa
and 46 kDa. This would suggest that the 115 kDa, 110 kDa, and the 105
kDa molecules all have the same protein backbone and that processing
probably does not involve addition of 0-linked sugars. These data
also suggest that the 2 new molecules of 60 kDa and 46 kDa are the
digestion products of the 78 kDa and 48 kDa proteins. After
consideration of these data we propose the following biosynthetic
pathway for the gp115 complex. The primary translation product gp110
is processed via a 105 kDa intermediate to a protein of 115 kDa. The
115 kDa molecule is cleaved into 2 proteins of 78 kDa and 48 kDa which
are linked by disulfide bonds. This disulfide linked heterodimer is
the mature form of the protein that is inserted into the virion
envelope.
These studies, although not exhaustive, have provided a
foundation for further evaluation of this apparently abundant protein
that undoubtedly has some functional significance to the virus and may
be useful as a reagent in diagnosis and possibly immunization. This
information will prove invaluable in pursuit of these goals.


CHAPTER 7
SUMMARY
An appreciation of the biological activity of herpesviruses is
dependent upon the understanding of the components, such as membrane
proteins, that are important to the process of infection. We have
been interested in examining the membrane proteins of Alcelaphine
herpesvirus 1 with several long term goals in mind.
First, the virus of malignant catarrhal fever is a significant
pathogen in cattle and results in a devastating disease. Studies of
the roles that membrane proteins play in the initiation of immunity
may in the future help to elucidate elements useful in the preparation
of subunit vaccines. We have produced one monoclonal antibody 12B5
that recognizes a complex of membrane glycoproteins that we have
designated gp115. The monoclonal antibody 12B5 is capable of
neutralizing virus. This result suggests that gp115, which is
expressed on the surface of the virion, warrants further evaluation as
a potential protective immunogen. With the potential importance of
this protein in mind, its processing was more thoroughly examined.
Second, we sought to help solve a problem that exists in
veterinary medicine. Prompt identification of animals that may serve
as carriers of AHV-1, and related viruses, is essential to effective
control and containment of malignant catarrhal fever. Our first
intention was to identify a protein unique to AHV-1 that was not
90


91
shared with the other herpesviruses infecting cattle. Our hypothesis
was that if we could identify such a protein, we could use antibodies
reactive with this protein as reagents in a diagnostic competitive
ELISA test. No single protein could be conclusively identified as
unique, therefore a different approach was taken. Monoclonal antibody
12B5 was confirmed to recognize an epitope unique to AHV-1, and
animals known to have been infected with AHV-1 possessed antibodies
recognizing this same epitope. The antibody was therefore shown to be
a suitable reagent for use in a competitive ELISA assay. Results
indicate that an assay of this type may prove to be useful in the
diagnosis of not only AHV-1, but also possibly the sheep associated
form of malignant catarrhal fever. One strategy for possible
improvement of the specificity of this assay might include the use of
portions of gp115 as antigen. Large amounts of gp115 could be
obtained by cloning the appropriate gene into plasmid expression
vectors. Since monoclonal antibody 12B5 recognized not one, but a
complex of proteins, the precursor/product relationships of these
molecules was further elucidated. Knowledge we obtained regarding the
relationships between these molecules will be useful in designing
cloning and selection stategies for genes encoding the protein
complex.
Third, we are intrigued by the pathological similarities between
AHV-1 and the human diseases associated with Epstein-Barr virus.
Alcelaphine herpesvirus 1 might in the future represent an interesting
animal model for the study of human gammaherpesviruses. This research
goal was not specifically addressed in this dissertation, but


92
the identification and characterization of the virus membrane proteins
that was accomplished will provide a useful foundation for the study
of the role of membrane proteins in gammaherpesvirus-induced disease.


REFERENCES
1. Adams, S.W., N. Balachandran, L.M. Hutt-Fletcher. 1988.
Proteins specified by bovine herpesvirus 2. Am. J. Vet. Res.
49:443-448.
2. Balachandran, N., S. Bachetti, W.E. Rawls. 1982. Protection
against lethal challenge of Balb/c mice by passive transfer of
monoclonal antibodies to 5 glycoproteins of herpes simplex
type 2. Infec. Immun. 37:1132-1137.
3. Balachandran, N., D.E. Oba, L.M. Hutt-Fletcher. 1987. Antigenic
cross reactions among herpes simplex virus types 1 and 2,
Epstein-Barr virus and cytomegalovirus. J. Virol. 61:1125-1135.
4. Batterson, W., D. Furlong, B. Roizman. 1983. Molecular genetics
of herpes simplex virus. VII. Further characterization of a ts
mutant defective in release of viral DNA and in other stages of
viral replicative cycle. J.Virol. 45:397-407.
5. Bonner, W.M., R.A. Laskey. 1974. A film detection method for
tritium-labeled proteins and nucleic acids in polyacrylamide
gels. Eur. J. Biochem. 40:83-88.
6. Brigden, A., A.J. Herring, H.W. Reid. 1986. The molecular
biology of Alcelaphine herpesvirus 1. Abstracts of the
Eleventh International Herpesvirus Workshop. Leeds, England.
7. Castro, A.E., G.G. Daley. 1982. Electron microscopic study of
the African strain of malignant catarrhal fever virus in bovine
cell cultures. Am. J. Vet. Res. 43:576-582.
8. Castro, A.E., G.G. Daley, M.A. Zimmer, D.L. Whiteneck, J.
Jenson. 1982. Malignant catarrhal fever in an Indian gaur and
greater kudu; experimental transmission, isolation, and
identification of a herpesvirus. Am. J. Vet. Res. 43:5-11.
9. Castro, A.E., D.L. Whiteneck. D.E. Goodwin. 1981. Isolation
and identification of the herpesvirus of malignant catarrhal
fever from exotic ruminant species in a zoological park in North
America. Am. Assoc, of Vet. Lab. Diag. 24th Annual Proceedings
Chicago, IL p 67-68.
10. Daniels, R.S., J.C. Downie, A.J. Hay, M. Knassow, J.J. Skehel,
M.L. Wang, D.C. Wiley. 1985. Fusion mutants of the influenza
virus hemmagglutinin. J. Biol. Chem. 260:2973-2981.
93


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