Antigenic composition of five strains of herpes simplex virus type one that cause characteristic ocular disease

MISSING IMAGE

Material Information

Title:
Antigenic composition of five strains of herpes simplex virus type one that cause characteristic ocular disease
Physical Description:
vi, 146 leaves : ill. ; 29 cm.
Language:
English
Creator:
Noble, Alma Gwendolyn, 1950-
Publication Date:

Subjects

Subjects / Keywords:
Herpes Simplex   ( mesh )
Keratitis, Dendritic   ( mesh )
Antigens   ( mesh )
Rabbits   ( mesh )
Immunology and Medical Microbiology thesis Ph.D   ( mesh )
Dissertations, Academic -- Immunology and Medical Microbiology -- UF   ( mesh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1980.
Bibliography:
Bibliography: leaves 138-145.
Statement of Responsibility:
by Alma Gwendolyn Noble.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000898546
oclc - 22649003
notis - AEK7251
System ID:
AA00009122:00001


This item is only available as the following downloads:


Full Text











ANTIGENIC COMPOSITION OF FIVE STRAINS OF HERPES SIMPLEX VIRUS
TYPE ONE THAT CAUSE CHARACTERISTIC OCULAR DISEASE










By


ALMA GWENDOLYN NOBLE


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


UNIVERSITY OF FLORIDA


1980














ACKNOWLEDGMENTS

The author expresses appreciation to Drs. Ysolina M. Centifanto,

Herbert E. Kaufman, Kenneth I. Berns, George E. Gifford, and Donna H.

Duckworth for their suggestions while serving on her supervisory

committee. She is especially grateful to Dr. Ysolina M. Centifanto for

her constant intellectual stimulation, guidance, advice, support, and

patience during the course of this research and preparation of this

dissertation.

The author expresses appreciation to the Departments of

Ophthalmology at the University of Florida and Louisiana State

University Medical Center for the financial assistance provided. She

is grateful to Theresa Moorehead, the members of the Editorial Office

and the Department of Photography at the Louisiana State University Eye

Center, and the Department of Learning Resources at the Louisiana State

University Medical Center for their technical assistance in the

preparation of this dissertation. Special thanks go to the members of

Dr. Centifanto's laboratories at the University of Florida and

Louisiana State University Medical Center for their support and

assistance.


-ii-














TABLE OF CONTENTS

ACKNOWLEDGMENTS . ii

ABSTRACT v

INTRODUCTION 1
Virus Structure and Chemical Composition .. 2
Virus DNA 3
Virus Proteins 3
Virus Envelope . 4
Social Behavior of Virus Infected Cells 5
Virus Glycoproteins . 6
Virus Antigens . 8
Virus and'Ocular Disease 14

MATERIALS AND METHODS ........... 18
Virus Strains and Cell Culture ... 18
Preparation of Antigen ... 20
Preparation of Antisera. ....... 21
Crossed Immunoelectrophoresis 22
Microneutralization Assay . .. 24

RESULTS 26
Reference Patterns of Five Strains of
Herpes Simplex Virus Type One .. 26
Cross-Reacting Antigens of Five Strains of
Herpes Simplex Virus Type One .. 29
Glycosylated Antigen Patterns of Five Strains of
Herpes Simplex Virus Type One 31
Microneutralization Assay . .. 34

DISCUSSION 113
Source of Antisera . 113
Unknown Antigens .. ...... 115
Similarities among These Five Strains 116
Glycosylated Antigens 117
Correlation of the Antigens of These Five Strains
with Previous Studies .. .. 118
Relationship of Antigens with Glycoproteins 119
Ocular Disease Caused by These Five Strains 120
Correlation of the Antigenic Composition with the
Ocular Disease Pattern 121


-iii-










APPENDICES .. ... ... ...... 126
A TECHNICAL ASPECTS OF CROSSED IMMUNOELECTROPHORESIS 126

B ANTIGENIC COMPOSITION OF ONE STRAIN OF
HERPES SIMPLEX VIRUS TYPE TWO 131


REFERENCES


. . 138


BIOGRAPHICAL SKETCH .


. 146


-1V-














Abstract of Dissertation Presented to the Graduate Council of the
University of Florida in Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy


ANTIGENIC COMPOSITION OF FIVE STRAINS OF HERPES SIMPLEX VIRUS
TYPE ONE THAT CAUSE CHARACTERISTIC OCULAR DISEASE

By

ALMA GWENDOLYN NORLE

June 1980

Chairman: Ysolina M. Centifanto
Major Department: Immunology and Medical Microbiology


The antigenic composition of five strains of herpes simplex virus

(HSV) that cause characteristic ocular disease of varying severity in

New Zealand white rabbits has been studied by crossed

immunoelectrophoresis. Antiserum to each strain was raised by

intramuscular immunization and boosting by infection in the eyes of

rabbits. The immunoglobulin fraction was obtained by ammonium sulfate

precipitation. Crossed immunoelectrophoresis using homologous

antibodies showed that the Shealy and RE strains had thirteen

virus-specific antigens, the McKrae strain twelve, the F strain eleven,

and the CGA-3 strain eight. Experiments using heterologous antibodies

showed that three antigens were common to all five strains, ten were

shared by two or more strains, and ten were characteristic of one

strain only. The data indicated that there was a correlation between

the antigenic composition of the five strains and the kind and severity











of ocular disease produced. As the severity of disease increased, the

number of virus-specific antigens increased. Similarities in antigenic

composition among the strains also correlated with the kind of ocular

disease produced. It is hoped that the correlation of the antigenic

composition with other biological and biochemical properties may prove

helpful in understanding the pathogenesis of HSV in ocular disease.


-vi-














INTRODUCTION

Herpes simplex virus (HSV) can infect almost every organ of the

body and is involved in many diseases of man including, perhaps, some

forms of cancer. The virus has a predilection for the skin, genitals,

and eye, and infections of these tissues are common. Less often HSV

infects sites such as visceral organs and the central nervous system

and may result in fatal disease (43). Human isolates of HSV are

divided into two serotypes, type 1 and type 2, based on antigenic and

biological differences (53). Generally, it is said that strains of

HSV-1 are associated with infections above the waist and strains of

HSV-2 with infections below the waist (53).

Ocular infections caused by HSV are very troublesome. In the

United States, the most common and significant virus infection of the

cornea is caused by HSV (29). Isolates from eye lesions are usually

type 1, and both primary and recurrent infections can occur (53).

Ocular infections by HSV result in a range of disease from mild

conjunctivitis and dendritic keratitis to more severe disciform edema

and necrotizing stromal keratitis. The latter may result in blindness

(30).

HSV-induced ocular disease is an excellent model for the study of

causes of variation in the disease states produced by HSV. For many

years it was thought that differences in the hosts such as genetic (HLA

type), hormonal, and immunologic factors were responsible for the

varying forms of eye disease, and much work was done to try to confirm

1










this. Results from these studies have been disappointing. Recently,

differences in biological and biochemical properties of the infecting

viruses have been implicated as a factor in the variation in eye

disease, and investigators are now beginning to study these strain

differences in relation to disease.

Centifanto and associates (8,9) are presently investigating

differences in HSV strains by comparing their cytopathic effects in

vitro, plaque morphology (social behavior), sensitivity to antiviral

drugs, and their polypeptide composition with the kind, severity,

and virulence of disease produced in the rabbit cornea by these

strains. In the work to be presented here, the antigenic composition

of five strains of HSV type 1 was determined and correlated with the

kind and severity of eye disease that they caused. It is hoped that

the correlation of antigenic composition with other strain differences

and with the ocular disease pattern will help in the understanding of

the pathogenesis of HSV.

Virus Structure and Chemical Composition

HSV type 1 is a member of the Herpesviridae (22). The morphology

of the herpesviruses is the basis for their classification in this

family (80). Their virions are seen, by electron microscopic

examination of negatively stained preparations, to be divided into

three parts: the core, the capsid, and the outer membrane or envelope.

The core, 77.5 nm in diameter, contains the double-stranded DNA genome.

The capsid encloses the core, is 105 nm in diameter, is spherical or

hexagonal, and is composed of 162 capsomeres (150 hexagonal and 12

pentagonal) arranged to give icosahedral symmetry. The capsomeres are










approximately 100 A in diameter with a characteristic axial hole

about 40 A in diameter. The nucleocapsid is surrounded by an outer

membrane or envelope, and the enveloped nuleocapsid varies in size from

150 to 200 nm.

The major components of the virion are DNA, proteins, glyco-

proteins, polyamines, and lipids (58). The buoyant density of the

complete virion is 1.27 to 1.85 g/cm3 determined by equilibrium

centrifugation in cesium chloride (CsCl) density gradients (68,69).

Virus DNA

The DNA found in an HSV virion is a double-stranded, linear DNA

molecule (4,32). Kieff et al. (32) established the molecular weight of

the DNA of HSV-1 as (99 + 5) x 106 daltons, using its rate of

co-sedimentation with nonglycosylated T4 DNA. These results are

compatible with the measure of (95 + 1) x 106 determined by kinetic

complexity (13), with the measure of 95.7 x 106 established by

measuring the contour length of DNA (58), and with other determinations

(14,82). The guanine plus cytosine content of HSV-1 is 67 moles

percent, as demonstrated by equilibrium banding in analytical CsC1

density gradients and thermal denaturation (melting) temperature (32).

Virus Proteins

Based on kinetic, immunologic, and genetic criteria, approximately
48 virus-specific polypeptides ranging in molecular weight from 15,000

to 280,000 are separated on sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE) of HSV-1 infected cells (24,25). Twenty-

four of these 48 polypeptides are found in the virion and are










classified as structural (66); fifteen are nonstructural, and 9 are

unclassified polypeptides (24).

Naked nucleocapsids contain only four to eight polypeptides. This

suggests that most of the proteins and glycoproteins of the virion are

obtained during envelopment (67). One major protein of molecular

weight 140,000 to 160,000 daltons that is found in the nucleocapsid of

many herpesviruses is probably the major hexon protein. Since empty

capsids of HSV lack two of the nucleocapsid proteins, a role is implied

for these proteins in the packaging of the genome.

Virus Envelope

The envelopment of herpesviruses occurs at the inner nuclear

membrane (67). Although the virion buds through the membrane, the

virion envelope lacks most of the normal host cell proteins. This

occurrence implies a rearrangement of the membrane components (67). In

all herpesviruses studied, there are ten to fifteen nonglycosylated

proteins and approximately six glycoproteins which migrate from their

sites of synthesis in the cytoplasm to the inner nuclear membrane and

are acquired by the virus during envelopment. These virus-specific

glycoproteins are integral membrane proteins with their carbohydrate

moieties on the outer surface of the virion. The virus-specific

nonglycosylated proteins are important in the envelopment of the

nucleocapsid and are located between the nucleocapsid surface and the

inner surface of the lipid bilayer of the envelope (67).

The virion envelope, with its associated glycoproteins, plays a

pivotal role in the entrance of the nucleocapsid into a cell during the

initiation of infection (67). This conclusion is based on studies






5


indicating that naked nucleocapsids are not infectious, that

neutralizing antibodies are made to envelope components, and that

virion envelopes lacking the glycoprotein VP7(02) exhibit low

infectivity. The envelope of the virion apparently fuses with the

host cell membrane, and the nucleocapsid is released into the

cytoplasm, thereby initiating the infection (42).

Social Rehavior of Virus Infected Cells

Studies of the "social behavior" of HSV infected cells demonstrate

that the virus-specific structural glycoproteins are also important in

the interaction of infected cells with each other. The normal (wild

type or syn+) expression of this interaction is the clumping of

rounded cells (60). The variant expression (syn) is polykaryocytosis,

i.e. giant cell formation into syncitium (50). Since either clumping

or fusion requires the interaction of the plasma membrane of adjacent

cells, Roizman (56,57) proposed that changes in the glycoproteins

affect the plasma membrane of infected cells and that this results in

changes in immunological specificity and alteration in social behavior.

Later studies confirm this hypothesis (45,55,59).

The finding that viruses that cause different social behavior

express different membrane glycoproteins identified on SDS-PAGE (31),

as well as studies with the mutant strains, HSV-1(MP) and HSV-1(13VR4),

which cause polykaryocytosis and do not accumulate VP8(C2) (37,60),

demonstrate the role of membrane glycoproteins in social behavior. In

confirmatory studies on the relationship between the glycoproteins and

the interactions of HSV infected cells, Ruyechan et al. (60) mapped the

location on the HSV DNA of (a) three mutations at three different loci









that alter social behavior from wild type rounded cells in clumps to

polykaryocytosis, (b) one mutation which accumulates one of the major

structural glycoproteins, [VP8(C2)], and (c) the sequences for the
four major structural glycoproteins, VP8(C2), VP7(R2), VP8.5(A),

and VP19E(D2).

Two of the three loci that determine the social behavior of
infected cells, syn 1 and syn 2, do not map at the same location as the

structural genes encoding glycoproteins VPS(C2), VP7(B2), VPS.5 (A)

and VP19E(D2) (60). These mutations could possibly be in minor

glycosylated proteins which are not yet characterized, nonglycosylated

proteins, or enzymes which affect membrane proteins. The third locus,

syn 3, is found within the boundaries of the template for glycoproteins

VP8.5(A) and VP7(B2). Although it is not known whether syn 3 is

found within the templates specifying these glycoproteins or other un-

mapped proteins, glycoprotein VP7(B2) does play a significant role in
cell fusion (37,61). The Cr locus, which determines the accumulation

of glycoprotein VP8(C2) maps to the right of the structural gene for

VP8(C2). Loci syn 1 and syn 2 are found at or near this Cr locus but
can be segregated from it. Since both spontaneous HSV-1(MP) and muta-

gen-induced HSV-1(13VB4) mutants show the Cr- syn phenotype, perhaps

there is a relationship between these loci. It is possible that the
template specifying VP8(C2), the Cr locus, and the syn 2 locus may be
within a transcriptional unit or in overlapping templates.

Virus Glycoproteins
Virions of HSV-1 exhibit five antigenically distinct glycoproteins
called VP8(C2), VP7(B2), VP8.5(A), VP18(D2), (67) and E2 (3).










Virally-induced glycoproteins are incorporated into the cellular

membranes of infected cells (20,21). These glycoproteins in the cell

membrane cannot be differentiated from the structural virion

glycoproteins on the basis of their number or electrophoretic mobility

(26,64). The structural glycoproteins which are exposed on the virion

surface are responsible for virus adsorption and penetration and are

targets of neutralizing antibodies (67).

Studies with mutants have indicated the specific function for

several of the glycoproteins. Glycoprotein VP8(C2) is not important

in viral morphogenesis or adsorption as shown by one spontaneous

mutant, HSV-1(MP), which yields infectious virions but does not make

VP8(C2). This glycoprotein is associated with the suppression of

HSV-induced cell fusion as shown by the mutant HSV-I(MP) which causes

polykaryocytosis and other studies which indicate that mutants that do

not produce this glycoprotein also have this effect (37). Studies

using the temperature-sensitive mutant HSV-1[HFEM]tsB5 show that

glycoprotein VP7(B2) is not required for viral morphogenesis or virus

adsorption but is necessary for penetration (67). At the nonpermissive

temperature, 390C, this mutant virus does not produce functional

VP7(B2) and yields noninfectious virions which lack this

glycoprotein. These virions adsorb to cells but do not start

infection; after addition of polyethylene glycol, which promotes

membrane fusion, the infectivity of this mutant increased (61). Other

studies indicate that this glycoprotein plays a significant role in

HSV-induced cell fusion presumably by promoting fusion between the

envelope of the virions and the membrane of the host cell (37,61).









Glycoprotein E2 binds to the Fc part of the immunoglobulin G

molecule, and anti-E2 has neutralizing activity in a complement-

dependent reaction (Spear, personal communication). The specific

functions of VP18(02) and VP8.5(A) are not known.

In summary, glycoprotein VP8(C2) is not required for

infectivity, virus morphogenesis, or penetration, and mutants that do

not produce VPS(C2) usually cause polykaryocytosis. VP7(B2) is

required for infectivity but not for virus morphogenesis or adsorption;

it is necessary for penetration and plays a role in HSV-induced cell

fusion. Glycoprotein E2 binds to the Fc portion of the immuoglobulin

G molecule. The roles of glycoproteins VP8.5(A) and VP18(D2) are not

known. Glycoprotein VP8.5(A) is related to glycoprotein VP7(B2) and

may be a separate form or breakdown product of VP7(B2). Three of the

glycoproteins [VP7(82), VP18(D2), E2] elicit the production of

neutralizing antibodies in immunized New Zealand white rabbits.

Virus Antigens
As seen from the preceding discussion, virus infection results in

the formation of many new proteins and glycoproteins. These may all

serve as antigens. Several factors determine if an antigen is

biologically significant: its location within the cell, whether or not

it is a structural or nonstructural protein, its exposure on the

surface of the virion or the infected cell membrane, its interaction

with host cell components, its immunogenicity, and its role in the

immunopathological process (7). Many serological techniques including

neutralization, immunofluorescence, immunoferritin staining,

immunoperoxidase staining, immune agglutination of virus particles,









complement fixation, immunodiffusion, crossed immunoelectrophoresis,

and immunocytolysis are used in characterizing antigens in HSV infected

cells both in vitro and in vivo (16).

Using one of these methods, agar gel immunodiffusion, Watson et

al. (81) demonstrated 12 virus-specific antigens of HSV-1 infected

cells, and Honess et al. (23) demonstrated 11 and showed that six of

these antigens were type specific and five type common. This study

demonstrated that seven of the 11 were structural antigens (three type

specific and four type common) and that two of these structural

antigens were involved in type specific neutralization and one in type

common neutralization. One of the nonstructural type specific antigens

was thymidine kinase, which was not glycosylated (27). The functions

of the other antigens were unknown.

Using other techniques, Cohen et al. (10) described the CP-1

antigen, which is a type common, glycosylated antigen that elicits the

production of neutralizing antibody and is detected in soluble form in

infected cells, in detergent extracts of infected cell membranes, and

in the envelope of the HSV virion. Cohen has correlated his results

with those of other laboratories, stating that CP-1 is possibly related

to Spear's glycoprotein VP18(D2) (64), VP17, 18, 19E of Heine et al.

(19), Norrild and Vestergaard's Ag8 (49), and 8/9 of Powell et al.

(52).

In recent studies using crossed immunoelectrophoresis (clEP), the

antigens of HSV-1 and HSV-2 infected cells have been characterized by

their electrophoretic mobility in antibody-containing agarose gels

(71). The membranes of HSV infected cells have been solubilized with










the nonionic detergent Triton X-100, which preserves the native macro-

molecular protein conformation and the immunologic reactivity of the

antigens (5). Eleven antigens were originally identified by this meth-

od (71). Extensive studies were done to further characterize these an-

tigens including determination of the immunological significance with

respect to neutralization and immunocytolysis, polypeptide composition,

and location and time of appearance postinfection (46-50, 71-78).

One of the most important antigens is antigen 11 (Ag11). This

antigen is glycosylated (47,49,7a) and membrane-bound (73) and has both

type common and type-1 specific antigenic sites (71,76). It has also

been shown to be a virion protein by Commassie brilliant blue (CRB)

staining of clEP gels and has been shown on the surface of infected

cells 5 h postinfection (46). Antigen 11 eluted from the immunopre-

cipitates in antibody-containing gels of cIEP is composed of three

polypeptides as seen on SDS-PAGE, including glycoproteins VP8.5(A) and

VP7(B2) (48,49). When lactoperoxidase-catalyzed iodination was used,

only one polypeptide of the Ag11 complex of molecular weight 127 x

10-3 was found on the surface of 18 h infected cells (46), and this

polypeptide was shown to be glycoprotein VP7(B2) (47,49). Using cIEP

and rocket electrophoresis, Norrild et al. (47) demonstrated that this

antigen is a common antigen of cells infected with 4 herpesviruses:

HSV-1, HSV-2, bovine mammillitis virus, and R virus. Although the

composition of Ag11 differed in the four viruses, glycoprotein

VP7(B2) was demonstrated in all four herpesviruses, indicating that

the common antigenic site is on the glycoprotein.










Antigen 8 (Ag8) also has both type common and type-1 specific

antigenic sites (71,72,76). It is a glycosylated, membrane-bound

antigen found on the virion and on the surface of infected cells at 8 h

postinfection (46,47,49,71-74,76). This antigen is composed of several

polypeptides including various forms of glycoprotein VP18(D2)

(49,50). Three of these glycopolypeptides of molecular weight 80 x

10-3, 73 x 10-3, and 64 x 10-3 are exposed on the surface

of 18 h infected cells (46). Recent immunoelectron microscopy studies

using peroxidase-antiperoxidase (unlabeled antibody-enzyme method) and

monospecific anti-Ag8 demonstrated that Ag8 is localized at both the

nuclear and plasma membranes of HSV infected cells and is detected both

early (2 h postinfection) and late (19 h postinfection) (17). These

studies support an earlier finding that showed that most of the

virus-specific proteins are present from early to late stages of

infection, but in different amounts (24).

Monoprecipitin antisera to Agll and Ag8 have high neutralizing

antibody titers against both HSV-1 and HSV-2 (78) and mediate

immunocytolysis in the presence of complement (antibody-dependent

complement-mediated cytotoxicity) or peripheral blood mononuclear cells

(antibody-dependent cell-mediated cytotoxicity or ADCC) (48). High

antibody titers against these two antigens were found in 100 human sera

tested and these titers correlated with HSV neutralizing antibody

titers (72).

A third type common antigen is antigen 3 (Ag3) which is

water-soluble, nonglycosylated and composed of several polypeptides

(49,71,73). This antigen is not detected on purified HSV-1 virion









preparations by clEP and is not exposed on the surface of infected

cells (46). Vestergaard (72) speculates that Ag3 may be a major capsid

antigen, but the nature of this antigen is presently unknown.

Antibodies to Ag3 have also been demonstrated in human sera (72), and

rabbits produce antibodies against Ag3 rapidly (73); however, the

immunological significance of these antibodies is presently unknown.

An interesting antigen which is HSV type 1-specific is antigen 6

(Ag6). This antigen is a virion protein that is glycosylated,

membrane-bound, and located on the surface of infected cells between 8

and 18 h postinfection (46,71,74). The major polypeptide is

glycoprotein VP8(C2) as seen on SDS-PAGE (48,77). This is confirmed

by studies showing that this antigen is not detected in cIEP of

HSV-1(MP) infected cells that lack glycoprotein VP8(C2).

Furthermore, monoprecipitin antiserum to Ag6 did not react in the ADCC

reaction with either Chang liver cells or HEp-2 cells infected with

HSV-I(MP) (48).

When lactoperoxidase-catalyzed iodination of HSV infected cells

was used, Ag6 of HSV-1(VR3) infected Chang liver cells was shown on the

surface of 8 h infected cells (48) in contrast to the same antigen of

HSV-I(F) infected HEp-2 cells which was shown on the surface only late

in infection (8 to 18 h postinfection) (46). Although two different

strains of virus were used, the difference in the surface expression of

this antigen appears to be host cell dependent (48). In addition,

anti-Ag6 resulted in a lower ADCC reaction in HEp-2 infected cells than

in Chang liver infected cells (48). These studies agree with the









demonstration that mutations in the syn1 and syn2 loci specifying

social behavior of HSV infected cells are host cell dependent (60).

Monoprecipitin antisera to Ag6 has both neutralizing and

immunocytolytic activities against HSV-1 infected cells (48,78).

Antibodies to this antigen were shown in human sera, and the titers

correlated better with HSV-1 than HSV-2 neutralizing titers (72).

Another antigen which is possibly a HSV type 1-specific antigen is

antigen 7 (Ag7) (71). It is a virion protein which is membrane-bound,

glycosylated (76; Norrild, personal communication) and exposed on the

surface of HSV-1 infected cells between 8 and 18 h postinfection (46).

Another antigen which has recently been identified on clEP is antigen 5

(Ag5), which is composed of the major capsid protein, ICP-5 (19,48).

Two antigens which are HSV type 2-specific are antigen 4 (Ag4) and

antigen 9 (Ag9) (73,76). Both of these antigens are membrane-bound and

glycosylated (71,74,76). Anti-Ag4 and anti-Ag9 both neutralize HSV-2

to a greater extent than they do HSV-1 (78). Also, antibodies to these

two antigens were demonstrated in human sera and the titers correlated

better with HSV-2 neutralizing titers than HSV-1 titers (72).

In summary, antigens of HSV infected cells have been characterized

by several serological techniques including immunodiffusion and crossed

immunoelectrophoresis. The immunological significance of these

antigens has been demonstrated by neutralization and immunocytolysis.

When clEP in agarose gel was used, seven antigens were characterized in

various strains of HSV-1 (3,3A,5,6,7,8,11) and eight antigens in

various strains of HSV-2 (1,2,3,4,8,9,10,11). Antigens 3, 8, and 11

are type common; Ag6 and possibly Ag7 are type 1-specific, and Ag4 and










Ag 9 are type 2-specific. Antigens 6, 8, and 11 are virion proteins

which are glycosylated, membrane-bound, and found on the surface of

infected cells. Antigen 6 is composed of glycoprotein VP8(C2); Ag8

of glycoprotein VP18(D2), and Agll of glycoproteins VP8.5(A) and

VP7(B2). Antigen 7 is a glycosylated, membrane-bound, virion protein

located on the surface of infected cells. Antigens 4 and 9 are

glycosylated and membrane-bound. Antigens 1, 3 and 5 are not

glycosylated. Monoprecipitin antisera to antigens 4,6,8,9 and 11 have

neutralizing activity and anti-6, -8 and -11 can also mediate

immunocytolysis. Vestergaard (72) proposed that in human sera the

serological cross-reactivity of HSV-1 and HSV-2 is due to antibodies to

Ag8 and Agll, that antigens 6, 8 and 11 are responsible for the

production of antibodies to HSV-1, and that antigens 4 and 9 elicit

antibodies to HSV-2.

Virus and Ocular Disease

Antigens on the HSV virion and on the surface of infected cells

play an important role in HSV-induced ocular disease. Infections by

HSV cause both a humoral and a cellular immune response in which a

variety of entities can interact with these antigens. It is also

significant that HSV can infect cells involved in the immune response,

such as T and B lymphocytes, macrophages, and polymorphonuclear leuko-

cytes (43).

Varying forms of ocular disease are produced by HSV, and the

different disease states have been reviewed by Kaufman (30), Metcalf

and Kaufman (40), Metcalf et al. (41), Rawls (53) and Nahmias and

Norrild (43). In acute conjunctivitis there are ulcers on the










conjunctiva and vesicle formation on the lid margin and skin. This

primary infection may be limited to the conjunctiva, or the cornea may

also be involved. The virus replicates in the epithelial layer of the

cornea in epithelial keratitis, resulting in characteristic dendritic

ulceration; punctate erosions or small dendritic ulcers may progress to

larger, geographic defects. The disease is often mild, localized, and

self-limiting and can be controlled by topical antiviral drugs. After

a primary infection in which infectious virus can be recovered, the

virus persists in a latent form in the trigeminal or other ganglia (11,

44,70). Infectious virus is not detected in the ganglia but can be

demonstrated after explanting the ganglia or cocultivation with rabbit

kidney cells (11,44,70; Centifanto, personal communication). This

latent form of the virus is the source of possible recurrences and in

recurrent keratitis, the virus is reactivated and causes deep,

unbranched, marginal ulcers and large areas of erosion that can result

in irreversible corneal scarring and opacification.

A more serious sequela of epithelial keratitis is the development

of deep stromal disease with disciform edema (30,41,83). The milder

form includes two or three months of localized edema and opacification

which then heals, leaving little or no scarring. In some patients, a

more severe form of the necrotizing keratitis develops with dense,

cheesy opacities in the corneal stroma, corneal vascularization, and

necrosis of stromal collagen fibers. This severe form of the disease

results in permanent scarring and blindness.

In the rabbit cornea, different strains of HSV have been shown to

produce different disease patterns, ranging from mild epithelial










keratitis to severe stromal necrotizing keratitis (63,79), and these

varying disease states show many similarities to human infections (41).

Virus replication occurs in epithelial keratitis, and HSV antigens have

been demonstrated within the nucleus, on nuclear membranes, and on

membranes of epithelial cells (39). When the disease progresses to

necrotizing keratitis, viral particles are not found in the stroma;

however, virus antigens are found on the surface of stromal

keratocytes. Several studies indicate that viral replication

is important in initiating, but not in maintaining, the disease state

(38,62). In stromal keratitis an initial inflammatory response of

polymorphonuclear leukocytes and macrophages is followed by lymphocytes

and plasma cells (40,41), and these lymphocytes are often found in

close association with the degenerating stromal keratocytes (40). The

histology and pathology of stromal keratitis demonstrated in the rabbit

model and the fact that disciform edema in humans is treated with

corticosteroids (29,30) both indicate that immunopathogenesis has a

role in the maintenance of the disease state.

The variation in ocular disease is dependent upon the differences

in the host response to the infecting virus and the properties of that

strain, and both have been studied for their effect on the type of eye

disease caused by HSV. Since antigens have been shown to be important

in infectivity, immune response, and disease, the antigenic composition

of five strains of HSV type 1 that cause varying forms of ocular

disease has been determined in this study by clEP in agarose gels.

Crossed immunoelectrophoresis using homologous antiserum demonstrated

the virus-specific antigens of each strain. The similarities among






17


these strains were shown using heterologous antisera. Since

glycoproteins are involved in disease, neutralization, and kind of

cytopathic effect, the glycosylated antigen pattern was also

determined. The antigenic composition of these five strains was found

to be related to the severity and kind of eye disease they produced.

It is hoped that this correlation will help in the understanding of the

pathogenesis of HSV-induced ocular disease.













MATERIALS AND METHODS

Virus Strains and Cell Culture

Five strains of herpes simplex virus type 1 were used. The

Shealy strain was obtained from Dr. Andre Nahmias (Emory University).

The McKrae strain was isolated in Dr. Ysolina M. Centifanto's labora-

tory from a patient of Dr. Herbert E. Kaufman. The F strain was a gift

from Dr. Bernard Roizman (University of Chicago). The RE strain was

obtained from Dr. Chandler R. Dawson (Francis I. Procter Eye

Institute, San Francisco). The CGA-3 strain was a gift from Dr. R. F.

McNair Scott (University of Pennsylvania).

To obtain stock virus, Vero or HEp-2 cells were grown to

confluency in 32 oz tissue culture flasks using complete growth medium

composed of Basal Medium Eagle [BME (Microbiological Associates,

Walkersville, Maryland)] or Minimum Essential Medium (Eagle) [MEM

(Grand Island Biological Company, Grand Island, New York)], 10%

heat-inactivated fetal calf serum [FCS (Microbiological Associates)],

10% sodium bicarbonate (7.5% solution) [NaHCO3 (Flow Laboratories,

Rockville, Maryland)], 1% glutamine (Grand Island Biological Company),

penicillin-streptomycin mixture (Microbiological Associates) and

fungizone (E. R. Squibb and Sons, Incorporated, Princeton, New Jersey).

Virus was seeded on cultures and adsorbed for 1 h at 370C. Complete

maintenance medium, including the same components as growth medium with

2% NaHCO3 and 2% FCS, was added. After 24 to 48 h of infection at

37*C, infected cells were frozen, thawed, dispensed, and stored at

-760C.









To grow virus stocks of very high titers, HEp-2 cells were grown

to confluency in glass roller bottles (Bellco Glass, Inc., Vineland,

New Jersey) with MEM, 5% non-heat-inactivated FCS, 1% NaHCO3, 1%

glutamine, and antibiotics. The cell layer was washed extensively with

phosphate-buffered saline (PBS), pH 7.2. Stock virus was added in 5 ml

volume and adsorbed for 1 h at 370C. Medium 199 (Grand Island

Biological Company) with 1% non-heat-inactivated FCS dialyzed in

NaHCO3, antibiotics and fungizone were added, and cells were

incubated at 370C. After 24 to 48 h of infection, the medium was

decanted, and the cells were washed extensively with PBS (three to five

times), scraped, pelleted by centrifugation, washed with PRS by

resuspension and sedimentation, and frozen at -760C.

To grow virus for radioactively labeled antigen for

immunoelectrophoresis, HEp-2 cells were grown to confluency in

150 cm2 tissue culture flasks (Corning Glass Works, Corning, New

York) with complete growth medium. Cells were washed extensively with

PBS, virus grown in roller bottles (as described above) was adsorbed

for 1 h, and medium containing the radioactively labeled components was

added. To radioactively label proteins, Hanks Balanced Salt Solution

[HBSS (Microbiological Associates)] with 1% MEM vitamin solution (Grand

Island Biological Company), 2% non-essential amino acids (one tenth the

normal concentration) (Flow Laboratories), 1% NaHCO3, 1% glutamine,

1 uCi/ml each 14C-leucine, -isoleucine, -valine (New England

Nuclear, Boston, Massachusettes) to equal 15 uCi of each amino acid,

antibiotics and fungizone were added. To radioactively label

glycoproteins, Medium 199 with 1% non-heat-inactivated FCS dialyzed in









NaHCO3, antibiotics, fungizone, and 2 uCi/ml 14C-glucosamine

hydrochloride (New England Nuclear) to equal 40 uCi were added. After

24 to 48 h of infection, the cells were scraped, pelleted by

centrifugation, washed with PBS three times, and frozen at -760C.

To grow virus for immunogen, cultures of rabbit kidney-13 [RK-13

(Flow Laboratories)] were grown to confluency in 32 oz tissue culture

flasks using MEM complete growth medium. The cultures were inoculated

with virus which had been passage two times (Vero, RK-13) without FCS.

After 1 h of adsorption, BME with 10% NCTC 135 (Grand Island Biological

Company), 1% NaHC03, 1% glutamine, 1% non-essential amino acids,

antibiotics, and fungizone were added. After 24 to 48 h of infection,

the cells were frozen, thawed, dispensed, and stored at -760C.

Preparation of Antigen

To prepare antigens for immunoelectrophoresis, the procedure of

Norrild and Vestergaard was followed (49,71; Norrild, personal

communication; Vestergaard, personal communication). To obtain

radioactively labeled infected cells, 1.0 x 107 HEp-2 cells were

infected with each of the five viruses at multiplicities of infection

of ten or more plaque-forming units/cell (PFU/cell). Virus was

adsorbed for 1 h. To radioactively label proteins, medium containing

amino acids at one tenth the normal concentration, supplemented with

14C-leucine, -isoleucine, -valine, was added. To radioactively

label glycoproteins, medium containing 14C-glucosamine

hydrochloride was added. After 24 to 48 h of infection, cells were

scraped and harvested by centrifugation; the pellet was washed

extensively with PBS to remove components of the medium. The protein









was solubilized in 0.0076 M tris(hydroxymethyl)aminomethane (Tris) -

0.02 M glycine containing 5" (vol/vol) Triton X-100 (octyl phenoxy

polyethoxyethanol) (Sigma Chemical Company, St. Louis, Missouri) in

four times the volume of the wet cell pellet. The pellet was disrupted

by sonic treatment at 20,000 cycles/sec four times for 1 min at 10 min

intervals. The mixture was centrifuged at 100,000 x g for 1 h, and the

supernatant fluid containing the antigen was stored at -760C. HEp-2

cells were mock infected and labeled, and antigen was prepared.

Antigens with radioactively labeled proteins contained the following

counts per minute, cpm/0.1 ml: Shealy- 349,528; McKrae- 69,220; F-

63,094; RE- 280,776; CGA-3- 148,204; HEp-2 mock infected- 404,070.

Antigens with radioactively labeled glucosamine contained the following

cpm/0.1 ml: Shealy- 128,102; McKrae- 116,814; F- 107,667; RE- 132,636;

CGA-3- 243,084; and HEp-2 mock infected- 163,821.

Preparation of Antisera

Rabbit kidney-13 cells were infected with each of the five viruses

passed twice without FCS. After 1 h of adsorption, BME with no FCS was

added to each infected culture. After 24 to 48 h, the infected cells

were frozen at -760C. The infected cells were thawed and used as the

immunogen. One milliliter of a 1:1 mixture of immunogen to Freund's

Complete Adjuvant (Cappel Laboratories, Incorporated, Cochranville,

Pennsylvania) was injected intramuscularly (i.m.) into each of the

hind legs of a New Zealand white rabbit. Four rabbits were immunized

per virus. Each rabbit was immunized weekly for three weeks, boosted

by infection in the eye ten days later, and bled 6, 10, 17, and 20

days postinfection. Sera from the four rabbits immunized with the










same virus were pooled from each of the four separate bleeding dates.

All five viruses were neutralized by the homologous antisera from the

first and last bleeding dates. Therefore, the sera from all the

bleeding dates were pooled for each antiserum.

The procedure of Harboe and Ingild (18) was adapted to obtain an

immunoglobulin-rich fraction of antisera. An aliquot of the pooled

serum was precipitated by ammonium sulfate overnight at 40C. The

mixture was centrifuged at 4000 rpm for 30 min, and the pellet was

resuspended in the amount of deionized water approximately equal to

one fifth the original volume. The mixture was dialyzed against

deionized water overnight at 40C and dialyzed against acetate buffer

(0.05 M sodium acetate 0.0?2 M acetic acid), pH 5.0, overnight. The

antiserum was centrifuged at 10,000 rpm for 10 min to remove the lipid.

The supernatant fluid was dialyzed against PBS overnight. The

immunoglobulin fraction was tested against various antigens for

immunoelectrophoresis by Ouchterlony agar diffusion and was used in

crossed immunoelectrophoresis.

Crossed Immunoelectrophoresis

The procedure for crossed immunoelectrophoresis was modified from

Norrild and Vestergaard (49; Norrild, personal communication). For

electrophoresis in the first dimension, 15 ml of 1% agarose made from

agarose immunoelectrophoresis tablets (Bio-Rad Laboratories, Richmond,

California) containing 1% Triton X-100 was cast on a 90 mm by 110 mn

glass plate. Four wells 4 mm in diameter were cut in the agarose

approximately 20 mm from one edge of the plate. The antigen was

applied to each well in 5, 10, or 15 ul volumes. If more than 15 ul










was necessary, either antigen was added in 15 ul aliquots and the

plates placed in a moist chamber at 370C for diffusion into the

agarose, or more than one 4 mm well was cut and 15 ul of antigen added.

Five microliters of 0.05% phenol red in 0.0076 M Tris 0.02 M glycine

containing 5% Triton X-100 was added to each well as a marker. The

antigen and phenol red were subjected to electrophoresis from the

negative to the positive pole at high voltage (100 to 110 V, 5 V/cm)

for 90 or 120 min. After the proteins were separated in the first

dimension, the agarose gel was cut into 20 mm slabs. Each slab was

transferred to a 70.7 mm by 70.7 mm glass plate. An intermediate gel

of agarose was cast between the first dimension slab and a brass bar

barrier (2). After the gel hardened, the barrier was removed and the

intermediate gel was cut to 20 mm with a sharp blade. Agarose was

mixed with antibody and was poured onto the remainder of the plate (30

mm). The antigen was subjected to electrophoresis from the negative to

the positive pole at low voltage (50 to 60 V, 1.5 to 2 V/cm) for 18 h.

After electrophoresis, the agarose gel was covered with filter paper

and pressed under a heavy weight. The gel was washed in 0.1 M sodium

chloride (NaC1) to remove nonprecipitated proteins, rinsed in deionized

water to remove NaCl, and pressed. The gel was dried under a stream of

warm air, stained in Coomassie brilliant blue (Bio-Rad Laboratories) in

45% ethyl alcohol 10% glacial acetic acid, and destined in 45% ethyl

alcohol 10% glacial acetic acid to remove the background staining.

After electrophoresis in the first dimension, the anodic edge of the

phenol red marker of each well was measured and the value was set equal

to 100%. The 100% value was used as a standard to calculate relative









migration velocity (RMV) of the other peaks. The immunoprecipitation

peaks of the experimental gels were traced on tracing paper and

measured from the anodic edge of the well to the center of each peak.

The RMV was calculated and the peaks were numbered according to their

relationship to the phenol red marker.

To visualize immunoprecipitation peaks from radioactively labeled

antigens, the fluorographic procedure for thin layers was used (6).

Two-methylnaphthalene [2-MN (Aldrich Chemical Company, Incorporated,

Milwaukee, Wisconsin)] containing 0.4% 2,5-diphenyloxazole (Sigma

Chemical Company) was melted at 34 to 360C on a hot plate. The glass

plate with the stained dried agarose was soaked in this mixture for

approximately 30 to 45 sec until saturated. After the 2-MN had dried,

the agarose was placed in contact with flashed X-Omat R X-ray film

(Eastman Kodak Company, Rochester, New York) in a Kodak X-ray exposure

holder between two pieces of masonite for pressing. The film was

flashed following the procedure of Laskey and Mills (35), using a Kalt

280B electronic flash (Kalt Corporation, Santa Monica, California) with

Kodak Wratten No. 22 gelatin filter and two pieces of Whatman No. 1

filter paper at a distance of 25 mm. The flashed side of the film was

adjacent to the soaked agarose. The film was exposed for various

intervals at -760C and developed in an automatic processor.

Microneutralization Assay
In this procedure adapted from Rawls et al. (54), the immuno-

globulin-rich fraction of each of the five antisera was tested against

the homologous virus. The antisera were originally diluted 1:10 in

maintenance medium followed by 11 twofold dilutions to a final dilution










of 1:2048. Virus dilutions made in maintenance medium contained 106

to 108 PFU/ml. From the virus dilution, 0.2 ml was added to 0.8 ml

of maintenance medium for the virus control and to each antiserum

dilution. For the antiserum control, 0.2 ml of the 1:10 dilution was

added to 0.8 ml of maintenance medium. The mixtures were incubated in

a 370C water bath for 30 min. After incubation, 0.1 ml of each virus-

antiserum mixture was added to RK-13 cells grown in 24 multiwell tissue

culture plates (Falcon, Oxnard, California). After the antiserum-

medium mixture, virus-medium mixture, and each virus-antiserum mixture

were added to three wells, the plates were incubated at 370C in a

humidified atmosphere with 5% CO2 for 15 min. After virus

adsorption, 1 ml of maintenance medium was added to each well and

plates were incubated for 24 to 48 h or until one half of the virus

control exhibited cytopathic effects. The cultures were examined and

scored on the basis of 0 to 4+ cell destruction. The neutralization

titer of each antiserum was calculated using the Kaerber Method (1).














RESULTS
To determine if there is a correlation between the antigenic

composition of different strains of HSV and the ocular disease they

cause, the antigenic composition of five strains of HSV-1 which cause

characteristic and reproducible disease in the corneas of New Zealand

white rabbits was determined. The kind of disease produced varied from

epithelial (surface) only to a combination of epithelial and stromal

(deep) disease (79). The severity of disease varied from mild to

severe (8). The five HSV-1 strains used in this study caused

epithelial disease with a variation in the severity: Shealy and RE,

severe; McKrae and F, moderate; and CGA-3, mild. Four of the five

strains also caused stromal disease and again there is variation in the

severity: Shealy and RE, severe stromal necrosis with total corneal

vascularization; McKrae and F, moderate stromal disease. CGA-3 does

not cause stromal disease. The following experiments were done to see

if a correlation between the antigenic composition of the five strains

of HSV-1 and the ocular disease pattern exists.

Reference Patterns of Five Strains
of Herpes Simplex Virus Type One

Cultures of HEp-2 cells were infected with each of the five

strains that cause varying forms of eye disease and were radioactively

labeled with 14C-valine, -leucine, -isoleucine. The antigen

preparation of each strain was subjected to electrophoresis against the

homologous antiserum in cIEP and the immunoprecipitation peaks were










visualized by CBB staining and fluorography. Different volumes of

antigen and antibody were used to determine the best resolution, and

reproducible patterns were obtained with these different

antigen/antibody ratios. As the ratio changed, the density and

separation of the immunoprecipitation peaks also changed. The best

resolution based on density and separation of immunoprecipitation peaks

was established for the five strains using 20 to 45 ul antigen and 6 to

12 ul/cm2 antibody. Immunoprecipitation peaks compared on the basis

of RMV, shape and location were assigned the same antigen number.

To determine which of these antigens were virus-specific, cultures

of HEp-2 cells were mock infected and radioactively labeled with

14C-valine, -leucine, -isoleucine. The antigen preparation (15 ul)

was subjected to electrophoresis against 10 ul/cm2 of each of the

five antisera, and a total of five peaks (two to five peaks with each

antiserum) were resolved on the CBB stained gels and the fluorographs.

These five peaks, designated A,B,C,D,E, were nonspecific. In the

reference patterns of all five strains, all of the peaks resolved on

the CBB stained gels were also resolved on the fluorographs except one

or two peaks that matched peaks on the CBB stained gels of the HEp-2.

mock infected cells with the same antiserum. Note A and B on Table 1

and Figure 2. In the reference patterns of all five strains, all of

the peaks resolved on the fluorographs were virus-specific antigens

except one to three peaks that matched peaks on the fluorographs of the

HEp-2 mock infected cells with the same antiserum. Note C and D on

Table 1 and Figure 2.









Twenty-three virus-specific antigens were resolved in the five

strains and were classified as common, shared, and distinguishing.

Three common antigens, designated c, were resolved in all five strains.

Ten shared antigens, designated s, were resolved in more than one

strain. Ten distinguishing antigens, designated d, were resolved in

only one reference pattern. Antigens of the reference patterns of each

of the five strains comparing the CBB stained gel and fluorography of

each strain with that of the HEp-2 mock infected are shown in Figures

1-10. Tables 1-5 compare the RMV of these antigens and Table 6

summarizes these data.

The Shealy strain was designated as the prototype of HSV-1, since

its reference pattern consists of the largest total number of antigens,

13, the largest number of shared antigens, ten, and no distinguishing

antigens (Figures 1, 2; Table 1). Shealy, RE and McKrae strains showed

very similar reference patterns of 13, 13, and 12 virus-specific

antigens, respectively (Figures 1-6). RE and McKrae strains each

shared ten antigens with Shealy and eight antigens with each other; RE

strain had three distinguishing antigens and McKrae had two (Table 6).

F strain, with 11 antigens, was less similar, sharing only eight

antigens with Shealy strain, seven antigens with RE strain, and five

antigens with McKrae strain, and having three distinguishing antigens

(Figures 7, 8; Table 6). CGA-3 strain, with the fewest virus-specific

antigens, eight, had two distinguishing antigens and the most distinct

reference pattern. CGA-3 strain shared only six antigens with Shealy

and McKrae strains, four antigens with RE strain, and three common

antigens with F (Figures 9, 10; Table 6).









Cross-Reacting Antigens of Five Strains
of Herpes Simplex Virus Type One
To further characterize the common, shared, and distinguishing

antigens, experiments were done to determine cross-reacting antigens

in which the 14C-valine, -leucine, -isoleucine labeled antigen

preparation of each strain was subjected to electrophoresis against the

other four heterologous antisera. Each experiment was composed of one

control and four experimental gels. The ratio of antigen and antiserum

which was used to resolve the virus-specific antigens of each reference

pattern was duplicated in the controls of these five experiments. In

each experiment, the volume of antiserum used in the control gel was

repeated in the other four experimental gels. The volume of antigen in

the experimental gels was the same as that used to determine the

reference patterns. The same nomenclature designating antigens of the

reference patterns was chosen. Thirteen antigens which were not

matched with one of the 23 virus-specific antigens were designated "U"

for unknown. Only antigens resolved by fluorography were considered

significant. Figures 11-20 show the cross-reacting antigens of these

experiments. Tables 7-11 show the RMV of these antigens, and Table 12

summarizes these data.

These data show that the majority, 21 of the 23 virus-specific

antigens, cross-react. Each antiserum resolved all but one or two of

the virus-specific antigens of its homologous strain in at least one of

the other four strains. Anti-Shealy did not resolve 5s or 6s (Table

7), anti-RE did not resolve Id (Table 8), anti-McKrae did not resolve

9d (Table 9), and anti-F did not resolve 6s or 20d (Table 10) in any of










the four heterologous strains. Anti-CGA-3 resolved all of the virus-

specific antigens of CGA-3 in at least two of the other four strains

(Table 11). The three antigens resolved in each strain by all four

antisera are the three common antigens resolved in all five reference

patterns (2c, 21c, 22c) (Tables 7-11). The one exception, 2c, was not

resolved by anti-F in the RE strain (Table 10). The ten shared

antigens resolved in more than one reference pattern were resolved with

more than one heterologous antiserum (Tables 7-11). Seven of these

antigens (7s, 8s, 10s, 13s, 14s, 19s, 23s) were resolved in half or

more than half of the 20 fluorographs, and three were found in less

than half of the fluorographs (5s, 6s, 16s). Eight of these antigens

were distinguishing antigens found in less than half of the 20

fluorographs (Tables 7-11). These distinguishing antigens (3d, 4d, 9d,

11d, 12d, 15d, 17d, and 18d) were Iound in the reference pattern of one

strain only by the homologous antiserum or in that same strain with

heterologous antisera or if present in a different strain, were

resolved by the original homologous antiserum. Two distinguishing

peaks, ld of RE strain (Table 8) and 20d of F strain (Table 10), did

not cross-react with any heterologous antisera.

Two points are important in the analysis of the cross-reactivity

of one strain with one heterologous antiserum. First, what antigens

that belong to the reference pattern of the control (homologous)

antigen were resolved with the heterologous antiserum (antigens that

cross-react), and second, what antigens that belong to the reference

pattern of the heterologous antigen were resolved with the heterologous

antiserum. Table 12 summarizes the data of the cross-reacting antigens










and supports the previous observation from analysis of the reference

patterns showing the similarities and differences among these five

strains. The four heterologous antisera resolved the largest number of

cross-reacting antigens in the Shealy strain and the largest number of

virus-specific antigens of the reference patterns in the Shealy strain.

RE and McKrae strains were second and third respectively, showing many

cross-reacting antigens. Strain F showed fewer cross-reacting

antigens, and heterologous antisera with CGA-3 resolved the fewest

cross-reacting antigens and the fewest antigens of the reference

patterns in the CGA-3 strain. The data also showed the largest number

of cross-reacting antigens between Shealy, RE and McKrae strains,

several cross-reacting antigens between F strain and Shealy, RE, and

McKrae strains and the fewest cross-reacting antigens between CGA-3 and

the other four strains.

One interesting point is that each antiserum resolved antigens of

a heterologous strain hut did not resolve the same antigens in the

reference pattern of the control strain. For example, 7s was resolved

by anti-CGA-3 in all four heterologous strains but not in CGA-3. The

corollary is also true, in that the other four antisera resolved 7s in

the antigen preparation of the other four strains but not in the CGA-3

antigen preparation.

Glycosylated Antigen Patterns of Five Strains
of Herpes Simplex Virus Type One

Since glycoproteins are important in vivo in disease and neutral-

ization and in vitro in the kind of cytopathic effect (60,67,72,78),

glycosylated virus-specific antigens were determined. Cultures of









HEp-2 cells were infected with each of the five strains and

radioactively labeled with 14C-glucosamine hydrochloride. Each

strain was subjected to electrophoresis against the homologous

antiserum, and reproducible patterns were obtained with different

antigen/antibody ratios. Glycosylated antigen patterns were

established using 60 to 120 ul antigen and 6 to 12 ul/cm2 antibody.

The same nomenclature designating antigens of the reference patterns

and the cross-reacting patterns was used. Figures 21-30 illustrate

these antigens, Tables 13-17 show the RMV of these antigens, and Table

18 summarizes these data.

To determine which of these antigens are virus-specific, cultures

of HEp-2 cells were mock infected and radioactively labeled with

14C-glucosamine. Sixty microliters of this antigen was subjected

to electrophoresis against each of the five antisera, and a total of

eight peaks were resolved on CBB staining (three to six with each

antiserum). Five of these peaks were the same nonspecific A through E

designated with the radioactive amino acid labeled HEp-2 mock infected

cells plus three new peaks designated nonspecific, F, G and H. No

peaks were resolved on the fluorographs of the HEp-2 mock infected

cells. All the peaks resolved on CBR stained gels of the glycosylated

antigen patterns were detected on fluorographs except two to five peaks

that were nonspecific, i.e., that matched peaks on the CBB stained gel

of the HEp-2 mock infected cells with the same antiserum. Note

A,B,C,F,H of Table 13 and Figures 21 and 22. All of the peaks resolved

by fluorography of each strain were virus specific except one or two

peaks that matched peaks on the CBB stained gel of the HEp-2 mock










infected cells with the same antiserum. Note C of Table 14 and Figures

23 and 24.

Characteristic patterns were established with the homologous anti-

serum, and these patterns resemble the reference patterns of the

14C-amino acid labeled strains. Strains F and CGA-3 were more like

their own reference patterns than were Shealy, RE, and McKrae strains,

in that seven glycosylated antigens of the 11 virus-specific antigens

of F strain were resolved and five glycosylated antigens of the eight

virus-specific antigens of CGA-3 were resolved. The majority, 14, of

the virus-specific glycosylated antigens were identifiable with the 23

virus-specific antigens of the reference patterns (Table 18). The same

point was illustrated by the ratio of the number of glycosylated virus-

specific antigens matching the antigens of the reference pattern to the

total number of glycosylated virus-specific antigens resolved in each

strain: Shealy 8/9, RE 5/8, McKrae 8/11, F 7/7 and CGA-3 5/7. There

were nine antigens designated "U" which were not identifiable on either

the 14C-amino acid labeled reference pattern of each strain or the

14C-amino acid or -glucosamine labeled HEp-2 mock infected cells

with each antiserum (Table 18). Two of the three common antigens, 21c

and 22c, were glycosylated in all five strains, were resolved in all

five reference patterns of 14C-amino acid labeled strains, and were

recognized by all four heterologous antisera in the cross-reacting

experiments (Tables 6, 7-11, 18). Six of the ten shared antigens were

glycosylated; 10s, 13s, 14s and 19s were resolved in at least three

strains and 16s and 23s were resolved in two strains (Table 18). Six

of the ten distinguishing antigens were glycosylated: 9d and 18d of






34


McKrae strain; 12d, 15d, and 20d of F strain and 3d of CGA-3 strain

(Table 18).

Microneutralization Assay

The neutralization titers of the five antisera used in cIEP were

calculated using the Kaerber method. The log of the neutralization

titer for each antiserum was anti-Shealy, -3.5; anti-McKrae, -3.5;

anti-F, -5.5, anti-RE, -4.5; and anti-CGA-3, -0.833.











FIGURE 1
Crossed immunoelectrophoretic analysis of the reference pattern of the
Shealy strain. Cultures of HEp-2 cells were infected with Shealy or
mock infected and grown in medium supplemented with C-valine,
-leucine, -isoleucine. Antigen was applied in the first dimensional
electrophoresis, the intermediate gel contained agarose only, and the
reference gel contained the immunoglobulin (Ig) rich fraction of
antiserum to the Shealy strain grown in rabbit kidney-13 cells without
FCS. Crossed immunoelectrophoresis was performed as described in
Materials and Methods. (A) Coomassie brilliant blue staining of 20 ul
of Shealy antigen and 6 ul/cm- anti-Shealy Ig. (8) Fluorography of
A. (C) Coomassie brilliant blue staining of 15 ul of HEp-2 mock
infected antigen and 10 ul/cm4 anti-Shealy Ig. (D) Fluorography of
C.

















0












FIGURE 2
Tracing of Figure 1 of the reference pattern of the Shealy strain.
(A) Coomassie brilliant blue staining of 20 ul of Shealy antigen and 6
ul/cmL anti-Shealy Ig. (B) Fluorography of A. (C) Coomassie
brilliant blue staining of 15 ul of HEp-2 mock infected antigen and 10
ul/cm' anti-Shealy Ig. (D) Fluorography of C. Immunoprecipitation
peaks labeled: A,B,C,D,E are nonspecific peaks; c is common antigen,
s is shared antigen.












r \
SS %

S :

6 : ~


21c .. : as
0-23s 16s ....
--. .
J-~ :.. ....


2c


I \
I :
i :- I
I -
I I
I s
I 1


.4.
I -


0





D


I
I

I
I
I


8


C..- C


O


O


0


"^A










TABLE 1. REFERENCE PATTERN OF SHEALY


IMMUNOPPT.
PEAK
DESIGNATION"


HEp-2-SHEALY11
RMV RANGEt
CBB FLUOROGRAPHY


HEp-2-MOCK INFECTED0
RMV VIRUS-
C8B FLUOROGRAPHY SPECIFIC


67-69
54-56
45-55
55
45-52
37
33-36
36
32
26-32
9-26
22-27
12-24

17-19
12
12
4-12


54-79
50-56
45-48
31-36
33-35
32-33
31
29-36
5-29
25-27
13-24

16-19
10-14
10-12
4-10


55 54

32


+

+

+
+
+
+
+
+
+

+
+
+
+
13/18


A
B
2c*
C
5s
D
6s
7s
8s
lOs
13s*
14s
16s*
E
19s
21c
22c
23s*


*A,B,C,D,E are nonspecific peaks; c is common antigen, s is shared
antigen
'120 ul 14C-val, -leu, -isl labeled HEp-2 infected with Shealy IEP
antigen and 6 or 8 ul/cm' anti-Shealy Ig
Relative migration velocity of immunoprecipitation peaks of 3 gels
Coomassie brilliant blue
015 ul 14C-val, -leu, -isl labeled HEp-2 mock infected IEP antigen
and 10 ul/cm2 anti-Shealy Ig
*Greater range due to broad peak


22 27











FIGURE 3
Crossed immunoelectrophoretic analysis of the reference pattern of the
RE strain. Cultures of HEp-2 cells were infected with RE or mock
infected and grown in medium supplemented with C-valine,
-leucine, -isoleucine. Antigen was applied in the first dimensional
electrophoresis, the intermediate gel contained agarose only, and the
reference gel contained the immunoglobulin (Ig) rich fraction of
antiserum to the RE strain grown in rabbit kidney-13 cells without FCS.
Crossed immunoelectrophoresis was performed as described in Materials
and Methods. (A) Coomassie brilliant blue staining of 30 ul of RE
antigen and 12 ul/cm6 anti-RE Ig. (B) Fluorography of A.
(C) Coomassie brilliant blue staining of 15 ul of HEp-2 mock infected
antigen and 10 ul/cm anti-RE Ig. (D) Fluorography of C.






41



A B
















0 0O





C D














4

.41











FIGURE 4
Tracing of Figure 3 of the reference pattern of the RE strain.
(A) Coomassie brilliant blue staining of 30 ul of RE antigen and 12
ul/cmS anti-RE Ig. (B) Fluorography of A. (C) Coomassie brilliant
blue staining of 15 ul of HEp-2 mock infected antigen and 10 ul/cm
anti-RE Ig. (D) Fluorography of C. Immunoprecipitation peaks labeled:
A,C,D,E are nonspecific peaks; c is common antigen, s is shared
antigen, d is distinguishing antigen. Immunoprecipitation peaks ld and
5s were not resolved in the gel in Figure 3A and 38 but were detected
in at least two other gels of the reference pattern of the RE strain.



















8s

7s

21c

S13s
1"^


0


l i



SIl
I I



17d a
I i
I I


.* .. 4
dI 5s



1922c 1 ..6s


19s


2c


0


i
I
I
I
I
I
I


E ". """.,


0










TABLE 2. REFERENCE PATTERN OF RE


IMMUNOPPT.
PEAK
DESIGNATION*


HEp-2-RE '
RMV RANGEt
CBB FLUOROGRAPHY


HEp-2-MOCK INFECTED0
RMV
CBB FLUOROGRAPHY SI


VIRUS-
'ECIFIC


82
68-71
46-56

33

31-33
31-33

22-31
25-30
26-29


15-20
14-15
13-16


67-81

49-53

32-43
31-32
28-32
31-32
31-32
18-31
29-31
28-30

21-23
13-22
13-16
14-19


54 57

43


+

+
+

+
+

+
+
+
+
+
+
+
-


+
13/17
13/17


"A,C,D,E are nonspecific peaks; d is distinguishing antigen, c is
common antigen, s is shared antigen
'30 ul 14C-val, -leu, -isl labeled HEp-2 infected with RE IEP
antigen and 8 or 12 ul/cm2 anti-RE Ig
Relative migration velocity of immunoprecipitation peaks of 3 gels
Coomassie brilliant blue
015 ul 14C-val, -leu, -isl labeled HEp-2 mock infected IEP antigen
and 10 ul/cm2 anti-RE Ig
*Greater range due to broad peak


Id*
A
2c*
C
5s*
D
7s
8s
1ld
13s*
14s
16s
E
17d
19s
21c
22c











FIGURE 5
Crossed immunoelectrophoretic analysis of the reference pattern of the
McKrae strain. Cultures of HEp-2 cells were infected with McKrae or
mock infected and grown in medium supplemented with C-valine,
-leucine, -isoleucine. Antigen was applied in the first dimensional
electrophoresis, the intermediate gel contained agarose only, and the
reference gel contained the immunoglobulin (Ig) rich fraction of
antiserum to the McKrae strain grown in rabbit kidney-13 cells without
FCS. Crossed immunoelectrophoresis was performed as described in
Materials and Methods. (A) Coomassie brilliant blue staining of 30 ul
of McKrae antigen and 6 ul/cm anti-McKrae Ig. (B) Fluorography of
A. (C) Coomassie brilliant blue staining of 15 ul of HEp-2 mock
infected antigen and 10 ul/cm anti-McKrae Ig. (D) Fluorography of
C.


















-"9_


'*~llu~J~;i;~sl~llIrO


gr











FIGURE 6
Tracing of Figure 5 of the reference pattern of the McKrae strain.
(A) Coomassie brilliant blue staining of 30 ul of McKrae antigen and 6
ul/cm anti-McKrae Ig. (B) Fluorography of A. (C) Coomassie
brilliant blue staining of 15 ul of HEp-2 mock infected antigen and 10
ul/cm anti-McKrae Ig. (D) Fluorography of C. Immunoprecipitation
peaks labeled: A,B,C,D,E are nonspecific peaks; c is common antigen,
s is shared antigen, d is distinguishing antigen.














S7 s

21c 4

22c 9d
195 ,


0


I
I 'c
I c


B
***,


0


0


0










TABLE 3. REFERENCE PATTERN OF McKRAE


IMMUNOPPT.
PEAK
DESIGNATION'


HEp-2-McKRAE'I
RMV RANGEt
CBB FLUOROGRAPHY


HEp-2-MOCK INFECTED0
RMV VIRUS-
CBB FLUOROGRAPHY SPECIFIC


42-65

40-46
28
28-36
27-35
30-34
29-33
21-26

19-21
14-19
12-15
12-15
11-16


54 54

32


22 19


12/17


"A,B,C,D,E are nonspecific peaks; c is common antigen, s is shared
antigen, d is distinguishing antigen
115 or 30 ul 14C-val, -leu, -isl labeled HEp-2 infected with
McKrae IEP antigen and 6 ul/cm2 anti-McKrae Ig
Relative migration velocity of immunoprecipitation peaks of 3 gels
Coomassie brilliant blue
015 ul 14C-val, -leu, -isl labeled HEp-2 mock infected IEP antigen
and 10 ul/cm2 anti-McKrae Ig
*Greater range due to broad peak


A
B
2c*
C
5s
D
7s
8s
9d
10s
14s
E
18d
19s
21c
22c
23s


65-70
52-58
52-56

42-44

33-37
30-37
30-37
30-37
22-24

16-18
16-18
14-16
14-15
13











FIGURE 7
Crossed immunoelectrophoretic analysis of the reference pattern of the
F strain. Cultures of HEp-2 cells were infected with F or mock
infected and grown in medium supplemented with C-valine,
-leucine, -isoleucine. Antigen was applied in the first dimensional
electrophoresis, the intermediate gel contained agarose only, and the
reference gel contained 10 ul/cmz of the immunoglobulin (Ig) rich
fraction of antiserum to the F strain grown in rabbit kidney-13 cells
without FCS. Crossed immunoelectrophoresis was performed as described
in Materials and Methods. (A) Coomassie brilliant blue staining of 30
ul of F antigen. (B) Fluorography of A. (C) Coomassie brilliant
blue staining of 15 ul of HEp-2 mock infected antigen. (D) Fluoro-
graphy of C.






51


A B









*:. ..i ... .....
.. .
.a








C D






i
j ~~~F --i .."

rP











FIGURE 8
Tracing of Figure 7 of the referRnce pattern of the F strain. The
reference gel contained 10 ul/cm4 anti-F Ig. (A) Coomassie
brilliant blue staining of 30 ul of F antigen. (B) Fluorography of A.
(C) Coomassie brilliant blue staining of 15 ul of HEp-2 mock infected
antigen. (D) Fluorography of C. Immunoprecipitation peaks labeled:
A,B,C,D,E are nonspecific peaks; c is common antigen, s is shared
antigen, d is distinguishing antigen.



















8s
|1 i
21c 8s

S I

S.... :,, .......... o .'.o
"* -

20d 13s 12d
20 15d


,, 22c


i i
I I
I I

I'
I' I

I 7

6s

.. *^ 6.,


2c


0


I
I
I
I
I


I
I
ID

1


... C


0


0


0


"......A










TABLE 4. REFERENCE PATTERN OF F


IMMUNOPPT.
PEAK
DESIGNATION'


HEp-2-F1
RMV PANGEt
CBB FLUOROGRAPHY


HEp-2-MOCK INFECTED0
RMV
CRBB FLUOROGRAPHY SI


VIRUS-
PECIFIC


66-67
56
44-54

33-37
33-35
33
33-35
26-27
19-33
23-32
17-23

14
7-16
(-3)-0


48-59

33-37
32-35
32-33
32-35
25-32
19-33
20-29
22-25


54 56
42


+


+
+
+
+
+
+
+

+
+
+
11/16


A
B
2c*
C
D
6s
7s
8s
12d
13s*
15d
16s
E
20d
21c
22c*


"A,B,C,D,E are nonspecific peaks; c is common antigen, s is shared
antigen d is distinguishing antigen
*130 ul 14C-val, -leu, -isl labeled HEp-2 infected with F IEP
antigen and 10 ul/cm2 anti-F Ig
Relative migration velocity of immunoprecipitation peaks of 3 gels
Coomassie brilliant blue
015 ul 14C-val, -leu, -isl labeled HEp-2 mock infected IEP antigen
and 10 ul/cm2 anti-F Ig
*Greater range due to broad peak


15
9-16
(-4)-11











FIGURE 9
Crossed immunoelectrophoretic analysis of the reference pattern of the
CGA-3 strain. Cultures of HEp-2 cells were infected with CGA-3 or mock
infected and grown in medium supplemented with C-valine,
-leucine, -isoleucine. Antigen was applied in the first dimensional
electrophoresis, the intermediate gel contained agarose only, and the
reference gel contained the immunoglobulin (Ig) rich fraction of
antiserum to the CGA-3 strain grown in rabbit kidney-13 cells without
FCS. Crossed immunoelectrophoresis was performed as described in
Materials and Methods. (A) Coomassie brilliant blue staining of 45 ul
of CGA-3 antigen and 10 ul/cm anti-CGA-3 Ig. (B) Fluorography of
A. (C) Coomassie brilliant blue staining of 15 ul of HEp-2 mock
infected antigen and 10 ul/cm anti-CGA-3 Ig. (D) Fluorography of
C.









A B

















C D












FIGURE 10
Tracing of Figure 9 of the reference pattern of the CGA-3 strain.
(A) Coomassie brilliant blue staining of 45 ul of CGA-3 antigen and 10
ul/cmL anti-CGA-3 Ig. (B) Fluorography of A. (C) Coomassie
brilliant blue staining of 15 ul of HEp-2 mock infected antigen and 10
ul/cm anti-CGA-3 Ig. (D) Fluorography of C. Immunoprecipitation
peaks labeled: A,B,C,D,E are nonspecific peaks; c is common antigen,
s is shared antigen, d is distinguishing antigen.













i i
I
I
II
I

I I
I \
I I

S=21c\


.O. s


0






D


I 1
I
I
I 19


C.... --
C ...- "


0


14s


S 3d


i~;


B
- --A










TABLE 5. REFERENCE PATTERN OF CGA-3


IMMUNOPPT.
PEAK
DESIGNATION"


HEp-2-CGA-3 3
RMV RANGEt
CBR6 FLUOROGPAPHY


HEp-2-MOCK INFECTED
RMV VIRUS-
CRB FLUnROGRAPHY SPECIFIC


66-72
53-58
52-60
59-62
55-56
41-46

27-32
27-33
24-25
15-19
11-15
11


65-73

55-61

51-57
37-46
31-39
26-30
24-31
22-30
14-20
12-15
8-9


54 57


40


21 23


8/13


'A,B,C,0,E are nonspecific peaks; c is common antigen, d is
distinguishing antigen, s is shared antigen
45 ul 14C-val, -leu, -isl labeled HEp-2 infected with CGA-3 IEP
antigen and 10 ul/cm2 anti-CGA-3 Ig
Relative migration velocity of immunoprecipitation peaks of 3 gels
Coomassie brilliant blue
015 ul 14C-val, -leu, -isl labeled HEp-2 mock infected IEP antigen
and 10 ul/cm2 anti-CGA-3 Ig


A
8
2c
C
3d
4d
D
10s
14s
E
21c
22c
23s






60



TABLE 6. SUMMARY OF VIRUS-SPECIFIC ANTIGENS FROM FLUOROGRAPHY
OF REFERENCE PATTERNS OF 14C-VAL, -LEU, -ISL LABELED STRAINS

IMMUNOPPT.
PEAK
DESIGNATION SHEALY RE McKRAE F CGA-3

Id* X
2c X X X X X
3d X
4d X
5s!l X X
6s X X
7s X X X X
8s X X X X
9d X
10s X X X
lld X
12d X
13s X X X
14s X X X X
15d X
16s X X X
17d X
18d X
19s X X X
20d X
21c X X X X X
22c X X X X X
23s X X X
Virus-specific
Antigens 13 13 12 11 8


*d is distinguishing antigen
c is common antigen
:s is shared antigen











FIGURE 11
Crossed immunoelectrophoretic analysis of the pattern of cross-reacting
antigens of the Shealy strain. Cultures of HEp-2 cells were infected
with Sealy, RE, McKrae, F, or CGA-3 and grown in medium supplemented
with C-valine, -leucine, -isoleucine. Antigen was applied in
the first dimensional electrophoresis, the intermediate gel contained
agarose only, and the reference gel contained 6 ul/cm6 of the
immunoglobulin (Ig) rich fraction of antiserum to the Shealy strain
grown in rabbit kidney-13 cells without FCS. Crossed
immunoelectrophoresis was performed as described in Materials and
Methods. Fluorography of (A) 20 ul Shealy antigen, (B) 30 ul RE
antigen, (C) 30 ul McKrae antigen, (D) 30 ul F antigen, (E) 45 ul CGA-3
antigen.













()
C)


A


0)


77~7&T-g


)


)











FIGURE 12
Tracing of Figure 11 of the pattern of cross-reacting antigens of the
Shealy strain. Fluorography of 6 ul/cm anti-Shealy Ig and (A) 20 ul
Shealy antigen, (B) 30 ul RE antigen, (C) 30 ul McKrae antigen, (0) 30
ul F antigen, (E) 45 ul CGA-3 antigen. Immunoprecipitation peaks
labeled: C and D are nonspecific peaks; c is common antigen, s is
shared antigen, d is distinguishing antigen. Immunoprecipitation peak
C was not resolved in the gel in Figure 11A but was detected in at
least two other gels of the reference pattern of the Shealy strain.




















21c
.,,," ,


Ss
I
I
I 5 ID
I %




5

Is
s .

14s


* ,* i::.:tS "
s" ....



19s


I
I \D

I %
lid "
I
21c I --s





23s- 16s .
13s


21c : "
2\\\\\ \ 14



19s
..': 18d


0


D A



I D
I
I



23s 2c


<, 13s


I
I
I
I
I
I
I
/


21c 14s

223s

" 23 .


0


iD
I
I
I
I
I
I

t
I
%
.9










TABLE 7. CROSS-REACTING ANTIGENS WITH
HETEROLOGOUS ANTISERUM: ANTI-SHEALY Ig


IMMUNOPPT.
PEAK
DESIGNATION


ANTIGENS THAT
CROSS-REACT
WITH
ANTIGENS OF
THE REFERENCE
PATTERN OF
THE HOMOLOGOUS
VIRUS
[SHEALY]


2c*
C
5s
D
6s
7s
8s
Ins
13s*
14s
16s*
1.9s
21c
22c
23s


SHEALYt REt


40-590
( )0
450
310
350
320
310
300
5-290
250
10-190
160
140
120
40


43-60


330
330
29
24-290
240
19-290
9
160
160
8


McKRAEt Ft
43-600 44-560


320
320
220

220

140
160
160


33
18-290
24

20
130
140
10


ANTIGENS OF 3d
THE REFERENCE 9d
PATTERN OF THE lid
HETEROLOGOUS 18d
VIRUS


TOTAL NUMBER OF PEAKS: 14

VIRUS-SPECIFIC ANTIGENS
THAT CROSS-REACT WITH SHEALY:

VIRUS-SPECIFIC ANTIGENS
OF EACH REFERENCE PATTERN: 13/13


13 10


11/13 10/12 5/11


*C and D are nonspecific peaks; c is common
antigen, d is distinguishing antigen


8


6


7/8


antigen, s is shared


tRelative migration velocity of the immunoprecipitation peaks of
14C-val, -leu, -isl IEP antigen against 6 ul/cm2 anti-Shealy Ig
resolved on fluorography: 20 ul Shealy; 30 ul RE, McKrae, F; 45 ul
CGA-3 antigen.
OAntigens of the reference pattern of each virus
()Antigen of the Shealy reference pattern not detected in this gel
*Greater range due to broad peak


CGA-3t
52-63


260


230-

210


130
130
80


520


170











FIGURE 13
Crossed immunoelectrophoretic analysis of the pattern of cross-reacting
antigens of the RE strain. Cultures of HEp-2 cells were infected with
, Shealy, McKrae, F, or CGA-3 and grown in medium supplemented with
C-valine, -leucine, -isoleucine. Antigen was applied in the
first dimensional electrophoresis, the intermediate gel contained
agarose only, and the reference gel contained 12 ul/cm of the
immunoglobulin (Ig) rich fraction of antiserum to the RE strain grown
in rabbit kidney-13 cells without FCS. Crossed immunoelectrophoresis
was performed as described in Materials and Methods. Fluorography of
(A) 30 ul RE antigen, (B) 20 ul Shealy antigen, (C) 30 ul McKrae
antigen, (D) 30 ul F antigen, (E) 45 ul CGA-3 antigen.



















C











E







0











FIGURE 14
Tracing of Figure 13 of the pattern of cross-reacting antigens of the
RE strain. Fluorography of 12 ul/cm2 anti-RE Ig and (A) 30 ul RE
antigen, (B) 20 ul Shealy antigen, (C) 30 ul McKrae antigen, (D) 30 ul
F antigen, (E) 45 ul CGA-3 antigen. Immunoprecipitation peaks
labeled: C and D are nonspecific peaks; c is common antigen, s is
shared antigen, d is distinguishing antigen. Immunoprecipitation peaks
which are not labeled are unknown antigens.










A
I
I ID


17d I
I ild \

*Ss %
/ ^Id


.9 \ .: .

19s
zli _~i~Ic
:li iC
ii;gsib


B


\

/
17d


6s ~ 5s






198
21c ." 14s /os


,> //1' I. 1, ."









D

/'


21c
12d

22c 13s


..." 2c


lid
17d S


: -- 2c
.
21c :1


22C .. 9d
~ 8d

19s





0

E


I
I
I
I
'I


/ 5s


I0
I
I
I
9,
9,
9,


2c


21c

i/_ *. 14s
/ 22c
19


0


:":"i,: : ::"3d
^"










TABLE 8. CROSS-REACTING ANTIGENS WITH
HETEROLOGOUS ANTISEPUM- ANTI-RE Ig


IMMUNOPPT.
PEAK
DESIGNATION'


REt SHEALYt McKRAEt Ft CGA-3t


ANTIGENS THAT
CROSS-REACT
WITH
ANTIGENS OF
THE REFERENCE
PATTERN OF
THE HOMOLOGOUS
VIRUS
[RE]


Id
2c*
5s
D
7s
8s
lld
13s*
14s
16s*
17d
19s
21c
22c


67-710
44-580
430
320
320
310
310
18-310
290
290
230
180
130
170


ANTIGENS OF C
THE REFERENCE 3d
PATTERN OF THE 6s
HETEROLOGOUS 9d
VIRUS 10s
12d
18d


47-620
510

390
330

11-290
310
11-220
22
200
110
110


44-620 580
35


310
290
31

310

22
160
140
160


310
310

23-330


19
19
140
190


580

370

310


UNKNOWN
ANTIGENS


TOTAL NUMBER OF PEAKS:

VIRUS-SPECIFIC ANTIGENS
THAT CROSS-REACT WITH RE:


VIRUS-SPECIFIC ANTIGENS
OF EACH REFERENCE PATTERN: 13/13


12/13 10/12 7/11 6/8


"D and C are nonspecific peaks; d is distinguishing antigen, c is
common antigen, s is shared antigen, U is unknown antigen
tRelative migration velocity of the immunoprecipitation peaks of
14C-val, -leu, -isl IEP antigen against 12 ul/cm2 anti-RE Ig
resolved on fluorography: 20 ul Shealy; 30 ul RE, McKrae, F; 45 ul
CGA-3 antigen.
OAntigens of the reference pattern of each virus
*Greater range due to broad peak


58-700

370




300


190
190
190


13 10


9 9











FIGURE 15
Crossed immunoelectrophoretic analysis of the pattern of cross-reacting
antigens of the McKrae strain. Cultures of HEp-2 cells were infected
with %Krae, Shealy, RE, F, or CGA-3 and grown in medium supplemented
with C-valine, -leucine, -isoleucine. Antigen was applied in
the first dimensional electrophoresis, the intermediate gel contained
agarose only, and the reference gel contained 6 ul/cm/ of the
immunoglobulin (Ig) rich fraction of antiserum to the McKrae strain
grown in rabbit kidney-13 cells without FCS. Crossed immunoelectro-
phoresis was performed as described in Materials and Methods.
Fluorography of (A) 30 ul McKrae antigen, (B) 20 ul Shealy antigen,
(C) 30 ul RE antigen, (D) 30 ul F antigen, (E) 60 ul CGA-3 antigen.












0


)


"300
tz.--


II
)


I I


A


F4


I











FIGURE 16
Tracing of Figure 15 of the pattern of cross-reacting antigens of the
McKrae strain. Fluorography of 6 ul/cm2 anti-McKrae Ig and (A) 30 ul
McKrae antigen, (B) 20 ul Shealy antigen, (C) 30 ul RE antigen, (D) 30
ul F antigen, (E) 60 ul CGA-3 antigen. Immunoprecipitation peaks
labeled: D is a nonspecific peak; c is common antigen, s is shared
antigen, d is distinguishing antigen. Immunoprecipitation peak 18d was
not resolved in the gel in Figure 15A but was detected in at least two
other gels of the reference pattern of the McKrae strain.
Immunoprecipitation peaks which are not labeled are unknown antigens.










A \,,
/ 8s %
I I



21cA
I 7s t


/ 14s 2c

I s
22c .

19s
18d




O
B s"

I s
%

/ s. -


21c
,/ -" l 2c
235
22c ,
16s/











4s 2c
1s d





O
44j



I9
I2; -- t





^ I28d



19s



0


I Ild D



14s ,
'\
235 ,, ,



23s
13s
,- 19s


0


E s i
I
I D
I
I t
I
I '4
I \



I os
2 2 2c
21c 14s ..........
... .... lOs
"'22c .- ... .,, .


0










TABLE 9. CROSS-REACTING ANTIGENS WITH
HETEROLOGOUS ANTISERUM: ANTI-McKRAE Ig


IMMUNOPPT.
PEAK
DESIGNATION'


ANTIGENS THAT
CROSS-REACT
WITH
ANTIGENS OF
THE REFERENCE
PATTERN OF
THE HOMOLOGOUS
VIRUS
[McKRAE]


2c*
5s
D
7s
8s
9d
10s
14s
18d
19s
21c
22c
23s


McKRAEt SHEALYt


44-650
460
( )0
350
350
340
350
260
( )0
190
150
150
150


ANTIGENS OF 6s
THE REFERENCE 11d
PATTERN OF THE 12d
HETEROLOGOUS 13s
VIRUS 15d
16s

UNKNOWN U*
ANTIGENS U

TOTAL NUMBER OF PEAKS:


VIRUS-SPECIFIC ANTIGENS
THAT CROSS-REACT WITH McKRAE:

VIRUS-SPECIFIC ANTIGENS
OF EACH REFERENCE PATTERN: 11/12


37-560
370
330
320
320

250
200
15
150
110
90
9

320


REt


40-60 6


15-220 26-340

11-170 26-340


Ft rCGA-3t
i40 630


330

330

330


61-82
31


15 13 11


9 7


13/13 10/13 7/11


*D is a nonspecific peak; c is common antigen, s is shared antigen, d
is distinguishing antigen, U is unknown antigen
tRelative migration velocity of the immunoprecipitation peaks of
14C-val, -leu, -isl IEP antigen against 6 ul/cm2 anti-McKrae Ig
resolved on fluorography: 20 ul Shealy; 30 ul McKrae, RE, F; 60 ul
CGA-3 antigen.
Antigens of the reference pattern of each virus
()Antigens of the McKrae reference pattern not detected in this gel
*Greater range due to broad peak


8


5


5/8











FIGURE 17
Crossed immunoelectrophoretic analysis of the pattern of cross-reacting
antigens of the F strain. Cultures of HEp-2 cells were infected with
4 Shealy, RE, McKrae, or CGA-3 and grown in medium supplemented with
C-valine, -leucine, -isoleucine. Antigen was applied in the
first dimensional electrophoresis, the intermediate gel contained
agarose only, and the reference gel contained 10 ul/cm2 of the
immunoglobulin (Ig) rich fraction of antiserum to the F strain grown in
rabbit kidney-13 cells without FCS. Crossed immunoelectrophoresis was
performed as described in Materials and Methods. Fluorography of (A)
30 ul F antigen, (B) 20 ul Shealy antigen, (C) 30 ul RE antigen, (D) 30
ul McKrae antigen, (E) 60 ul CGA-3 antigen.












/


iP











FIGURE 18
Tracing of Figure 17 of the pattern of cross-reacting antigens of the
F strain. Fluorography of 10 ul/cm2 anti-F Ig and (A) 30 ul F
antigen, (B) 20 ul Shealy antigen, (C) 30 ul RE antigen, (D) 30 ul
McKrae antigen, (E) 60 ul CGA-3 antigen. Immunoprecipitation peaks
labeled: A and D are nonspecific peaks; c is common antigen, s is
shared antigen, d is distinguishing antigen. Immunoprecipitation peak
20d was not resolved in the gel in Figure 17A but was detected in at
least two other gels of the reference pattern of the F strain.
Immunoprecipitation peaks which are not labeled are unknown antigens.







A \ i 79
.\ ID
l -\ '- I
21c Ir 1'\






3s








8 s 8

21c D \7s-, 7s


~ ..
-23s 5s
lb 15d
2c
1313s





0 0

s I SD
S8s 21c I
", I ', I


21 2 s 2c c
S. "* .. los 2c
/ / '.\ : ..,.,- O s.
9 d.- ..**; :--U Afl
18d 3



0









CROSS-REACTING ANTIGENS WITH


HETEROLOGOUS ANTISERUM: ANTI-F Ig


IMMUNOPPT.
PEAK
DESIGNATION'


ANTIGENS THAT
CROSS-REACT
WITH
ANTIGENS OF
THE REFERENCE
PATTERN OF
THE HOMOLOGOUS
VIRUS
[F]


2c*
D
6s
7s
8s
12d
13s
15d
16s
20d
21c
22c


Ft
590
350
350
320
350
250
220
220
220
( )0
90
110


SHEALYt
33-560
310

310
310
28
240
26
240

100
110


REt McKRAEt
40-620
310


CGA-3t
42-700
350


310
310


ANTIGENS OF
THE REFERENCE
PATTERN OF THE
HETEROLOGOUS
VIRUS


220
3


UNKNOWN
ANTIGENS


TOTAL NUMBER OF PEAKS:

VIRUS-SPECIFIC ANTIGENS
THAT CROSS-REACT WITH F:

VIRUS-SPECIFIC ANTIGENS
OF EACH REFERENCE PATTERN:


11 12


10/11


9 11


9/13 7/13 8/12


*D and A are nonspecific peaks; c is common antigen, s is shared
antigen, d is distinguishing antigen, IU is unknown antigen
tRelative migration velocity of the immunoprecipitation peaks of
14C-val, -leu, -isl IEP antigen against 10 ul/cm2 anti-F Ig
resolved on fluorography: 20 ul Shealy; 30 ul F, RE, McKrae; 60
CGA-3 antigen.
"Antigens of the reference pattern of each virus
()Antigen of the F reference pattern not detected in this gel
*Greater range due to broad peak


TABLE 10.











FIGURE 19
Crossed immunoelectrophoretic analysis of the pattern of cross-reacting
antigens of the CGA-3 strain. Cultures of HEp-2 cells were infected
with Y A-3, Shealy, RE, McKrae, or F and grown in medium supplemented
with C-valine, -leucine, -isoleucine. Antigen was applied in
the first dimensional electrophoresis, the intermediate gel contained
agarose only, and the reference gel contained 10 ul/cmr of the
immunoglobulin (Ig) rich fraction of antiserum to the CGA-3 strain
grown in rabbit kidney-13 cells without FCS. Crossed
immunoelectrophoresis was performed as described in Materials and
Methods. Fluorography of (A) 45 ul CGA-3 antigen, (B) 20 ul Shealy
antigen, (C) 30 ul RE antigen, (D) 30 ul McKrae antigen, (E) 30 ul F
antigen.





























0


0


O











FIGURE 20
Tracing of Figure 19 of the pattern of cross-reacting antigens of the
CGA-3 strain. Fluorography of 10 ul/cm anti-CGA-3 Ig and (A) 45 ul
CGA-3 antigen, (B) 20 ul Shealy antigen, (C) 30 ul RE antigen, (D) 30
ul McKrae antigen, (E) 30 ul F antigen. Immunoprecipitation peaks
labeled: A,D,E are nonspecific peaks; c is common antigen, s is
shared antigen, d is distinguishing antigen. Immunoprecipitation peaks
which are not labeled are unknown antigens.












A I
I
I
I
I




0s


E


I



I 2
\.





4.-

Ad ,-"- A


I \




I \
64 s0
* .': 6 ';


'7


0
D


2c
. : ^


ID
I
I


1


*
* 9d


2c

A


23s 16s


16s s ,
/


ID
I I
I \
I \

-- 85 s


I : 1Os 2c
14s 7s
.s.r" .*- J^


0


I, %
\o

S \
0o


',..
A


e










TABLE 11. CROSS-REACTING ANTIGENS WITH
HETEROLOGOIS ANTISERUM: ANTI-CGA-3 Ig

IMMUNOPPT.
PEAK
DESIGNATION' CGA-3t SHEALYt REt McKRAEt Ft
A 650 67 69 67 65
ANTIGENS THAT 2c* 51-610 53-590 43-640 52-630 50-560
CROSS-REACT 3d 510 54
WITH 4d 370 46
ANTIGENS OF 0 290 370 400 330 330
THE REFERENCE 10s 270 180 28 28
PATTERN OF 14s 240 250 26 26
THE HOMOLOGOUS E 220
VIRUS 21c 140 100 180 130 130
[CGA-3] 22c 140 100 180 130 140
23s 100 5s 60 10

5s 450 440
ANTIGENS OF 6s 330
THE REFERENCE 7s 330 340 330 310
PATTERN OF THE 8s 330 340 310
HETEROLOGOIJS 9d 330
VIRUS 13s 30 250
16s 180 30 250

U 47
UNKNOWN U 33
ANTIGENS U 29
U 15
U 13

TOTAL NUMBER OF PEAKS: 11 14 13 12 13

VIRUS-SPECIFIC ANTIGENS
THAT CROSS-REACT WITH CGA-3: 8 5 4 6

VIRUS-SPECIFIC ANTIGENS
OF EACH REFERENCE PATTERN: 8/8 10/13 9/13 7/12 7/11

*A,D and E are nonspecific peaks; c is common antigen, d is
distinguishing antigen, s is shared antigen, U is unknown antigen
tRelative migration velocity of the immunoprecipitation peaks of
14C-val, -leu, -isl IEP antigen against 10 ul/cm2 anti-CGA-3 Ig
resolved on fluorography: 20 ul Shealy; 30 ul RE, McKrae, F; 45 ul
CGA-3 antigen.
oAntigens of the reference pattern of each virus
*Greater range due to broad peak






86



TABLE 12. SUMMARY OF VIRUS-SPECIFIC ANTIGENS FROM FLUOROGRAPHY
OF CROSS-REACTING ANTIGENS WITH HETEROLOGOUS ANTISERUM

SHEALY1 REt McKRAEt Ft CGA-3t


Virus-specific antigens
of each reference pattern
with homologous antiserum


Virus-specific antigens
that cross-react*


anti-SHEALY
anti-RE
anti-McKRAE
anti-F
anti-CGA-3


13 13 12 11 8


11 8
q


Virus-specific antigens
of each reference pattern
with heterologous antiserum


anti-SHEALY
anti-RE
anti-McKRAE
anti-F
anti-CGA-3


11 10
10


tl4C-val, -leu, -isl labeled antigen subjected to
electrophoresis against four heterologous antisera.
Immunoprecipitation peaks were resolved by fluorography.
*Antigens that belong to the reference pattern of the control
antigen that were resolved with heterologous antiserum.












FIGURE 21
Crossed immunoelectrophoretic analysis of the pattern of glycosylated
antigens of the Shealy strain. Cultures of HEp-2 cells were infected
ith Shealy or mock infected and grown in medium supplemented with
C-glucosamine. Antigen was applied in the first dimensional
electrophoresis, the intermediate gel contained agarose only, and the
reference gel contained 6 ul/cm' of the immunoglobulin (Ig) rich
fraction of antiserum to the Shealy strain grown in rabbit kidney-13
cells without FCS. Crossed immunoelectrophoresis was performed as
described in Materials and Methods. (A) Coomassie brilliant blue
staining of 120 ul of Shealy antigen. (B) Fluorography of A.
(C) Coomassie brilliant blue staining of 60 ul of HEp-2 mock infected
antigen. (D) Fluorography of C.









A B












C; D




C D











FIGURE 22
Tracing of Figure 21 of the pattern of glycosylated antigens of the
Shealy strain. The reference gel contained 6 ul/cm2 anti-Shealy Ig.
(A) Coomassie brilliant blue staining of 120 ul of Shealy antigen.
(B) Fluorography of A. (C) Coomassie brilliant blue staining of 60
ul of HEp-2 mock infected antigen. (D) Fluorography of C. Immuno-
precipitation peaks labeled: A,B,C,D,F,H are nonspecific peaks; c is
common antigen, s is shared antigen; U is unknown antigen. Immuno-
precipitation peak F was not resolved in the gel in Figure 21A but was
detected in at least two other gels of the reference pattern of the
glycosylated antigen pattern of the Shealy strain.





























21c

..'"22c :
22c


I
I
I
I
ID
I





10s


A/.


i






I4s


23 ... -

19s-' .' Ui


D


H /... --"
















TABLE 13. GLYCOSYLATED ANTIGEN PATTERN OF SHEALY


HEp-2-SHEALY
RMV RANGEt
CBB FLUOROGRAPHY


64-70
63
52-59
48-52
26
25-33
25-32
25-27
25-32
21-29

9-17
9-17


VIRUS-
HEp-2 MOCK INFECTED SPECIFIC
RMV GLYCOSYLATED
CBB FLUOROGRAPHY ANTIGENS


67-69*
61
57*
52


28
27-33
28-30
27-30
26-30

17-22
9-18
9-18
7
17-20


9/15


*A,B,C,D,F,H are nonspecific peaks; s is shared antigen, c is common
antigen, IJ is unknown antigen
30, 90, 120 ul 14C-glucosamine labeled HEp-2 infected with
Shealy IEP antigen and 6 ul/cm2 anti-Shealy Ig
tRelative migration velocity of immunoprecipitation peaks of 3 gels
Coomassie brilliant blue
60 ul 14C-glucosamine labeled HEp-2 mock infected IEP antigen and
6 ul/cm2 anti-Shealy Ig
*RMV range of complex of 2 or 3 peaks


IMMUNOPPT.
PEAK
DESIGNATION"











FIGURE 23
Crossed immunoelectrophoretic analysis of the pattern of glycosylated
antigens of the RE strain. Cultures of HEp-2 cells were infected with
Sor mock infected and grown in medium supplemented with
C-glucosamine. Antigen was applied in the first dimensional
electrophoresis, the intermediate gel contained agarose only, and the
reference gel contained 12 ul/cm of the immunoglobulin (Ig) rich
fraction of antiserum to the RE strain grown in rabbit kidney-13 cells
without FCS. Crossed immunoelectrophoresis was performed as described
in Materials and Methods. (A) Coomassie brilliant blue staining of
120 ul of RE antigen. (B) Fluorography of A. (C) Coomassie
brilliant blue staining of 60 ul of HEp-2 mock infected antigen.
(D) Fluorography of C.









A B












c )



C 0












FIGURE 24
Tracing of Figure 23 of the pattern of glycoyylated antigens of the
RE strain. Reference gel contained 12 ul/cmL anti-RE Ig.
(A) Coomassie brilliant blue staining of 120 ul of RE antigen.
(B) Fluorography of A. (C) Coomassie brilliant blue staining of 60
ul of HEp-2 mock infected antigen. (D) Fluorography of C.
Immunoprecipitation peaks labeled: A,C,D,H are nonspecific peaks; c is
common antigen, s is shared antigen; U is unknown antigen.




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID ECUSEKPQY_7452VE INGEST_TIME 2012-03-02T21:39:02Z PACKAGE AA00009122_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES