Membrane proteins of alcelaphine herpesvirus 1


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Membrane proteins of alcelaphine herpesvirus 1
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vi, 99 leaves : ill. ; 29 cm.
Adams, Steven Wade
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Membrane Proteins   ( mesh )
Herpesviridae   ( mesh )
Cattle   ( mesh )
bibliography   ( marcgt )
non-fiction   ( marcgt )


Thesis (Ph. D.)--University of Florida, 1989.
Includes bibliographical references (leaves 93-98).
Statement of Responsibility:
by Steven Wade Adams.
General Note:
General Note:

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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oclc - 20895185
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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 influences--social, philosophical and scientific--should 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.



ACKNOWLED ENTS.................................................ii

ABSTRACT .........................................................



Historical Perspective ................................... 1
Clinical and Pathological Manifestations.................. 3
Transmission............................................. 5
Virus Characteristics.................................... 6
Diagnosis................................................ 9
Importance of Membrane Proteins .......................... 12


Materials and Methods....................................... 16


Materials and Methods.....................................32


Materials and Methods....................................... 52


Materials and Methods...................................... 65
Results .................................................... 67
Discussion ............................................... 71



Introduction.............................................. 75
Materials and Methods .......................................75

7 SUMMARY....................................................90

REFERENCES .....................................................93

BIOGRAPHICAL SKETCH...........................................99

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



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

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 are 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.



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

lymphoproliferative disease with a mortality approaching 100%


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

mortality in endangered species as a result of transmission of AHV-1


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).

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 serious 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

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

in gut-associated lymphoid tissue. This lesion may represent a

potential model for herpes virus-induced oncogenesis (24).


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 from 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

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.

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 ateles. 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

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 from 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.ateles, 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 ateles. The M component is thought to consist of unique coding

regions surrounded by flanking repeated sequences of varying length.


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).


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 conmonly 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


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.

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 mammnillitis 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 titerr) 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


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 CR2 (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

upon a decrease in pH within the endosome 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


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

inmunogens for prophylaxis. Further, more detailed characterization

of these proteins will provide information useful to better understand

the pathogenesis of infection.




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 35S-

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 identified the times we might maximize

radiolabeling of particular proteins of interest. Since glycoproteins

present in cell and virus membranes are highly immunogenic and

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 34'C 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

in Vero cell monolayers at 10 days post infection. Titers of WC11

ranged from 1.5 x 103 to 1 x 10 PFU per ml. Titers of 1982 pools

ranged from 1 x 106 to 1 x 107 PFU per ml.


Vero cells were infected with WC11 virus at an MOI of 1 PFU to

100-1000 cells, and labeled with 35S-methionine or 3H-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 -20"C. Bovine embryonic lung cells were infected with strain 1982

virus at an MOI of 5-10 PFU/cell and labeled with 35S-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 1251 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 1251 by the 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 1l samples.

Labeled samples were either suspended immediately in 100 4l

sample buffer containing 0.65 M Tris, 1% sodium dodecyl sulfate (SDS),

10% glycerol and 1% 2-mercaptoethanol, or prepared for immuno-


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 from

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 1251

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.)


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 NaC1, 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 41 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 A1

of sample buffer to dissociate immunoprecipitated proteins. The

amount of radioactivity in a 5 4l sample was determined and the

proteins were either immediately analyzed or stored at -20 C for

analysis at a later time.


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. Lanunoprecipitated 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 destined 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 125I 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 35S or 3H 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 dH20

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).


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 35S-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 cellproteins

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

1 2 3 4
'UMW low lwnwPW

205K -

116K- ~-145



Figure 2.1 SDS-PAGE analysis of 35S-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 (x1000).

1 2 3 4 5 6 7 8
-~" -- _`~? -"

~--I "-V r-
S" .-.,
P'~~~~~ ~ "^*t1 *"^* -< tt. -si"* .^_ -*-
&.. .; *** -- < 7 --
ft;kY~ IPL je ,,**,_ t4

9 10
qw Iw --

S 135
g 105

= -37

Figure 2.2 SDS-PAGE analysis of 35S-methionine labeled proteins
immunoprecipitated by rabbit anti-AHV-1 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 (x000).







WC11, cells were infected with each strain, labeled with

35S-methionine, harvested and immunoprecipitated with polyclonal

rabbit antibodies as described above. The 35S-methionine labeled

protein profiles of WC11 and 1982 are virtually identical with one

exception. A 37 kDa protein is imnunoprecipitated from WC11-infected

Vero cell lysate, but not from lysate of 1982-infected BEL cells. All

other viral induced proteins identified in WC11 appear to have

counterparts in the 1982 strain with similar molecular weights (Figure


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 125I, lysed, 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 1251 lysed 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 1251

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

WC11 1982

0[ -+-175 200K- -175

-145 1
S --135 1
.125 -c135
116K- 115 --125
-0-110 116K- 115
S-105 1-i10
97K- -*95 97K- --95
-*-78' .*--78

-+55 l' -*"55

-,-44 ,.,.-,-.- -44
42K-4 42K-

Figure 2.3 SDS-PAGE analysis of 35S-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).






200k- -




w 4130
116k- A*,110








Figure 2.4 SDS-PAGE analysis of 1251 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 (x1000).


(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
125I (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 1251 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.


U N-


- *-.


..4, 130




116K .-

97K- .

66K- '*.




.- 4130
., .. 115
4I .110


4 62



Figure 2.5 SDS-PAGE analysis of 3H-glucosamine labeled proteins
inrrunoprecipitated from uninfected (Vero, BEL) and AHV-1 strain WC11
(WC11) and AHV-1 strain 1982 (1982) cells. Numbers to the right
indicate virus induced proteins and their apparent molecular weights






-' -

Table 2.1- Proteins Identified by Immunoprecipitation with
Polyclonal Antibodies to AHV-1.


M.W.a 35S-methionine 1251 3H-glucosamine

WC11 1982 WC11 1982 WC11 1982






















a- Molecular weight (x1000).
b- A glycoprotein of 44 Kd was not precipitated with polyclonal
antibody, but was identified with monoclonal antibody 11 C3
(see Chapter 4).


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 35S-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 35S

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 1251.

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 1251. 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 3H-

glucosamine or 35S-methionine. If we assume that the proteins


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 1251. 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.




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


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 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 /l

SDMEM with 20% fetal bovine serum (FBS) per well. After 24 hours, 100

Ai 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.


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

each incubation with 0.05% Tween 20 in 0.85% NaCI; 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 4"C 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

neutralizing activity. Subclones of hybridoma 12B5 were further

screened with this technique to confirm neutralizing activity.



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 Ag

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


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






0.2 1

30 6 3 1.5 .75 .38 0

Antigen concentration (ug/ml)

Figure 3.1 Titration curve of AHV-1 infected and uninfected Vero
cell ELISA antigen. 0 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.

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 125I or 35S-methionine.

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 immunoprecipitate a labeled protein from the lysate of 1251

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.

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

imnunoprecipitate this 95 kDa molecule from 1251 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 35S-methionine and 3H-glucosamine labeled virus-infected cells that

had been immunoprecipitated with polyclonal rabbit antibodies to AHV-

1. Monoclonal antibodies 24C1 and 21B1 did not imnunoprecipitate this

protein from 3H-glucosamine labeled 1982-infected cells (Figure 3.6),

but did imnunoprecipitate a 95 kDa protein from 3H-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-

infected cells metabolically labeled with 35S-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 3H-glucosamine. It

was immunoprecipitated weakly by polyclonal serum, but was readily

identifiable after immunoprecipitation with monoclonal 11C3. It was

particularly prominent in infected cells that had been labeled with

35S-methionine, but could not be identified by labeling of virions

with 1251. 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 3H-glucosamine and 35S-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 immuno-

precipitated only 2 proteins of the group from lysate of 125I

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).


Production of monoclonal antibodies allows analysis of individual

AHV-1 proteins. A summary of the data presented in this chapter




97k- I





d .478

~J4 48

- 44

Figure 3.2 SDS-PAGE Analysis of 35S-methionine labeled proteins
immunoprecipitated from cells infected with WC11 by rabbit polyclonal
anti-AHV 1 antibodies (R-AHV), and monoclonal antibodies 14B6, 12B5,
21B1 and 11C3. Arrows to the right indicate proteins irmmuno-
precipitated from infected cells and their apparent molecular weights



m 4145



Va 105

4 195



Z 4*48

O 4144

Figure 3.3 SDS-PAGE analysis of 35S-methionine labeled 1982 proteins
irrmunoprecipitated 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).

I m




iM 478


Figure 3.4 SDS-PAGE analysis of 1251 labeled proteins of WC11
virions, inmmunoprecipitated by polyclonal rabbit anti-AHV I 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).







Figure 3.5 SDS-PAGE analysis of 1251 extrinsically labeled proteins
of 1982 virions, irmunoprecipitated 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).








- 4145


97k- ,





-#4 44

Figure 3.6 SDS-PAGE Analysis of 3H-glucosamine 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).

Table 3.1 Summary of Characteristics of Hybridoma Antibodies
Reactive with Virus Induced Proteins.

ELISA Reactivity
MWa Hybridoma Infected Uninfected Floresc. Immunoppt.b Neut.c
Antibodies Antigen 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.

ELISA reavtivity
Surface Virus
MWa Hybridoma Infected Uninfected Flouresc. Immunoppt.b Neut.c
Antibodies Antigen 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 imnunoprecipitated by antibodies.
b positive indicates hybridomas that were capable of immunprecipitating
proteins radiolabeled with 125I and/or 35S-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

21 B2

Goat (-)
Calf (-)
R21 (+)

(-) Known negative sera
(+) Known positive sera

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 1251. 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 35S-methionine or 3H-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 35S-

methionine, and 3H-glucosamine labeled infected cells. This protein

was not appreciably labeled by 1251.

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

the plaques appeared to be smaller in diameter. The 5 proteins

labeled very well with 35S-methionine and 3H-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 1251 labeled virions. The evaluation of the

relationships between the proteins of the group is the subject of

Chapter 6.




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, 3, 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 are 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, DNA homology as well as antigenic relationships are 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

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 35S-

methionine (40 jCi/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


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 37'C until CPE was extensive. Cells were harvested and

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 5 x 107 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 (10mM Tris

chloride, 1mM 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.

The purified virus pellet was suspended in 0.5 ml of TE buffer

and protein was digested by the addition of Proteinase K (100,Ag/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 4'C.

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 ul 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 NaC1 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 80'C 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

/Ag/ml of denatured herring sperm DNA in a sealed bag at 56'C for 3

hours. The 32P labeled AHV-1 DNA with total counts per minute (CPM)

of 2.2 x106 was added and allowed to hybridize for 12 hours at 56'C.

The nitrocellulose was then washed 3 times, for 30 minutes at 56'C, 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 Ig

of AHV-1 DNA was denatured at 95"C for 10 minutes, then cooled on ice.

Unlabeled nucleotides dGTP, dTIP, 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 pl 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 Ag/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.


Cross-reactivity of AHV-1 Proteins

Virus-induced proteins (strain WC11) radiolabeled with 35S-

methionine were immunoprecipitated with equal concentrations (35 Ag)








45k- vw 4

- -1


Figure 4.1 SDS-PAGE analysis of 35S-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 iimunoprecipitated from infected cells and their
apparent molecular weights (x000).

AHV Infected

AHV1 Infected





> M
j: |

Figure 4.2 SDS-PAGE analysis of 35S-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).


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 lg anti-AHV-1, 48 /g anti-SHV-

1, 55 Ag anti-BHV-1, 115 uAg anti-BHV-2, and 175 Ag 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 32P 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


> g> >
o o 0 X







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 32P, by
random primer extension. Numbers to the right indicate positions of
lambda-Hind III fragment standards, and their sizes in kilobases.

exposure of the probed nitrocellulose to radiographic film (Figure


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 35S-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 immuno-

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.


42K- ;

Figure 4.4 SDS-PAGE analysis of 35S-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


200K- ,






116K- -





o M
CO W M n c
0 C) 32 Cc



116K- 116K-

97K- 97K-

66K- 66K-

42K- 42K- -

Figure 4.5 SDS-PAGE analysis of 35S-methionine labeled Movar
proteins (left 4 lanes), and SHV-1 proteins (right 4 lanes)
inmunoprecipitated with their respective polyclonal antibodies (anti-
Movar or anti-SHV-1), monoclonal antibodies 12B5 and 31B3 and non-
innune antibodies (PB). Numbers to the left indicate positions of
molecular weight standards and their apparent molecular weights


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



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.




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%

NaC1. 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 i g 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

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 H2S04,

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 g to 100 pg 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


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

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



Titration of 12B5

The optimum concentration of monoclonal antibody 12B5 for use in

the competitive ELISA was found to be 0.1 pg 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 /Ag 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 jAg and 20 Ag

of each polyclonal antibody was used in this assay, the AHV-1 antibody

was better able to compete with binding of antibody than any of the









0 a b
S 90







c d
10 i t i i I '

0 20 40 60 80 100 0 20 40 60 80 100

Reciprocal of sera dilutions

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 /pg/ml b) 10 /ig/ml
c) 1 Ag/ml d) 0.1 Ag/ml.



0I I I



20 2 1 .5 .25.12 .06

Antibody concentration (ug/well)

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-
imnune rabbit antibodies x 100. o- rabbit anti-AHV-1, rabbit
anti-Movar, a rabbit anti-BHV-1, 0- rabbit anti-BHV-2, a -
rabbit anti-SHV-1.

other polyclonal antibodies. When the anti AHV-1 antibody concen-

tration dropped below 0.5 Ag, 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

reduce binding to AHV-1 antigen. One known positive sheep serum

sample reduced binding only 8% (Figure 5.4).


Although very preliminary, the results thus far generated with

the prototype competitive ELISA strongly suggested that it has promise

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 compete for binding with monoclonal 12B5 was more variable,

but no samples were able to compete 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.



0 30
0 0
o 30-

20- 8
S 20



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. 0- Bovine and
Alcelaphine serum samples known to be negative by virus neutralization
and Bovine samples from sentinel herds presumed to be negative.


-I *

0 30- o

20- 0

o 0
0 0

Bovine & Ovine

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. 0- Samples known to be positive by virus neutralization.
O- Samples known to be negative by neutralization. Borderline
result in virus neutralization assay.


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 from 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.




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 molecules) 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.

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 ACi/ml of 35S-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 35S-methionine was removed and replaced with fresh

media containing 100 /g of cycloheximide and 100X 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 -20'C.

N-glycanase digestion

Strain 1982 infected BEL cells were labeled with 35S-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 #l1 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

41 0.67 M phosphate buffer pH 8.6, 10 1l 0.1 M phenanthroline, 16.7 4l

7.5% NP-40 and 10 Al of distilled water. The mixture was vortexed.

To one sample was added 2.5 U of N-glycanase in 10 Jl 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



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 125I 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 35S-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

115 116K- I 4115
105 4l110


C440 78


Figure 6.1 SDS-PAGE analysis of 35S-methionine labeled proteins
iniunoprecipitated 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).






116k- 4115






Figure 6.2 SDS-PAGE analysis of proteins irmunoprecipitated from
125I labeled 1982 virions immunoprecipitated with monoclonal antibody
12B5 and electrophoresed under reducing (R), and non-reducing (NR)
conditions. Arrows to the right indicate proteins inmmunoprecipitated
from virion lysate and their apparent molecular weights (x1000).

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 35S-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

were infected and labeled with 35S-methionine and immunoprecipitated

with 12B5. The inmunoprecipitate 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).

CL. g 9 U) -o
T_ V to(









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 35S-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 proteins irmmunoprecipitated with
monoclonal antibody 12B5.



0 z
-- 0 X. 0 0


- M" I1





ai^ _4115



-_ ~4 48

-10o -MMM

Figure 6.4 SDS-PAGE analysis of proteins immunoprecipitated by
monoclonal antibody 12B5, and electrophoresed under reducing
conditions. Lanes show infected cells (P) pulsed with 35S-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.

" o "- -


10 9
E 10
n 11

0 13 _"_//___/____/,__

0 100 200 300 400


Figure 6.5 Histogram indicating counts per minutes in 3 mm punch
samples that were used to identify the location of 35S-methionine
labeled proteins of AHV-1 immunoprecipitated by monoclonal antibody
12B5 and electrophoresed in a 9% polyacrylamide gel under non-reducing



i: 4115
m -. 4 110



,... 48


Figure 6.6 SDS-PAGE analysis of 35S-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).




97k- *105




14 78


Af-4b6 0

4 548


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).


N-Glycanase digestion

Digestion of the inmunoprecipitated 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).


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

the mature form of the protein. Further support for this assumption

was provided by the fact that when lysate of 1251 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


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


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.



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


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


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


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.


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