ANTIGENIC PROPERTIES OF CALIFORNIA

ENCEPHALITIS VIRUSES

by

Joseph Anthony Crookston

A dissertation submitted to the faculty of the University of Utah in partial fulfillment of the requirem9nts

for the degree of

Doctor of Philosophy

Department of Microbiology

University of Utah

June I 1975

SUP E R V I S OR y eo 1\1 1\} 1 T TEE 1\ J) I' R 0 V A L

of a dissertation submitted by

Joseph Anthony Crookston

I h;ln� read this (lisscriatiolJ d�cto:,t�!de�ree.

_J/:Z' 7 � ________ . . Date

I 11:1\,C read this dissertation cIoct oral degree.

f/ · , I , . I , el ! / 7 ; - - -�' -'�';."-":..." .-------"

Date'

;)f1<!

l'Iicmbcr., Super __ i .. ",.·:; COfllmittee

I kn'c re:ld this dis:ilTt:Jt:(JIJ and han� found it to he' of satisfactory quality fnr :\

dlxtoral degree.

. __ )!2{! >�. ______ . __ _

Date

I !J;!\,c read this diss!'rl:l�io" d()(�t ral degree .

q .' 7 . -- -:lJrL�----�--.---

Date

I have re:1d this diss"rlatin:; docloral degree.

('/ /� / '" _ __ � ___ £J __ . __ L.L _________ -- -

Date

� P' /' '/,1 .. J -- .. -----.----.�---- - - --- --._-- .----.- ... -----�--- ---_.-._- .

Pa1..l1 S. 'Ln1iibardi, Ph. D. ]vfcmOCT, SUP("[vjs:)r/ COlnrnittcc

J..1cmb{'!·, Supervisory COfllll:illec

and In\'l� fmlnd it tn h� of sa;isfac[Qry quality [or a

....Paul S. �icholcs. Ph.D.

Mt�:nber, Supe�vi,{)t·y· Committee

dissnt:lliolt :tnd h;\\'l' f(lund it to hi' of �,lIl't,,( tOIY qll:llity

---fJ1({,'�U<;�h".k 0 (;rC��-�l

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'.A � ' ,� ] . .> '.,. . , ... I .. T '1) Ii-/ I) l'!"rVl., \(' . " " " -' , �y . . �i(;Jnh('r .. SIIP'T\'i';(Il), COJl!lIlit 1('1'

.

UNIVERSITY OF UTAH GRADUATE SCHOOL

FINAL READING APPROVAL

To the Graduate Council of the University of Utah:

I have read the dissertation of Joseph Anthony Crooks tOE in its final form and have found that (1) changes suggested by the Supervisory Committee have been completed in the manuscript; (2) reference citations and bibliography are consistent and in an acceptable form; (3) all illustrative materials including figures, tables, and charts are in place; and (4) the final manuscript is satisfactory and ready for submission to the Graduate School.

� -----------------Paul S. Lombardi, Ph.D. Member, Supervisory Committee

Approved for the Major Department

�eP CF'.�---� Lowell A. Glasgow, M.D.

Chairman /Dean

Approved for the Graduate Council

I '- -,, ' //( \ ( ( i (if'" ,/;.;' ;» )) . - , i • _ ,- t�_._/

S�terling M�/ McMurrin, pH.D. braduate Dean

ACKNOWLEDGMENTS

The author wishes to express his appreciation to

Dr. Douglas W. Hill for the contribution he made to th2

author's education. While the effort he extended to pro­

vide guidance and counsel were of inestimable worth in

conducting this investigation, the opportunity to observe

and share his philosophy toward the pursuit of knowledge

and one's education was invaluable in the evolution of the

author's academic attitudes. A feeling of indebtedness

is also held for the suggestions and efforts of Drs. L. P.

Gebhardt, S. Marcus, P. S. Nicholes, and M. Rogolsky. The

time, advice and stimulating discussions offered by Dr. P. S.

Lombardi are especially acknowledged.

The author would also like to express his gratitude

for and acknowledge the support and assistance offered by

his wife, Janice.

Finally, the financial assistance received from the

National Science Foundation in the form of a predoctoral

traineeship is gratefully acknowledged.

TABLE OF CONTENTS

PAGE

LIST OF FIGUHES . . . . . . . . . . . . . . vii

LI ST O}' TABLES . • • · . . . · . x

AB8TRACT • • • . . · . . . . xii

INTRODUCTION . . . . . · . · . . . 1

LITE~ATunE REVIEW . . . . . . . . . · . . . . . 3

I. HISTORY OF THE CALIFORNIA ENCEPHALITIS ARTHROPOD-BonNE VIRUSES • • • • • • • •• 3

I I. A:t'YEIGEIJIC PROPERTIES A!\T]) RELATIONSHIPS AJ,!ONG CALIFO~,ijIA ENCEPHALITIS ARTHROPOD·-

III.

IV.

BORNE VI~USES • • • • • • • • • • · . . . PLA~UE REDUCTION (NEUT~ALIZATION) AS A SEROLOG I CAL TECHNI r~UE • • • • • • • • • •

PLAQUE FOJ1HATION BY CALIFORNIA ENCET-'HA­LITIS VIRUSES • • • .

MATERIALS AND METHODS · . . . . . . I . VIHUS STRAINS . · . . . . . .

II. CELL CULTUllES • · . . . . . . III. ~IEDIA AND SOLUTIONS . · . .

IV. ASSAY OF VIRAL INFECTIVITY

V. ANIMAL IMMUNIZATIONS · . . . . . VI. VIRUS NEUTRALIZATIO:1 STUDIES · . . .

VII. VIRUS PURIFICATION . . . . . . . . . . . VIII. PROTEIN DETEHHINATION •• · . . . . .

6

13

18

20

20

24

28

29

30

31

32

PAnE

34

I. PLAQUE FORMATlnN 31

II. PURIFICATION OF CALIFORNIA E:rCEPIIALITIS VIRUSES • • • • • • • • • • • • • • • 60

III. VIRUS-A~rTIBODY IHTEnACTION STUDIES 66

IV. NEUTRALI~ATION STUDIES 92

DISCUSSION • . . . . . . 116

RE}i'EnE:~CES • . . . . . . . . . . . . . . . . . . 131

VIrrA • • • • • 142

vi

LIST OF FIGURES

FIGURE

1. California encephalitis virus plaque nurruJers as a function of sample dilution .

2. ion of BFS-283(LP) virus in primary embryo fibroblasts . .

3. Replication of BFS-283 (Sp) v ir . .ls in primary chick embryo fibroblasts .

4. Repl ion of San Angelo virus in prlmary chick embryo fibroblasts . . . .

5. R9plicat of Tahyna virus in prlmary chick

6.

embryo fibroblasts .

Comparison California e Melnick's and med ia .. .

single step growth rates of litis virus (30521) in

's minimum essential

7. Replication of BFS-283(LP) virus in a con-

PAGE

40

42

42

43

43

45

tinuous line of baby hamster kidney cells 55

8. Replication of LaCrosse virus in a contin-uous line of baby hamster kidney cells . 56

9. Distrib'..ltion of infectivity following clari­fication of suckling mouse brain-propagated Trivittatus virus by sed tation through 25% sucrose onto a 65% sucrose cushion . 62

10. Distribution of sucrose cushion-clarified, mouse brain-derived v virus infec-tivity following sedimentation to equilibrium in a 25-65 percent sucrose gradient 64

11. Partial purification of mouse brain-propagated San Angelo virus by centri on of a crude suspension through 25-45 percent sucrose (A) gradients and rccentri tion to equilibrium (8) of a peak fraction (#18) through 25-65 percent sucrose 65

FIGURE

12. Influence of the host cell used to propagate San Angelo virus on neutralization by rabbit anti-San Angelo virus serum . 68

13. Neutralization of BFS-283(LP) virus by homolo­gous "early immune II rabbit serum collected 7 days after intramuscular injection of virus . 73

14. Neutralization of San Angelo virus added to previously incubated San Angelo virus-homolo-gous antibody mixtures 75

15. Neutralization of chick cell-propagated San Angelo virus by antiserum pre-incubated with heat inactivated mouse brain-derived San Angelo virus or normal mouse brain material 77

16. Comparison of neutralization of mouse-brain and chick cell-derived San Angelo viruses by rabbit anti-San Angelo virus serum, assayed on primary chick embryo and modified baby hamster kidney cells 82

17. Neutralization of mouse brain-derived LaCrosse virus by rabbit anti-LaCrosse virus serum. 84

18. The effect of initial virus concentration the rate of neutralization of chick cell­propagated San Angelo virus by homologous immune rabbit antiserum.

19. Neutralization of baby hamster kidney and embryo cell-derived San Angelo viruses by bit anti-San Angelo virus serum ..

on

hyper-

chick rab-

20. Neutralization of California encephalitis viruses by mouse anti-BFS-283 virus ascitic fluid . . • .

21. Neutralization of California encephalitis viruses by mouse anti-BFS-283 virus ascitic fluid .

22. Neutralization of California encephalitis viruses by antisera collected from two rab­bits submitted to similar schedules of immunization with BFS-283 virus .

23. Influence of antiserum concentration on the kinetics of homologous and heterologous Ca fornia encephalitis virus n(~utralization

viii

93

95

99

99

100

102

FIGURE

24. Neutralization of California encephalitis viruses by rabbit antiserum to mouse brain-de~ived BFS-283(LP) virus. . . • . . 105

25. Neutralization of California encephalitis viruses by rabbit antiserum to mouse brain-derived LaCrosse virus ..•.. 107

26. Neutralization of California encephalitis viruses by rabbit antiserum to mouse brain-derived Tahyna virus .•..•.•.. • .. 109

27. Neutralization of California encephalitis viruses by rabbit antiserum to mouse brain-derived Jerry Slough virus .••. . •• III

28. Neutralization of California encephalitis viruses by rabbit antiserum to mouse brain-derived Trivittatus virus . • . . . . .• 112

29. Neutralization of California encephalitis viruses by rabbit antiserum to mouse brain-deri ved San Angelo virus •..• .•.. 114

ix

LIST OF 'IIABLES

TABLE

1. Viruses of the Californ encephalitis group included in the study • .

2. Inoculation schedule for preparation of lIearly immune I', lIimmune" and IIhyperimmune " rabbit anti-

21

sera to members of the CEV group 30

3. Influence of temperature during virus sample dilution and adsorption to cells on California encephal is (30521) virus plaque numbers . 37

4. Time required for adsorption of California encephal is virus (30521) to primary chick embryo cells at 25 C 38

5. California encephalitis virus plaque size and quality as a function of sodium bicarbonate con-centration . • . 46

6. Plaque formation by California encephalitis viruses on primary chick embryo cells . 49

7. Effect of init 1 overlay pH on the morphol::Jgy of plaques formed on pri1nary chick embcyo cells by Californ encephalitis group viruses 51

8. Plaque formation by California encephalitis viruses on primary chick embryo cells . 52

9. Comparison of plaque-forming unit titers of Ca lifornia encephali tis gr::Jup virus sllspen:3io!1s when assayed on chick embryo fibroblasts and baby hamster kidney cells . 57

10. Neutralization of San Angelo virus propagated in mouse brain and chick embryo cells by rab-­bit antisera to normal chick cells and chick cell-propagated San Angelo virus 69

11. Neutralization oE mouse brain- and chick embryo cell-derived San Angelo and BFS-283 viruses by rabbit anti BFS-283 virus serum . 71

TABLE PAGE

12. Influence of antiserum dilution on the neu­tralization of mouse brain-propagated San Angelo virus by rabbit anti-San Angelo virus serum . .

13. Enhancement of neutralization of monse bra propagated San Angelo virus by goat anti­rabbit globulin serum following incubation with

78

rabbit anti-S3n A~gelo virus serum 80

14. Effect of trypsin treatment on the infectivity of partially purified LaCrosse virus propa ted in BRK cells and the suckling mouse . 88

15. Effect of phospholipase C treatment on the infectivity of partially purified mouse brain-derived LaCrosse virus 90

16. Residual antiviral antibody activity of rabbit sera after 100-fold dilution beyond pre-deter­mined optimal dilution for neutralization of homologous virus 101

17. Effect of extended time on the neutralization of selected California encephalitis viruses by rabbit anti-mouse brain-derived LaCrosse virus serum .

18. Neutralization of California encephalitis

108

viruses by rabbit anti-Tahyna virus serum 110

19. Influence of extended time and increased con­centration of antiserum on the neutralization of California encephalitis viruses by rabbit antiserum to Jerry Slough virus propagated in suckling mice . 113

xi

ABSTRACT

The kinetics of inactivation by antiserum of several

strains of Cal ornia encephalitis virus were examined by

the technique of plaque reduction. The antisera employed

were ob-tained from rabbi ts following multiple injections of

virus strains pro?agated in the brain tissues of suckling

mice. The viruses used in the plaque reduction studies

were propagated in cultures of baby hamster kidney cells.

This was done to maximize the likelihood that the inter­

action between virus and antibody would involve virus­

specific determinants and minimize the possibility of

interference by antibodies directed against host cell com­

ponents. An antiserum dilution was predetermined for each

homologous virus-antiserum combination so that similar rates

of inactivation resulted. That is, when the results of

each of the homologous neutralization reactions of several

viruses were compared graphically, the curves were found to

coincide. When attempts were made to neutralize heterologous

virus strains with each of these sera at their unique dilu­

tions, three types of reaction were apparent. In some, the

rates of inactivation of certain heterologous viruses were

similar to that of the homologous virus, that is, inactiva­

tion was immediate u.nd without a lag following u.dmixture.

In the second type of cross-reaction, a significant lag

preceded neutralization, while in the third type of reac­

tion no indication of cross-reaction could be detected.

The demonstration of a kinetic lag preceding neutralization

supported the concept of multihit inactivation kinetics

rather than single hit kinetics. The differences between

the two types of heterologous cross-reactivity suggested the

significance of the configuration of the antigenic deter­

minants in determining the ability of a cross-reacting

antibody to "recognize" a portion of the determinant.

In the course of preliminary experiments it became

apparent that phenotypic differences arose in genotypically

identical viruses when different types of host cells were

used to propagate the viruses. A significant persistent

fraction of surviving virus was noted when neutralization

experiments were conducted with viruses propagated in

suckling mice. This fraction of non-neutralizable virus

could not be reduced by any treatment of the virus suspension

or the antiserum or by using different assay cells. Attempts

to modify the surfaces of mouse brain-derived viruses by

enzymatic digestion with trypsin, phospholipase C and

neuraminidase did result in the detection of differences in

the surfaces of viruses grown in these cells and in baby

hamster kidney cells. However, treatment of mouse brain­

propagated viruses with these enzymesilid not enhance their

neutralizability.

xiii

As a part of this study, experiments were also con­

ducted in which variables influencing plaque formation by

California encephalitis viruses were investigated. The

plaquability of the viruses was found to be extremely

sensitive to the pH of the agarose overlay. In contrast

to other viruses that exhibit pH sensitivit s, California

encephalitis viruses did not produce plaques when the

initial pH of the overlay was greater than 7.5. Optimum

conditions for plaque formation by most of the viruses Were

noted when the initial agarose overlay pH was between 7.0

and 7.2.

xiv

INTRODUCTION

The data obtained from the techniques previously

appl d to the examination of antigenic differences and

similar ies between viruses of the Ca fornia encephalitis

group of arboviruses have been rpr~ted as an indication

that only minor differences exist between many of the

viruses. Considerable antigenic overlap was apparent when

immunodiffusion and immunoelectrophorectic techniques were

util to examine and compare members of the group:

Several virus strains Were indistinguishable by these methods.

Complement fixation comparisons were more discriminatory,

although heavy reliance had to be placed upon the reproduci­

bility of single two-fold serum dilution titer differences.

Other attempts desi d to further the understanding of the

antigenic relationships of these viruses have relied upon

modification or refinement of the abovementioned techniques

instead of the application of methods in which cross­

reactivities are minimized. This situation prompted an

attempt to gain further ight into the nature of the

viruses' intragroup antigenic relationships by studying the

e ct of specific homologous and heterologous antiviral

antibodies on the ability of different virus strains to

success fully infect cells. AeJdi tionally , conditions which

affected neutralization of the viruses as well as factors

that influenced the production of plaques by the viruses on

different cells were studied.

2

LITERATURE REVIEW

I. HISTORY OF THE CALIFORNIA ENCEPHALITIS ARTHROPOD-BORNE VIRUSES

-The first lation of a virus (Bakersfield Field

Specimen #91, BFS-91) belonging to what has subsequently

been named the Ca fornia encephalit (CE) group of

arthropod-borne viruses was made in 1943 from mosquitoes

during an encephalitis epidemic in southern California

involving Western equine encephalitis and St. Louis enceph-

alitis viruses (Hammon et al., 1945). The recovery of this

virus in North America was unique that no known disease

could be associated with the agent at that time, although

the sera from several animal species, including man, possessed

antibodies which neutralized the ctivity of the virus.

In 1944 two additional isolations of the same virus (BFS-283

and BFS-395) were made from mosquitoes in the same locality

(Hammon and Reeves, 1945). Early serological surveys

implicated small mammals (ground squirrels and rabbits)

as reservoirs of infection in nature (Hammon & Reeves,

1947) . Infections produced experimentally in these

animals were inappareni, although a demonstrable viremia

occurred (Hammon & Reeves, 1952b). These results, as well

as subsequent observations, indicated thilt, as a parasite,

the virus was well adapted to its hosts (Sudia et al.,

1971). Serological stud s of the seril from patients with

undiagnosed encephalitides from areas outside California

(Washington, Colorado, North Dakota, Kansas, and Japan)

4

did not provide evidence for the presence of this virus in

these areas, so the agent was assumed to have a very limited

geographic distribution (Hammon & Reeves, 1952a). Corrobora­

tive findings from the study of a number of domestic and

wild animal spec s supported this conclusion. In North

Dakota in 1948 an antigenically related but distinct virus

was recovered from Aedes trivitattus mosquitoes (Hammon et

al., 1952b). This virus was given a designation reflecting

the species of mosquito from which it had been isolated;

most other California viruses have names associated with

their geographical origin. Evidence of more extensive

distribution of these viruses or viruses exhibi ng close

serological relationships was presented by the isolation of

a strain (Melao) from mosquitoes in Trinidad in 1955 (Spence

et al., 1962) and again in Brazil in 1957 (Causey et al.,

1961). Similarly, repeated isolations of another virus

were made from mosquitoes in Mozambique in 1959 and 1960

(Kokernot ct al., 1962). This strain received the designa­

tion Lumbo virus. The presence of CE virus in Europe was

ascertained in 1958 when Tahyna virus was isolated from

mosquitoes in Czechoslovakia (Bardos and Danielova, 1959).

SerologicCll surveys of humans in different Europe::1n areas

5

suggested an endemic status of a California virus throughout

central Europe (Bardos, 1960: Bardos & Sefcovicova, 1961).

In the United States, no isolations were made after Trivi­

tattus virus until 1958, when San Angelo virus was found

in mosquitoes in Texas (Grimes et al., 1962).

California encephalitis viruses were detected only in

mosquitoes until 1959, when the Snowshoe hare strain was

isolated from the blood of an animal described as II • •

an emaciated, rather sluggish snowshoe hare .•. 11 (Burg­

dorfer et al., 1961). The following year, isolations of

Tahyna virus were made from the sera of two apparently

healthy humans (Likar & Casals, 1963). These were originally

defined as new isolates and referred to as Trojica viruses,

although subsequently they were found to be identical to

Tahyna virus (Taylor, 1967). In the United States, the

first human isolate (LaCrosse) was recovered from the brain

t sues of a 4-year-old child who died of meningo-encephalitis

in 1960 (Thompson et al., 1965). Additional mosquito isola­

tions have given rise to other partially characterized

California viruses, e.g., Jamestown Canyon in Colorado

(1961; Taylor, 1967), Jerry Slough in Southern California

(1963; Taylor, 1967), and Keystone in Florida (1964; Bond

et al., 1966). Of interest is the observation that no CE

viruses Were isolated in California during the 20 year

period following the initial isolation, although a large

nUI~er of mosquitoes Were trapped and examined each year

6

(Gresikova et al., 1964). Since 1965, California group

viruses have been found in at least 26 additional states

and the number of isolations have continued to increase

each year (Sudia et al., 1971).

Coincidental with the increasing frequency of virus

isolations has been the number of human infections detect-

ed, d gnoseo and reported. Hammon and Reeves (1952a)

described three clinical cases presumably attributable to

these viruses in 1943-44; the next human case was not

recorded until 1963, a significant span of 20 years (Quick

et al., 1965). Available data suggest that the incidence

of human involvement was, at best, limited before 1964.

Beginning in that year, epidemics were reported from

Indiana (Marshall, 1964) and Ohio (Spencer, 1965) and

subsequently from Wisconsin, Minnesota and Iowa (Sudia et

al.,1971).

II. ANTIGENIC PROPERTIES AND RELATIONSHIPS AMONG CALIFORNIA ENCEPHALITIS ARTHROPOD-BORNE VIRUSES

When the increasing prevalence of Cal ornia enceph-

alitis viruses became apparent, attempts were made to

clarify the antigenic relationships of these viruses among

themselves and other arthropod-borne viruses. The earliest

efforts Were restricted by the techniques then available

and by a limitation shown by some of the viruses: Despite

repeutc:c1 efforts, hemagglutination could not be de1110n-

stratcd for some virus strains. Thus, none of the MelLio

7

strains (Spence et al., 1962: Whitman and Shope, 1962), the

Lumbo (Kokernot et al., 1962) or Snowshoe Hare (Burgdorfcr et

al., 1961) viruses were initially thought to "produce" hema­

gglutinins. Conversely, Tahyna (Bardos et al., 1961),

LaCrosse (Thompson et al., 1965) and BFS-283 (Whitman and

Shope, 1962) viruses were shown to exhibit hemagglutinating

activity, although the antigens were characteristically

low-titred or irregularly extractable. More recently,

techniques have been described which have resulted in the

isolation of hemagglutinating preparations from all Cali­

fornia viruses studied. These methods include trypsin

treatment of sucrose-acetone-prepared antigens (Hannoun,

1968) and sonication followed by trypsin treatment (Ardoin

et al., 1969). Less vigorous procedures have been required

to obtain satisfactory complement-fixing antigens (Hammon and

Sather, 1966). This property proved advantageous when com­

plement-fixation (CF) tests were observed to be more dis­

criminatory than neutraliza on (assayed by inoculation of

virus-serum mixtures into suckling or weanling mice) in the

differentiation of strains of CE virus. For example, anti­

serum to LaCrosse virus neutralized 2.9 logs of the homo­

logous virus, 2.8 logs of BFS-283, and 2.9 logs of the Snow­

shoe Hare virus. When assayed by CF, the titer of the

homologous virus-serum mixture exceeded those of the heter­

ologous mixtures by a factor of 4 (Thompson et al., 1965).

Other studies have corroborated and extended this observation

(Hi:.lmmon et ul., 1952b; Newhouse et a 1., 1964; Bond et ill.,

1966) .

The in vivo measurement of neutralization is not with­

out advantage, however. Although the greater apparent

specificity of CF resulted in reliance upon that test for

estimation of the antigenic relationships among CE viruses,

the relatively short time period over Which antibod s

spe fic for CF antigens can be detected in sera limits the

testis utility. Hammon and Reeves (1952a) found 32 of 294

human sera capable of neutralizing BFS-283 virus but none

had demonstrable complement-fixing activity. Similarly,

44 of 118 human sera examined by Gresikova et al. (1964)

exhibited significant neutralizing capacity While none was

able to fix complement. Recent clinical studies have pro­

vided evidence indicating that antibodies capable of inter­

acting with hemagglutinating antigens are short-lived,

increasing rapidly to maximum titer and then decreasing in

titer to undetectable levels after a short time (Quick et

al., 1965). Antibodies to complement-fixing CE virus

antigens increase in activity more slowly but are also

short-lived (Bond et al., 1966). In contrast, the neutral-

izing activity of sera increases rapidly and is maintained

at detectable levels for a much longer tim;:'! (Chun et al.,

(1968).

The unique antigenic character of the original CE

isolate wi t.h respect to other then known arboviruses was

8

first indicated by the experiments of Hammon, Reeves and

Gillindo (1945); no cross-reactivity was noted with any

other virus. Subsequently, Espana and Hammon (.l948)

demonstrated a subtle relationship between BFS-283 and

St. Louis and Japanese encephalitis viruses. In the CF

(but n,:)t neutralization) test, antiserum t() the California

virus reacted with undiluted, benzene-extracted an~igens

of the heterologous viruses; no relationship could be

detected in the recipr8cal tests.

9

The L' .. un'ba strain fro~ South America was foun:] to be

neutralized by Bwamba fever virus antisera (Kokernot et al.,

1962). The latter, isolated from humans in Afri2a, is the

prototype of a small but distinct serolog 1 arbovirus

group (Smithburn et al., 1941). The re ionsh was only

detectable by neutralization and was unidirecti~nal.

An antigenic similarity CE group viruses and members

of the B"dnyam\vGra virus l)"roup \Vas su!]ges ted 'tlhen G'Jaroa

virus was Sh·:)Wll to exh ibi t features of both groups (Whi tman

and Shope, 1962). Th virus has been classified as d

llle::n~"J2~ of the Bunyam\v[=cd group be:::;aus:~ of cr::-' cross-reactions

with other representatives of the same group. Neither

Guaroa virus nor its antiserum reacted with Bunyamwera virus

antisera or the respective viruses in hemagglutination

inhib ion or neutralization tests. Conversely, the

relationsh between Guaroa virus and the California viruses

could not be detected by CF studies (Sather and Harrunon,

10

1967); cross-reactions between the viruses and heterologous

sera were apparent only in neutralization and hemagglutina­

tion inhibition studies (Whitman and Shope, 1962).

Within the California group of arthropod-borne viruses

the antigenic relationships appear extremely complex when

examined by the CF technique (Sather and Hammon, 1967).

The high degree of cross-reactivity has been interpreted as

an indication of relatedness attributable to "antigenic

instability" (Hammon and Sather, 1966). In several

instances virus isolates have been designated as distinct

strains, although their specific antisera reacted to equal

titer in the CF test with some heterologous virus antigens

or d tinguished the homologous virus from some of the

heterologous viruses by only a two-fold dilution difference

(Sather and Ham~on, 1967). These d iculties notwithstand-

ing, the California virus group was tentatively organized

into 11 subgroups, eight representing the viruses alluded

to above that had been isolated in the United States.

Murphy and Coleman (1967) applied the Ouchterlony

technique of immunodiffusion to the serological study of

these viruses, using infected suckling mouse brain tissues

as the source of antigen. Their results were agreement

with the complement fixation studies of Sather and Hammon

(1967). Subsequently, 'Wel1ings et ale (1970) were able to

increase the speci city of the immunodiffusion technique

by e l.iminat.ing (through adsorptio:1) non-viral antigen-antibody

11

reactions and by using antisera obtained from inoculated

animals within two weeks of the primary injection. In 6

of 9 instances these sera were strain ("type") specific

while sera collected after subsequent injections possessed

broader group specificit s. These sera were also used to

examine the electrophoretic properties of the viral antigens

and the same specificities were noted (Wellings et al.,

1971) . Sera obta d after a series of virus injections

gave rise to multiple arcs of precipitation folloifJing

electrophoresis of the homologous or heterologous viruses.

Preliminary adsorption of these sera with a heterolog0~s,

cross-reacting virus did not necessarily eliminate arcs

present in the homologous virus-antibody reaction. Based

on the results of these experiments, it was suggested that

the classif tion of the California group be revised to

contain three subgroups, represented by BFS-283, Trivittatus

and Keystone viruses, the remaining members to be considered

as strains of BFS-283. Guaroa virus reportedly shared

"minor antige:1ic determinants" with all members tested

except Trivittatus, with which two "antigens" were found In

common.

Calisher and Maness (1970) screened imm'.lne sera from

several host sources representing a variety of imnunization

schedules by complement fixation, hemagglutination inhibi

tions, and neutralizatio:1 tests. Selecting sera [or titer,

specificity, availability and other features, the cross-

reacti v i tics of group members were co:npared by the d :::rJ.b Ie

diffusion-in-gel tech~ique. Reactivity w~s determined by

the density of the precipitin arc, "0" indicClting no

reaction, "4" indicating "maximum intens i ty and a narro'''''

precipitin arc". In these experiments S~owshoe Hlre virus

12

could n0t be distinguished from seven other viruses while

BFS-283 and LaCrosse viruses were only marginally different

from each other and five oth'2r viruses. The precipitin

lin·2s 0f th2 hO:1101090US react ions of these v iruses were

interpreted as 114+" arcs whereas the pre pitation inten-

s it ies of the six heterolog'')us v lruses in each caS~2 V/!2re

reported as "3+". Mela::::> and Trivittatus viruses exhibited

absolute specificity by lacking cross-reactivity with other

group me;:nbers and were sugg-ested as sub·-grol.lp prototy?es.

Three o:'her suh9:LOUPS 'Nere proposed: These consisted of

B23-283, SnJwshJ'2 Hare and LaCrosse viruses; Jamestown

Canyon and Jerry Slough viruses; and rrahyna and Lum:::>o

viruses. San Ange 10 v lrus "vas unique in pro\! ld ing an

"an-tigenic bcidge" between the lJroups containing BfS-283

anrJ Jamestown Canyon v iruse:3.

P:cesently, this group of viruses hilS be:::;!) separated

into three complexes accor-d ing ·to the Sl1bcolTI!,l:l. t te(~ on

Irrunl1nolo9ical R,~lationships Among CataloguGc] Arth_~:-t)pod-borne

Viruses (citecl in Sudia et al. I 1971). Th(~se c,)jnple:(c-~s

includ(~ Tri\l.ittatus, Melao and BFS-283 as prob::>types ,vh3_1<.:::

the rema. i-,lin(] (characl:eriz(~d) members are regc:u,"'(]ed as !3ubtypC:3

of BPS -283. Lllmbo ancl Jer ry S 10ugh virusc~s are cons ide! Led

to be var iants of Tahyna and lJamesto\Vl1 Canyon viruses,

respectively.

III. PL1\Q:JE REDUC'f ION (NEU'fIV\.LIZAT ION) AS A SEROLOGICAL TECHNIQUE

Casals (1957) demonstrated that in the study of arbo-

viruses, compleroont fixation occupies an intermediate

position between hemagglutination inhibition and neutral-

ization (assayed in mice) in specificity, i.e., lack of

cross-reactivity. Although each of these methods has as a

feature a quantal nature, neutralizatio~ was considered to

13

be the m8st specific or least cross-reactive. This increased

specificity is, in part, dependent on the route by which the

assay animals, usually mice, are challenged. Numerous

studies have also indicated the greater sensitivity of these

in. ~ivQ. neutralization tests, especially When mice are

inoculated intracranially rather than intraperitoneally

(Burgdorfer et al., 1961; Porterf Id, 1962a; Kunz et al.,

1964). Further, although no differences might be expected

when C:l virus-antiserum mixture is assayed in vivQ. or in

vitro (Porterfield, 1962a), the latter has generally been

reported to be more sensitive as well as specific for detec-

tion of antigenic similar ies and d ferences between viruses

(Porterf Id, 1962b; Daniels, et al., 1961).

Utilizing the quantitative techniques of plaque forma-

tion, Dulbecco ct al. (1956) demonstrated that the

14

ncutrillization of an infectious virus suspension could be

followed kinetically and, hence, the rate of inactivat.ion

could be ascertained. McBride (1959) presented evidence to

indicate that the rate of inactivation of a poliovirus

serotype by its homologous antiserum was always greater than

that observed when a heterologous serum was used. This

observation has been co~firmed by a number of other investi­

gators employing a variety of viruses and antisera (Ashe &

Scherp, 1963; Ozaki & Tabeyi, 1967). Inherent to this

approach is the idea that the degree of antigenic relatedness

will be reflected in the slope of the plotted neutralization

curve, the curve of the ho~~logous virus-serum reaction (as

a function of time) being characterized by a greater slope

than any heterologous reaction and the curves of heterologous

combinations reflecting the relative antigenic similarities

among different viruses. Because the slope of a curve can

be described mathematically, attempts have been made to sum­

marize the antigenic relationships of viruses in quantita­

tive terms (McBride, 1959).

Viral neutralization is presumed to be a bimolecular

event (or series of bimolecular events) in which one or more

molecules o~ antibody interact with an infectious virus

particle in some manner to bring about inactivation. Because

the serum concentration is usually great enough that the

chungQ in the number of antibody molecules is negligible,

ncutrQlization hilS generally been described in terms of

first order (monomolecular), rather than second order,

chemical kinetics. Thus,

dV - dt = kV (1)

That is, the change (decrease) in virus concentration with

respect to time -~~ is proportional to the concentration

of the virus (V) or equal to the product of the virus con-

15

centration and a velocity constant, k. When this relation-

ship is integra ted between two virus concentra tions V and o

Vt at zero minutes and t minutes, respectively, k can be

calculated from the following:

(2 )

Dulbecco et ale (1956) utilized equation (2) in modi-

fied form to include the influence of the antiserum:

K(t-to ) = In Vo ( 3 ) D Vt

Here, K represents the m~dified velocity constant and D,

the serum dilution. Graphically, this modified first order

equat.ion resembles its parent form, the plot being linear

and negative in slope (in the presence of excess antibody).

Whereas true first order reactions are characterized

graphica lly by linear exponential decline, m~st virus-

antibody systems depart radically from this by approaching

a horj.zontal asymptote as the reaction proceeds (Lewenton-

Kriss and Mandel, 1972). This departure from first order

kinetics has been described as non-neutralizable virus

(Mandel, 1961), a persistent fraction (Dulbecco et al.,

1956) a protected fraction (Lafferty, 1963a) and simple

equilibrium between dissociated and undissociated virus

and antibody (Burnet et al., 1937). The size of the per­

sistent fraction varies between antiserum preparations

(Lafferty, 1963b) as well as within single antiserum pre-

16

·paratio:1s collected at different times during the immuniza­

tion process (Lewenton-Kriss and Mandel, 1972); between

virus preparations (Lewenton-Kriss and Mandel, 1972); and

between cell species up:Jn which neutralizatio:1 is being

assayed (Kjellen and Schlesinger, 1959).

While the explanation of the persistent fractio:1 has

not been unequivocally ascertained, its existence is pre­

dicted by most models regarding virus-antibJdy interactio:1

(Lafferty, 1963a; Lewenton-Kriss and Mandel, 1972). Neu­

tralization is considered to be a tWo-step reaction 1n

which the virus particle first becomes reversibly sensitized

by interactiO:1 with one or more molecules of antibody and

then beco~es stabilized (neutralized) or continues to exist

as an infectious virus-antibody complex. Stabilization has

been attributed to binding of the second combining site of

an antibody molecule to a second antigenic determinant on

the same virus particle (Lafferty, 1963b); to the attach­

m~nt of an ilntiboc1y ffi:Jlccule t_o anyone of several "critical"

17

sites present on the virus surface (Rappaport, 1970); or to

the attachment of a final antibody molecule to a "critical

area" (composed of "groups of antigen-antibody complexes"

but otherwise similar to any other area of the virus sur­

face; Westaway, 1965b). The existence of infectious com­

plexes of virus and antibody has been demonstrated both in

vivo (Notkins et al., 1966) and in vitro (Ashe & Notkins,

1966). That antibody is associated with non-neutralized

virus is indicated by the secondary neutralization resulting

from the addition of specific antiglobulin.

A second point of departure from linearity, manifested

by an initial lag phase, has also been described but for

fewer systems. Lafferty (1963a) described a "shoulder" which

preceded the linear, exponential loss of infectivity follow­

ing mixture of virus and antibody. Although this lag was

only apparent at temperatures below 37 C, the same phenomenon

has been reported when antibody was present at low concen­

tration (Burnet et al., 1937; Kalmanson et al., 1942)

and when 195 antibodies were employed (Philipson, 1966).

The demonstration of a lag in the kinetic study of virus­

antibody interaction suggests "multiple hit" neutralization

kinetics, that is, the requirement for more than one

molecule of antibody to neutralize a virus particle (West­

away, 1965b). Experimental evidence for this has been

presented by Gard (1957), Philipson (1966), and Wallis and

Melnick (1970). The hypothesis that neutralization is

normally effected by a single molecule of antibody (single

hit kinetics) hus, however, received more popular support

(Rubin and Franklin, 1957; Lafferty, 1963b; Lewenton-Kriss

and Mandel, 1972).

IV. PLAQUE FORMATION BY CALIFORNIA ENCEPfffiLITIS VIRUSES

18

Several investigators have described conditio~s sup­

porting formation of plaques by members of the California

encephalitis group of viruses. Primary cultures of cells

and continu~~s cell lines have bea~ studied b~t limitatio~s

have been reported for most of these. Some of the diffi­

culties include length of incubation time required before

plaques large eno~gh to detect are formed, poor efficiency

of plaquing and inability to extend the use of a particular

cell to all CE strains.

Among primary cell cultures, thQse of chick embryo cells

have been used to plaque Tahyna virus (Porterfield, 1960)

and BFS-283 (Crookston et al., 1968): Duck embryo cells also

supported plaque formation of these viruses as well as Snow­

shoe Hare and a New Jersey isolate designated as South River

virus (Henderson and Coleman, 1971). Plaq~es were not

produced on duck embryo cells by Trivittatus '=>r Melao while

LaCrosse and Keystone viruses gave rise to faint, unco'-lntable

plaques. Henderson and Coleman (1971) also exa~in'2d

plaq'-1c forination on primary ham3ter embryo, h3.'nster kidney

und rh2sus monkey cells: With each of these at least one

19

of the above-mentioned viruses did not produce plaques while

the plaques of some of the strains that did form were too

faint to be counted. Hamster embryo cells were most suscep­

tible to plaque formation by these viruses (except Keystone),

however, the plaque titers observed were at least one log

lO'wer than those determined by suckling mouse intracranial

inoculation.

Continuous cell cultures have also been shown to sup­

port plaque form3tion by CE viruses. Berghold and Maz~ali

(1968) were able to plaque Melao virus on baby hamster kid­

ney cells (BHK-21), green monkey kidney cells (VERO), and

rhesus monkey kidney cells: no other members of the group

were studied. Stirn (1969) used VERO and rhesus monkey

kidney (LLC-MK2 ) cells and was able to demonstrate plaques

for every CE virus that he studied. The tim:; necessary

before plaques became visible varied between 2 and 10 days,

while maximum plaque diameters reached between 16 and 19

days was 1-15 mm. Henderson and Coleman (1971) studied

plaq',18 formatio:1 on BHK-21, porcine kidney (PK-13), HeLa,

green m~nkey kidney, KB and human diploid lung cells.

Only Blffi-21 cells supported plaque formation of all the

viruses tested. In each case suckling mouse inoculation

indicated higher titers than were obtained by plaque

quuntitation.

MATERIALS AND METHODS

I. V IRlJS S'fR,\ INS

The strains of California encephalitis virus used

throughout these stud s were obtained from several

sources. The virus designation, passage history at time

of receipt and the individual and laboratory from whom

each virus was obtained are indicated in Table 1.

The virus designated "3052111 was isolated on June 22,

1965, from Aedes dorsalis mosquitoes at Blue Lake, Utah.

Hemagglutination inhibition, complement fixation and neu­

tralization tests, the latter in mice, were used to

identify the virus as being closely related to BFS-283

(Crane et al., 1970).

Upon receipt, each strain of CE virus was inmediately

inoculated intracerebrally into suckling mice. When the

animals became moribund they were frozen at -70 C. After

thawing, the brains were removed and mixed with phosphate­

buffered saline (PBS) to obtain a 10 percent (vol/vol) suspen­

sion. Dilutions of this material were inoculated onto mono­

layers of chick embryo cells and assayed for plaque formation.

Plates containing fewer than 10 plaques were used to obtain

plugs from the agarose above characterist.ic plaques:

21

TABLE 1

VIRUSES OF THE CALIFORNIA ENCEPr~LITIS GROUP INCLUDED IN THE STUDY: VIRUS DESIGNATION, PASSAGE HISTORY AND SOURCE.

Virus

BFS-283

30521

LaCrosse (LaX)

Tahyna (stra 92, TAH)

Snowshoe Hare (SNH)

Jerry S18ugh (BFS-44 70, JS)

San Angelo (20230, SA)

Tr i vi-t ta t:1S (7941, TVT)

Jamesto#D Canyon (JC)

Melao (Tr9375, MEL)

Keystone (KEY)

Passage

MBP-8, HP-l*

MBP-3

MBP-8

MBP-20

MBP-14

MBP-5

?

MBP-3

MBP-5

MBP-3

?

Dugway Prov Grour}rJs I

Utah (K.L. Smart)

"

Rocky Mounta Lab Hamilton, MJntana (L.A. Thomas)

II

II

"

..

"

Publi2 Health Service Lab., Greeley, Colorado (J.S. Lazuick)

Center for Disease Con­trol, Atlanta, Georgia (G.E. Sather)

Walter Reed Army In­stitute of Research Washington, D.C. (J.M. Dalrymple)

* MBP mouse brain pass; HP = hamster pass.

22

these were mixed with PBS and inoculated into litters of

suckling mice. This plaque purification procedure was

repeated two additional times; after the third passage into

suckling mice, stocks were prepared by inoculating int~a­

cranially several litters of the same animals. These stock

pools consisted of 10 percent (vol/vol) infected mouse brain

suspensions in PBSi cellular debris was removed by low speed

centrifugation. The preparations were dispensed and stored

at -70 C. Viruses propagated in different kinds of cells

(chick embryo or baby hamster kidney) were obtained by

infecting cell cultures of each of these with a mouse brain

virus preparation and collecting the supernatant medium after

infection. These virus-containing fluids were clarified by

low speed centrifugation, dispensed into one ml aliquots and

stored at -70 C.

II. CELL CULTURES

The techniques described by Dolana (1968) were followed

in the preparation of primary chick embryo cells. Ten day

old chick embryos were aseptically removed from eggs and

placed in a sterile Petri dish containing cold phosphate­

buffered saline without magnesium or calcium salts (PD, see

belo\v). After removing the head, limbs and viscera from each

eniliryo, the remaining material was washed 4 times in cold

PD, drained and minced with scalpels. These minced tissues

were repeatedly washed with cold PD until red blood cells

could not be detected in the supernatant fluids. The

washed, minced tissues were digested by repeated 5 minute

exposures to 0.25 percent (wt/vol) trypsin dissolved in

23

PD. After each incubation with the enzyme the supernatant,

containing dispersed cells, was filtered through 6 layers

of cotton gauze and collected in chilled Melnick's growth

medium (see below) containing 20 percent (vol/vol) calf

serum. The latter was relied upon to prevent continued

trypsinization. When sufficient numbers of cells had been

collected, the suspension was centrifuged, the supernatant

decanted and the pellet of cells resuspended in fresh growth

medium. The cell concentration was determined by hemocyto­

meter count and enough growth medium was added to dilute

the cells to a final concentration of 1 x 106 cells per mI.

Five ml of this suspension were added to each 15 x 60 mm

plastic Petri dish (Falcon Plastics, Oxnard, California).

After 40-48 hour's incubation the cells were confluent and

were used to assay viral infectivity.

A mutant strain of the continuous line of baby hamster

kidney cells (BHK-2l/C13) was also employed. This cell,

designated BHK, was provided by J. C. Taylor and was obtained

by treatment of the parent clone with nitrosoguanidine

(N-methyl, N'nitro-N-nitrosoguanidine, Aldrich Chemical Co.,

Milwaukee, Wisconsin) and subaequent selection procedures

involving dependency upon added thymidine, hypoxanthine and

folic acid. The mlltant clone of cells was characterized

by ,lluch slower acidification of growth med iUIll than was th::!

wild type cell. Cells used for infectivity assays or

propagation of pools of virus were cultured in 250 ml

plastic tissued culture flasks (Falcon Plastics, Oxnard,

California). Confluent monolayers were remaved from the

plastic surface and dispersed by a trypsin-EDTA solution

(ATv, See below) and then suspended in Eagle's minim.llll

essential mediu:l1 (MEJi1, see below) supplemented with ').2

24

p2rcent Bactotryptose (Difco, Detroit, Michigan) a~d 10

percent (vol/vol) calf serum. The cell concentratio~ was

adjusted to contain 1.5 x 105 cells per mI. Five ml of the

cell suspension were added to each large flask. After 4)-

48 hO;..1rs of inc'ubation, cells had reached confluency.

III. MEDIA AND S'JLU'rIO~S

Melnick I S complete gcovvth ;l1ediulTI VJ3S ~..lsed for cl.llti­

vatio~ of chick embryo cells, while E3.g IS minim'..1:l1 essen­

tial medium (MEJv1) was '..1sed for propagatio~ of baby ha~;3tei:'

kid:1ey cells. Th2 latter mea iulU was reconst itLlteeJ from ':h2

?owelcred [orin (Grand Island Biolo9ical Co., Gra:1d Island,

Ne'w York) with rjistilled 'dater a~d was supplemented '/Jith 10

percent (vol/vol) calf serum, 0.2 percent (wt/vol) tryptose

(Difco, Detroit, Michigan) and 0.35 gil sodium bicarbonate.

Th,::! complete medium \-JdS steri 1 ized by Seitz filtratiO'1

tlll-OiJ(J~1 type ST ster i 1 iz ing f i 1 ters (Here ule s Fi 1 tor Corporu­

tion, S21n Francisco, California).

25

Each Ii tor of Melnick I s growth medium was prepared as

follows:

lOX Hank1s balanced salt solution.

Calf serum

Lactalbumin hydrolysate (Nutritional Biochemical Corp., Cleveland, Ohio)

NaHC03 (0.035 g/ml stock solution)

CaC12.2H20 (1.325 g/ml stock solution)

Penicillin

Streptomycin

Distilled H20 .

100 ml

50 ml

5.0 g

10 ml

1.4 ml

105 units

850 ml

Calf serum was collected at a lo~al slaughterhouse and

processed or was purchased from Grand Island Biological Co.

Each lot of serum was tested for cytotoxic and antiviral

activity before use. The completed medium was sterilized

by Seitz filtration.

Concentrated stocks of Hank1s balanced salt solution

contained the following chemicals in each liter of lOX

solution:

NaCl

KCl

MgS04 ·7H20 .

KH2 P04 •

Na 2 HP04

Glucose

Phcno 1 Reel •

80.0

4.0

2.0

0.6

0.48

10.0

0.2

9

9

9

9

9

9

9

26

For prop~gation of large pools of virus Mel~ick's

gI":)VlLh tn:l(li!.Hn V·F1S u~:;ed after slight modification: 'llhe calf

serum concentration was reduced to 2 percent (vol/vol) and

phenol red was not included. Tryptos~3 (2 gil) was added

when the med ium \,.,as to be used wi th baby hamster kidney

ce lIs .

. The overlay used during the assay of viral infectivity

consisted of MSM, 5 percent (vol/vol) calf serum, 0.2 per-

cent (wt/vol) tryptose, 0.02 M ·tris (tris (hydro~{ymeth:!l)

aminornethane I Nutri tional Bioclv::;mical Corp. I Cle-"eland I

Ohio» and 0.4 percent (wt/vol) agarose (Van Waters a~d

R0gers Scientific, San Francisco, California). The additi8n

of agarose contributed to acidification of the overlay; thi3

was anticipa.ted \vhi1e init lly adjusting the pH of the tris

SOl11tion.

Ph:::;,sphate-buEfered saline (PBS, pH ).2) was used to

wash cells and, after addition of 0.2 percent (wt/vol)

b:::;,vine serum albumin (BSA, Fraction V, Calbiochem, Los

A'1tj'21es, California) was also USGd as tht~ virus d i 11ent.

the following (per liter):

NaCl . 80.0 9

KCl 2.0 g

N ~-IP') . a 21 - . 4 11.4 '3

KH2 P04 . 2.0 9

fro prepare PBS, the following were added to 100 ml of

the above lOX solution:

MgC12·6H2° (0.1 g/ml stock solution) 1.0 ml

CaC12·2H20 (0.13 g/ml stock solution) 1.0 ml

27

Penicillin · . . 105 units

Streptomycin . . . · . . . . . . . . . . 105 ug

Distilled H2 O . . · . . . 900 ml

These solutions were also sterilized by Seitz filtra­

tion. Phosphate b~ffer (PD, pH 7.0) was prepared as above,

except that calcium and magnesium salts were not incorpor­

ated. This solution was used during trypsinization

procecLJ.res.

Tris-b'.J.ffered saline (TBS) was used in 2urificatio:1

procedures involving sucrose gradient centrifugation and

consisted of 0.01 M tris, 0.001 M EDTA (ethyle:::1,::=(diamin,::=)

tetraacetic acid, J.T. Baker Chemical Co., Phillipsburg,

New Jersey) and 5.85 gil NaCl. The desired pH was estab­

lished by the addition of 12 N HCl.

Chick e~)ryJ cells were freed from tissues and dispersed

from monolayers with a solution of 0.25 percent (wt/vol)

trypsin (Difco) in PDi the pH 'lias adjusted to 7.4 with 2 N

NaQn. Baby ha~ster kidney cells (BHK) were dispersed by a

solution containing trypsin and versene (ATV). Each liter

conta in2d th'2 following:

Ni.lCl

KCl .

8.0 g

0.4 g

28

Glucose . 1.0 9

NaHC03 . . . . 0.58 9

Trypsin 0.5 9

EDTA . . . 0.2 9

The presence of virus plaques on monolayers was detected

by the addition of a neutral red s01ution. Each liter of this

solution contained 0.2 9 neutral red and 8.5 9 NaCl.

IV. ASSAY OF VIR~L INFECTIVITY

Two techniques were used to quantitate th3 infectivity

of virus preparatio~s: Suckling mouse titration and plaque

assay. The assay in mice was limited to a determination ~f

th'3 di luti:Jll 1")£ a virus sample that was letha.l fo::- 50 ?'2r­

cent of the inoculated mice (LDSO). The LDSO was determined

by the method of Reed and Muench (1938). For each titration

litters containing at least 6 suckling mice were inoculated

intracranially with serial tenfold dilutions of the sample.

The plaque assay was the second m3th~d used for quan­

titating infectivity of virus samples. Determination of the

conditions that would pro.n~te formation of plaqu3s by CE

viruses constituted a significant portion of the work to be

presented; details of the procedures employed are discussej

in the Results section. Briefly, 4J-48 hCHJT old mon t )-

layers .~£ chick embryo or baby hamster kidney cells were

washed with PBS to remove cell debris and bicarbonate-

buffered growth .1\(~dium and th.3n appropr to d i lu':ioos OC

29

each virus sample were inoculated onto the washed cells 1n

0.2 ml amounts. Two, three or four monolayer cultures were

used for each dilution assayed. Virus 'IJas allowed to adsorb

to the cells for 60 minutes at room temperature or 37 C;

five ml of molten overlay were then pipetted into each 50 mm

Petri plate containing infected cells. The overlay medium

'Nas prepared by mixing double concentration solutions of

buffered MEM and molten agarose solution. B:Jth of th3se

were equilibrated to 42 C before ~ixture anj this temperature

was maintained during addition of the overlay. When the

medium had solidified, the plates were incubated at 37 C for

60 0:- 96 hours, at which time 2 ml of ne;J.tral red solutio::1

Were added to each plate. After 2 hours at 37 C the stain

was rem07ed and th3 cultures were left in a dark room Eo= 12

hours before plaques Were counted.

V. ANIMAL IMMUNIZATIONS

In early trials, antisera were prepared by variej 'nea,':1S

in rabbi ts a!ld mice. W'.lf3~e approp= iate, more d etai led

descriptio~s are provided in the Results section. Table 2

outlines the schedule follo'lled to obtain speci fie antisera

against 8 virus strains. This schedule was folloNed in the

hope that sera representing different specificities wO;J.ld

be available. Blood samples were removed follO'tJinl] cardiac

[J'.1ncture of the anesthetized animals. After the blood had

clotted at room temperature for 1-2 hours, it was

30

refrigerated an additional hour to facilitate separation and

removal of the serum. Sera Were collected and clarified by

low speed centrifugation and then stored at -20 C in 'J.5 :nl

aliquots.

Day

o

:)

7

8

15

43

52

TABLE 2

INOClJL7\T ION S':;HEDULE FOR PREPAR..l\T ION OF "E..I\RLY IMMUNE", "IMMUNE" AND "HYPERIMMUNE" RABBIT ANTISERA TO MEMBERS OF THE CEV GROUP.

Trea tml=!1t

Normal serum sample removed

First injection, 1 ml, intram~scular, each hind flank

First bleeding, "EARLY IMMUNE" serum

Second injection, as above

Second bleedin3', "IM1'11JNE" S'2~um

Third injectio~, as above

Exsanguination, "HYPERIMMUNE II serum

VI. VIR0S NEUTR1\LIZA.TION STUDIES

The techniques utilized were essentially those detailed

by Habel (1969) for polio,.,lrus. The virus in()clllum '."'3S

diluted in PBS-BSA to contain 1-5 x 106 plaque-forming units

(PFU) per mI. The viruses were propagated in the brain

tissues 'Jf sucklin9 ;nice and in cultures of chick ernbryo

and baby hamster kidney (BHK) cells. Sera were heated at

56 C for 30 minutes before use. An at tempt was ilkld~ to

31

determine a dilution for each antiserum that would result in

similar amounts and rates of neutralization by each antiserum

for the homologous mixtures (see Results). Aliquots of

serum and virus were equilibrated at 37 C for 20 minutes

before mixture. It was necessary to obtain zero tim!~

sa:nples from mix·tures of normal serum and virus because of

the rapidity of the virus-antibody interaction. At designated

tim3s, 0.1 ml s3'11£)les -were removed from -':.h8 incubatin3

mixtures and diluted immediately into 9.9 ml of chilled PBS­

BSA; these were maintained in an ice bath until all sam?les

had b2en taken. At this time additional dilu·tions, if

necessary, were made and then these were assayed for surviving

infectivity.

VII. VIRUS PURIFICATIO~

Viruses of th9 CE group ware partially purified by

sedimentation through sucrose gradients. Pools of virus

propagated in suckling mice or in cultures of chick embryo

or baby hamster kid~9y cells were Itered throug~ 1.2 un

pore diam'3ter membranes (Millipore Filter Corp. I Watertown,

Massachusetts) and th'3n clarified by sedimentation::::>nto

sucrose cush ions. Th'3se were prepared by layering 10 ml of

20 or 25 percent (wt/vol) sucrose (SchwarZ/Mann, Orangeburg,

New Jersey) onto 5 ml of: a high d'2ns i ty "pad" composed ::::>f

65 percent (wt/vol) sucrose. Ten to 12 ml of a filtered

virus sample were layered onto the lower density solution

32

of sucrose and these were centrifuged for 90 minutes at

20,OJJ RPM in a SW 25.1 rotor in a Spinco Model L pre­

parative ultracentrifuge. Fractions constituting the

sucrose-sucrose interface Were obtained by drop collection

fro:n the gradient tube bottom. This material was diluted

twofold with tris b~ffered saline and layered onto gradients

containing 25-65 percent sucrose. These Were prepared by

sequentially layering 3 ml of 5 percent increments of sucrose

solutions, beginning with th3 65 percent solution. Th

latter gradients ware centrifuged for 360 minutes under the

same conditions as above. Fractions were collected by

min-3ral oil drop'JI!ise dis?lacement of sucrose drops; th")se

containing the highest levels of infectivity were used in

subsequent experimental proced~res.

VIII. Pi{')TEIN D:2::TERMINATION

The :nethod of Lowry et al. (1951) was used after some

modification to estimate the amount of protein present in

various fluids. Cupric s~..llfate and SGdiu;ll ?:Jtassium tar­

trate were prepared separately as one and 2 percent solutions

(wt/vol), res?2ctively. Prior to use equal volumes of these

were combined and one ml of the resulting mixture was added

to 50 ml of a 2 percent (wt/vol) sodium carbJnate in ~.l N

s,")dium hydrox.i..d:::; solution. One ml of the second~ixture

was pipctted into each test tube containing a 200 microliter

prated n sample and rapio ly mixed. After 10 :ninute3 , 0.1 tnl

33

of Folin reag0nt (Fisher Scientific Co., Fairlawn, New Jersey)

was added to each sample with imillediate, vig8rous mixing.

This reagent hild previously been diluted with an equal

volume of d tilled water. After 30 minutes' incubation

at room temperature the absorbance of each sample was

determin2d at 660 :lm in a Beckman DU spectroph8tom'3ter. A

standard curve correlating absorbance to protein concentration

was constructed every time pro:.ein concentration det.ermina-

tions were made. Bovine serum albumin diluted in tris-

buffered saline was utilized as the reference pro:.ein.

RESULTS

I. PL.~QUE .FOR.!~~T ION

Initial attempts toward the development of a plaque

assay applicable to every member of the California enceph·­

alitis (CE) grou.p of viruses involved the use of primary

cultures of chick embryo cells (CEF). While plaque forma­

tion on CE~ has been reported for Tahyna virus (Porterf Id,

1960) using an agar-co~taining overlay, Bales et ale (1967)

were unable to plaque BFS-283 virus on these cells, althaug~

.?laqu~s 'tJere observed 'Nh~n green monkey kidney cells (VERO)

were used as a control. Using this second virus, regarded

as the group prototyp2, and Western equin,= encel.:)~alitis

(WEE) virus as a control, an effort was made to demonstrate

plaque formation on CEF monolayers. When the overlay

contained Melnick's 'jcowt.h medium, calf serum, s''Jdium bicar­

bonate and purified 0.9 percent (wt/vol) agar (Difco,

Detroi t, Michigan), plaqu'8s were consisten t ly obs-9rved 'Ni th

WE:~ virus 'Nhi le no evidence of cell destruction was apparent

in the presence of the CE virus.

Agar, which is a mixture of polysaccharides, inhibits

plaque fo:-mat ion:JE a variety ,")f envc loped (Colo:! et al.,

1965) and non-enveloped (Takemoto and Liebhaber, 1961)

viruses. Inhibition has been attributed to the neg~tively

35

charged sulfate and carboxyl groups associated with agaro­

pectin (Ventura, 1968) and can be largely prevented by the

use of agarose, a neutral galactose polymer component of

agar (Borden et al., 1972). Alternatively, the adverse

e of these polyanionic components can be lessened by

including polycationic substances in the overlay medium

(Val'le, 1971). Experimentally, neither the inclusion of

diethy noethyl dextran (DEAE-D, Sigma Chemical Co.,

St. Louis, Missouri) nor protamine sulfate (Sigma Chemical

Co.) at concentrations of 20-1000 and 20-400 micrograms,

respectively, faci tated the formation of CE virus plaques

under

Subst ion of agar by 0.5 percent (wt/vol) agarose

resulted in areas within the monolayers, although

the appearance was not that of a typical plaque. The

"plaques" were characterized by irregular size, diffuse

edges and an overall faint appearance that prevented quan-

titation. By ing Melnick's medium with Eagle's

minimum essent 1 medium (MEM) the number of "plaques ll

increased and the overall appearance (size, contrast, defini-

tion of edges) i Further refinement was achieved

by decreasing the concentration of agarose to 0.4 percent.

Plaques produced by a 1 mosquito isolate (#30521) under

these conditions were uniform in size (2mm) and well-defined

with respect to contrast and cd BFS-283 virus stock

sllspensions exhibited two (] istinct plaque size populations,

36

one larg'2 (4mm) and the other small (1 mm). The incubation

time required to attain these sizes was 60 hours.

Having determined the corditions that would permit

plaque formation and, hence, quantitation of at least two

CE viruses, the plaquing efficiency was compared to assays

performed by intracranial inoculation of suckling mice.

Litters of mice 36 to 43 hours old ~ere employed and each

.mouse was inoculated with 0.025 ml of one of several dilu-

tio:1s ofa stock preparation of the desert isolate. At th2

sa~l= time, 0.2 inl of the sam2 dilutions were inoculated onto

washed replicate CEF monolayers and assayed fa ..... plaque

fo:r-mation. In se~Je.ral comparisons the titer e;3tina:'ed by

assay i~ suckli~g ~e as the dos2 lethal for 50 perceDt of

the animals (LD- O: R~ed and Muench, 1938) was characteris-..)\

tically higher than that determined by the plaque assay,

although thi2 difference seld''Jm exceeded J.5 1095. Similar

results were noted when the titers of BFS-283 and Tahyna

were compared by these methods of assay.

Several characteristics of the CE virus-CEP cell

interaction were examined. These included the influence

of temperature and time on adsorption of virus, suscepti-

bility of the cells to infection by the viruses, a~d the

pattern of CE virus replication as a function of time.

'11he effect of temperature on th2 infectivi ty 0:: OIV:~

CE vjrus (3J521) during dilution and adsorption to the

assuy cells was fjrst studied. Separate samples of a

37

virus pool were equilibrated to 4 C or 25 C, diluted to

contain a countable number of plaque-forming units and then

maintained at these temperatures for 60 minutes. At this

time aliquots Were pipetted onto washed monolayers7 half

of the inoculated plates were incubated at 37 C for 60

minutes while the remaining mon81ayers were maintained at

25 C. At the end ~f this adsorption period all cultures

were overlaid with agarose-containing medium and incubated

for the time required for plaque formation. The results

(Table 3) indicated that the virus CQuid be manipulated at

room temperature without untoward effects and that the

differences in adsorption temperatures (25 C vs. 37 C) were

not sufficient to influence the number of plaques formed.

TABLE 3

INFLUEN2E OF TEM?E~z\TURE DURINI:; 'JIRU..3 SAM.PLE DILU­TIoN AND XDS')Ri?TION ro CELLS 0N CALIFORNIA ENCEPH­ALITIS (30521) VIRUS PLAQUE NUMBERS.a

-----.----- _._._._-----.-~.----- --- -. -~~~_.- -_._._-- - --~

Dilution Temperature (C)

4

25

Adsorption Temperature (C)

25

48± 7.0b

61± 6.7

37

482: 11

60± 5.0

a. Virus samples held at the dilution and adsorption temperature for 60 ~inutes each.

b. Average number of plaques and standard deviation of 7 replicate plates.

38

rl1he time required for adsorption of the desert isolate

(30521) to eEF cells was determined (Table 4). At the

indicated times after addition of the virus inoculum to

replicate monolayer cultures, unadsorbed virus was removed

and the plates washed 4 times before addition of molten

overlay. Maximum adsorption, determined by numbers of

plaques counted, was achieved 1 minute after pipetting

virus onto the cells.

TABLE 4

TINE REQUIRED FOR ADSORPTION OF CALIFORNIA ENCEPHALI'rIS VIRUS (30521) TO PRIt1ARY CHICK EMBRYO CELLS AT 25C.

Time (minutes) Titer (PFU x

0 2.4± 0.6*

1 4.9± 0.9

2 3.8± 0.8

5 4.7± 0.8

10 6.0± 0.4

20 5.4:!: 0.2

40 5.1± 1.0

60 5.1± 0.8

Inoculum by previous 5.0± 0.2 titration

* Standard deviation of four plate counts.

10 7 )

39

The susceptibility to infection of the cells which

com~Jrise a monolayer of chick embryo "fibroblasts" prepurQd

in the manner described (cf. Materials and Methods section)

was ascertained by two techniques. One approach, regarded

as a standard criterion to b '2 employed when evaluating the

susceptibility of a cell spec s to plaque formation by a

virus (Cooper, 1961), was to determine tha relationship

between incremental dilutions of a virus sample and the

number of plaques observed when assayed on th9 cells b'2in9

studied. In Fig~-1re 1 th3 nu·Th")er of plaques observed has

been plotted as a function of sample dilution between 10-5

and 10-6 . The results indicated a linear relationship

betwe<2n number of plaque-forming uni ts and virus concentra­

tion.

The second m9thod of determining the susceptibility of

chi~k cells to in£ecti~n by these viruses 4as to in~ect cell

mon~layers at a virus-to-cell ratio that would guarantee the

infection of every cell and to subsequently assay each ~ell

for infection by the added v (infective center assay).

This second step was accomplished by trypsinizing monolayers

of infected cells after adsorption at 37 C, determining the

number of cells present after dispersion and then plating

dilutions of these onto uninfected monolayers, similar to th9

steps follow·ad for a plaque assay.

The results indicated that at a mUltiplicity of in­

fection of 5 all cells were infected. According to th9

40

80

N . 60 0

~ (j)

Pi

W +' .r-l ~

1~0 :::>

till ~

.r-l S ~ 0 '~ ct-; I 20 i (j)

::5 ry ill r-l P ..

o

v s dilution

F 1. CALIFORNIA EPHALITIS VIRUS ( 283 ( » PLAQUE J>rUlVIBERS A FUNCrr I ON OF PLE DJ ION. REPRESENT 95 PERC CONFI E LIlVIITS.

41

Poisson equation, at least 99.3 percent of the cells would,

theoretically, have been infected. Similarly, at a virus

multipl ty of 0.005, less than 2 percent of the cells were

infected while, theoretically, less than one percent should

have been.

The replication of some members of the CE virus group

In CEF cells was investigated. Cultures contained in

plastic tissue culture flasks were inoculated with enough

virus to ensure infection of every cell. Simultaneous in­

fection of these cells permitted the study of a single cyc

of virus replication. Following adsorption at rODm tempt.::ra­

ture for 60 minutes, unadsorbed virus was removed before

adding fresh medium by washing monolayer cultures 3 times

with cold PBS. At va ous times aliquots of the growth

medium covering the infected cells were removed for ass of

infectivity and frozen until all samples had been collected.

The tures of replication are indicated for four CE iso-

lates in Figures 2 through 5. In para lIe 1 experim'2nts the

gro·.vth curves ·:Jf the S3.ffi'2 viruses in CEF cells pretreated

with actinomycin D (AcD, Calbiochem, LaJolla, California)

were determined. Cells were incubated In the presence of 2.5

ug AcD per culture (2 x 10 7 cells) for 90 minutes prior to

infection. These results are included in each graph.

The churacteristics of replication were simi r for

each of th(~ viruses examined (San Angelo, Tahynu and large

(LP) ilnd smull pluque (SP) variants of BFS-283). Virus,

9

AcD-treated cells 8

7

6

5

4, ________ ~ ________ ~ ________ ~ ______ ~ 6 12 18 24

Time ter infection (hours)

FIGURE 2. REPLICATION OF BF 283( ) VIRUS IN

Ui r4 r4 Q)

o

o r4 Of) o

IMARY C EMBRYO FIBROBLASTS.

9-Untreated c

H 4, __________ ~ ____ ----~--------~---------~

Time after infection (hours)

F' I G U H E 3. REP IJ I C A~ll ION 0 F S - 2 f) J ( S p) V I R U SIN PRIIVlAHY CHICK EMBRYO FIBROBLASTS.

42

9 --Untreated cells

rn r-f rl Q}

----------.---------8

8 o

7

6

5

4 6 12 18

Time ter infection (hours)

FIGURE 4. REPL]CATION OF SAN ANGELO VIRUS PRIMARY CHICK FIBROBLASTS.

rn

ill o

C"­o rl :x! (\J

H OJ ~

H Q)

..p ·ri ..p

o rl

hD o ~-l

9

8

7

6

5

4

ed cells

treated cells

6 12 18

Time after infection (hours)

24

FIGURE • REPLJCfiJ'ION OF 1~AIIYNA VIRUS IN PRI-MAHY CHICK YO FIBROBLASrrS.

43

44

presumably progl.:ny, was detected in the supernatant fluids

within 3 hours after infection. Exponential release of

virus was evident through the twelfth hour; further increase

in titer after this time was negligible. Cytopathic changes

did not beco~e apparent until 30-36 h~urs.

Cells pre-treated with AcD 3upported replication of all

CE strains studied. The activity of the AcD preparation was

tested by its ability to inhibit the replicati8fl of vaccinia,

a DNA-containing virus, under conditions similar to those

described above. Replication was mJre than 90 percent in­

hibited. Thi~se res~Jlts s'Jg3ested that DNA was nC)t involved

in the replication of CE viruses. In support of this,

McLerran and Arlingshaus (1973) have dete::;ted RNA in puri­

fied preparations of LaCrosse virus.

As noted above, the growth medium used in the plaque

assay determined I in part, plaqu9 :n::>rpholo9Y an::] nu:nbers.

The replication C)f the desert isolate (30521) in fluid cul­

tures, i.e., in the presence of Melnick's or Ea31e's MEM

growth ned ia lacking a-J.:l r 0::::- a]arose ,was determined. The

data of Figure 6 indicate that no significant differences

in virus propag.3.tion could be detected with either mediuiTI.

Having defined the ::;onditions necessary for plaque

formation by two CE virus group members and inves 1. igated

som3 basic aspects of the virus-cell interaction, the

a?plicahility of the assay to other viruses in the group was

examined. It was found that the several viruses studied

9 -;--m ill rl 8 rl OJ 0

C'-0 7 rl >< N

H 6 OJ PJ

H OJ

...p

5 .r-I 4.::>

0 rl

oD 4 0

H

6 12 18 24

Time after infection (hours)

FIGURE 6. COMPARISON OF s~rEP RATES IN IA S (#30521)

(G,) AND MIN IAL IV.EDIA.

45

46

vari.ed considcrubly in plnque morphology. Besides size

variation, the contrast between the plaques and uninfectcd

areas made detection of the plaques of some of the viruses

difficult. It had been noted that contrast could be improved

by doubling the bicarbonate concentration, so an experim2nt

was desig~ed to determine the concentration of bicarba~ate

that .would be optimal for the detection of Tahyna and "3052111

virus plaques (Table 5). The concentration of the buffer,

no.Lioully p:-esent in thf3 overlay at 0.35 g/1, was varied

between one-fo'Jrth and four times that amount. This range

was accompanied by differences in pH of from 6.9 to 7.9 -

apparent at the time th·3 o',erlay was added to the infected

cultures. During incubation, the infected, overlaid cells

Were maintain.3d in an atmosphe!:"e of apPY'oximately 5 percent

(vol/vol) carbon dioxide (C0 2 ).

TABLE 5

CALIFORNIA ENCEPfffiLITIS VIRUS PLAQUE SIZE AND QUALI'ry AS A FUNC'rrON OF 80DIU1"1 BICAR30UAT2 ·:OH·­CEN'rR}\.TION.

Virus BicarbJnate Concentration (g/l)

Tahyl1i1

#30521

.0875

(l)a,b

(1-2)

a. P1acIUC d iam(~ter (mm)

.175

( 1 )

2

.35 .70 1.05

( 1) 1-2 2-3

2-3 2-3 2-3

1.4-]

(2-3 )

2-3

b. ParcntlE~ses indicate poor qua1i ty, d iff icul t-i-.o-score plaque's.

47

Both Tahyna and the desert isolate appeared equally

sensitive to the conditions produced by low bicarbonate

concentrations (pH 6.9). At the highest bicarbonate

concentration the plaques of Tahyna virus were diffuse,

poorly contrasted and difficult to count, thereby restrict-

ing the range over which good quality plaques were observed.

The mQrphology of 30521 virus plaques did not change above

the lowest concentration of bicarbonate. In addition, the

number of plaques formed by the desert isolate did not

change as the concentration increased above 0.35 gil; for

Tahyna virus plaque numbers were highest at 0.7 and 1.05

gil. Because the pH and bicarbonate concentration increased

concomitantly, it was not possible to attribute the changes

in morphol09Y and numbers of plaques observed during this

experiment to a direct influence of the bicarbonate ion.

The acidity observed in growth media as the metabolism

of cultured cells proceeds has been attributed, in part,

to the accumulation of non-volatile 3-carbon acids (Cooper,

1961). Atmospheric carbon dioxide (C02 ) also contrib~tes

to acidification by combining with water to form carbonic

acid that subsequently dissociates into hydrogen and

bicarbonate ions:

+

An increase or decrease in C02 tension is accompanied by

a corresponding pH change. Experimentally this becam9

appitrent when bicarbonate-buffered overlay media were added

48

to infected cultures and left at roo(n temperatur.e dur:-i(}(]

:;:Jlidification of the overlay: The r.elatively IIJ\II p:::trt.ial

pres~_FlI~e of carbon dioxide prescnt in the atmJspherc caused

an increase in alkalinity. Because the pH fluctuated with

atmospht2ric changes between experiments, the initial pH

was difficult to control.

An awareness of the difficulty associated with the use

of bicarbonate in cell o7erlays led Porterfield (lQ60) to

replace this buffer with tris Hel (TRIS (hyjroxy~TI(~thyl)

amino:nethane). One of the viruses studied by Porterfield

was a CE ~irus strain, Tahyna, and the cells used were

from chick emb::yos. Hi.s results prompted an effort to

determine the applicability and use of this co:npound to

plaque oth~r CE v.irus8s. For these studies plates contain-

ing infected cells Were incubated in air-tight boxes (Labline

Instruments, Inc., Melrose Park, Illinois). This liu~ted

the concentration of the CO2 to ~hat amount present when

the containers were initially sealed and also to that pro-

duced by the respiration of th3 cells. Tris-Hel was used

at concentra tions betw'2(?n (). 01 M and 'J. 05 M; the pH of each

overlay was adjusted initially to 7.2 with H-::::l. By vis 11al

comparison rJur in9 incubation, the increase in acid i ty of th'3

tris-buffered overlay was slower than similar plates over­

laid with the bicarbonate-blJEEered ~U(:~d ium and incubated

in a 5 percent CO2 atmosphere. The plaques produced under

the tris-buffered overlay were similar to those noted [or

the bicarbol1ztte-buEEered media in all respects, e.g., diameter,

49

contrust tlnu sharply-defined cdges. It was concluded that

the usa of tris buffer was acceptable under the conditions

described.

Pltlque formation by BFS-283 and 30521 viruses was

studied using overlays containing both buffering substances,

i_e., tris Bel a!1t1 bicarbonate (Table 6). In addition to

TABLE 6

PLAQUE FORMATION BY CALIFORNIA ENCEPHALITIS VIRUSES ON PRIMARY CfIIC~ EMI3.:.~YO CE~LS: IN5'LUEI'JCE ~)g CAl{gON DIOXIDE 1

S0';)IU."1 BICARBONATE AND TRIS (HYDROXU1ETHYL) AMINOMETHANEa

-~------ ._--"- --- --------""- - ------.--"-HCO] pH~J #30521 B?S-233 --_. _._-----~-.. --.-----.----(g/l) CO2 No CO2 CO2 NJ C°2 _____ w_'_' _______

0.0975 6.9D 95c 61 TNCd 31

0.175 7.15 84 74 83 13

0.35 7.25 74 66 55 0

0.70 7.70 79 74 15 0

1.05 7.80 68 51 0 0

1.40 7.90 63 19 0 0

tris 7.30 84 85 TNC 59 alone

------'-~---- ----

a. OJerlny consisted oE 0.4 percent agaros~, Eagle's M~M! 0.02 M tris adjDsted to pH 7.3, and indicated amJunts of sodiun bicarbonate.

b. Determined at the time of overlay.

c. Plaqu(; forinin'~ units pj~ r O. 2 ml.

too numerous to coun t.

50

bicarbonate, tris-Hel was present in all overlay media at

0.02 M concentration and pH 7.3. The amount of bicarbonate

present increased in doubling concentrations fro~ 0.0875

gil to 1.40 gil. This resulted in a range of pH's. Dupli-

cate sets of plates were prepared for each virus so that one

could be incubated in an atm':)sphere of 5 percent CO2

and the

other without added CO2

. Significant differences between

the pH :ind/or bicarbonate sensitivities of the virus:~s

were noticed. As the bicarbonate concentration increased

in the presence or absence of added CO2

, the nu~)ers of

plaque=;; dec-cea3ed with both viruses, altholJr;3'h the sensitivity

of B2'5-283 -"'dS markedly greater. In the presence of added

CO2 , the bicarbQ~ate concentration range over which BFS-233

formc~d plaques was ext.ended. For the desert isolate, plaques

Were app3rent over the entire range, but the numbers did

increase when CO2

was prese7.lt. Th::=se results SU99Gst.ed a

d l2£inite inf luence of the small pH differences that eXLsted

at th::= tim::= the infected ffiJnolayers were overlaid but

because bicarbonate was present in relatively large am~unts

a definitive conclusion could not. be reached without first

eliminating th2 added bicarbonate.

'rris-HCl was previously sho'",n to support th'2 forma tion

of plaqu'2s by members of th8 CE virus g.COUp in thf:! abs;3nce

0:: added bicarbonate and carbon dioxide. Tris-buffered

ov~rlilYs of decreasing pH were prepared by adding increasing

amollnts of 6 N hy,Jrochloric acid to each oE severa 1 l.iquificd

51

overlays. Because the tris concentration was constant

while the initiol pH of each o'J'crlay was unique, the assump-

tiol1 was made that any effect on plaque formation would be

attributable to the pH. Tables 7 and 8 summarize the experi-

mental findings. Over the pH range studied (7.0 to 8.0)

variation was evident in plaque morphol:Jgy (Table 7) and

TABLE 7

EF.'BEC'r 01" INITI}\L OVERLAY pH ON THE MJRPHOLOSY OF PLAQTJES FORMED ON PRIMARY CHICK EMBRYO CELLS BY CALIF'OR~ IA ENCEPH.\LIT IS G~~OUP ~J IR~-;3ES. a

Virus OVerlay pH

7.0 7.3 7.5 7.7 7.9 ----

8.0 ----~.--.-"-------------------~----- ------.-.--

#30521 ++++b ++++ ++++ ++++ ++++ ++++

LaCrosse ++ +++ +++ ++ ++ a

B:"S-233 +tt ++ 0 0 a a

San Ang'~lo ++ t- t- + 0 0 0 0

Jerry Slou'3h + + 0 0 0 0

Sn:::)'Nshoe Hare + + 0 0 0 a

Trivittatus + 0 0 a 0 0

Melao 0 0 0 a a 0

a. Th'2 overlay contained 0.4 percent agarose, Eagle IS MEM and 0.02 M tris (hydroxym~thjl) d:ninometha:.le and initial pH was adjusted by addition of 6 N Hel.

b. +t-t-t-, greater than 2 m~ diam., well-defined edges, good contrast; +++, 1.5-2 1011 diam., well-defjned edg~:.3, gi,Jod contrast; +t-, 1-2 mm diam., diffuse ed;:;cs, fair con­trast: +, cytopathic changes of irregular shape, uncount­able; 0, no plaques.

52

numbers (Tub Ie 8). rrhc rna j<::>r i ty of the v lruses used in this

experiment formL:d plaques only over a narrow range of pH

values. An exception was the desert isolate (#30521) which

produced distinct, 3-4 mm r]iamc~ter plaques at each overlay

pH. While the data are not presented, it was noted that

the ability to form plaques was greatly reduced when the

ini t ~a lover lay pH was bE.~low 7. O. The resul ts in Tab le 8

indicate the countable plaques at different overlay pH's

of four of the viruses used in this experiment and suggest

the "recognition" by the viruses of an optimum pH fo:: plaq:ue

formation. In a parallel experiment, the effects of pH on

Virus

#30521

L~Cross:~

B:?S-233

TABLE 8

PLAQUE FORMATIO~ BY CALIFORNIA ENCE!?IL\LITIS VIRD3ES ON ?~ I MARY CHICK E.MBR-{O C£l..J.JS ~ IN­FLuENcE O? pH :)N NUMBERS OF PLAQUES.a

Overlay pH

7.0 7.3 7.5 7.7 7.9 8.0

30b 75 57 52 52 33

22 54 31 16 10 0

71 34 a a 0 0

San Angl.=lo 43 a a a 0 0

,--~.-~,-- - - -----

a. Oilerlay consisted of 0.4 percent agarose, Eagle's MEI1 and 0.02 M tris (hydroxymethyl) aminomethane adjusted to indicated pH values by addition of 6 N Hel.

b . A vcr a :-F:> n 'J mb e r 0 f P la '1.11 e s for s ~? V t:! n rep 1 i Cd t e p 1 ate:3 .

53

plaqu~ formation by four Group A Arboviruscs (Sindbis,

Western, Eastern and Venezuelan equine encephalitis viruses)

w(~re stur]ied: NJ dif£erenc(:;s could be detected in plaqu.2

numbers, size or contrast over the same pH range. The

plaques produced on chick embryo cells by Jerry SloUljh,

Trivittatl1s, S:1oyJ'shoc Hare, M'2lao or Jamesto\'Jn Canyon viruses

could not be improved by overlay pH adjustments: Keystone

and Lumbo 'viruses 'flere not studied.

Wh"~n Tahyna virus 'flas inoClllated onto chick emb.:::-yo cell

cultures aYld th'~n overlaid with the tris-buffered agarose

medium at a pH that pro'vided optimal conditions foe plaque

fO::-iOation I incorporation of 50-80 u9 Dill\E-Dextran into the

overlay increased the plaque size from 1-2 mm to 4-5 mm:

Inclusion of this p8lycation did not affect th9 :::lumbers of

plaques. Concentrations above 100 ug/ml were toxic for the

cells. The plaques of Trivittatus, Jamestown Canyon, Jerry

Slough and Melao viruses were :::lot improved by the addition

8£ DRA...E-D.

It was of some interest to determine the effect of pH

on the replicatio:1 of the viruses When the cells had n:)t

been covered with d:'1 a9d rose--containinC] ov2.r lay tn(~d i umw For

this experiment San Angelo virus, Which formed plaques at

pH 7.0 but not at pH 7.3, was selected. The conditions

9rowt::.h

(~I]rV~? :;tu(1ics in terHlS of mUltiplicity of infection, ad­

sorption and wilshings. The m9d ium (Melnick IS) was b;..1ffored

54

by tris with no added bicarbonate. The pH of throe uliquots

0f the culture mediu~ was initially adjusted to pH 7.0,

7.3 or 7.6. During th'2 24 hours of incubation after 1n-­

fecti~n, the pH did not change in any culture flask by wore

than 0.1 pH ~nit. N~ differences were noted in the infec­

tivity titers achieved between pH 7.0 and 7.6. The data

indicuted that, while pH affected plaque forioati:>J1 of San

A'19t~lo virus, it did not necessarily influence replicatio::1.

The applicability of a second cell for plaquing of CE

viruses was also investigated. Tnis was prompted by the

d if Eictll ties exper ienced wh'2n at tempts 'tlere made to demo::1-

strate plaques for some of the viruses. The cell (BHK)

was a strain:>f the continous line of bally hamster k lji1(~Y

cells (BH.<-21/C13) isolated by Stoker & McPherson (1964).

The origin of this clone is described in the Materials a':1d

Methads section. One desireable property o~ the cell,

compared to the wild type, was its slower rate of acidifica­

tion of culture media than the wild type. Several experi­

ments that had b(;(~n [:H.?r formed with chick emb::-yo ce Ils '.th~re

repeated, using the BHK line. For exa~ple, studies Were

co~ducted to determine the characteristics of replication ():

the large plaque variant o~ BFS-233 and LaCrosse viruses in

BH{ cells (Figurffi 7 and 8). While the general features of

replication resembled th'Jse using chick embryo cells, thl2

pL:.iquc~ fOrHl.ln(} t i tors dt~tected when Ba:< Ct)lls \rJere US'~:l (Nere

CY::i1(:riJ lly h igll(~r. As was noted with chick om~ryo cells I

W rl rl (I)

o

o r-;

oD o ~

9

8

7

6

5

4

FIGURE 7. C INUOUS

6 12

Untreated cells '- ------~ ......... - G1

AcD-treated celIE 0....--

18 24

Time after infection (hours)

CATION OF BFS-28J(1P) VIRUS IN A BABY HA1VlSTER KI C 11S.

55

rn rl rl (!)

o

C"­O rl X N

o

9

8

~ 5 o ~

4

Untreated cells

cells

6 12 18

Time after infection (hours)

FIGUHE 8. REPLICA11 ION OF LACROSSE VIRUS IN A CONT:! S IIJ BABY HAMSTER C

56

57

actino~~cin D had no de~~nstrablc effect on th2 replication

of th,? viruses.

A ~oiTIparison was also clade b'2tween the plaquing effi-

ciencies of chick embryo cells and BHK cells: San Angelo,

Tahyna and LaCrosse vIrus stock suspensions were used in

the compa.ris·'Jn (Table 9). Whi Ie the number of San Angelo

virus plaques was higher on chick cells than o~ baby h3~ster

Virus

TABLE 9

COMPARIS')0J 87 Ph"!\QUE-POR:qIN3 UNIT TITERS OF CALIFORNIA ENCEPHALITIS GROUP VIRUS SUSPEN­SIONS WHEN ASSAYED ON CRIer< EI13RYO ?I8,::<J)3~l\STS

AND 3AB'{ H}\MSTER KIDNE{ CELLS.a

Assay cell

BHK CEF

San Ang'210 2.1-3.1 x 10 7b 3.0-4.4 x 107

Ta"hy!la 1.8-2.5 x 108 2.0-2.6 x 10 8

LaCrosse 1.4-2.1 x 10 7 1.2-2.0 x 10 7

a. O'ler lay cons isted of 0.4 percent agarose, HEM and 0.02 M tris (hydroxym3thyl) amin'Jmethane; the pH of th(~ oV2rlay l..lsed -to plaqu!~ San An}2lr:::> and La­Crosse viruses was 7.1, and Tahyna was 7.4.

b. Range of PFO/ml at thf.::! 95 percent confid'2nc(~ limits.

kid~ey cells, the data were not regarded as significant

On01.19h to discontinue use of th,:; cells. Generally 1 the

than the corresponding virus plaques on chick embryo cells.

58

The effect of virus sample dilution on numbers of

plaques observed on BHK cells was studied, using BFS-283

(large plaque variant) propagated in suckling mice and in

BBK cells. The results were similar to those noted when

chick embryo cells were used as the assay cells: As dilu··

t.iol1 of the YJirus-containing sample increased, the n~.lmbers

of plaques appearing decreased. Again, the data suggested

the ability of single, infectious virus particles to infect

cells. Infective center assays were not performed.

The variation in numbers and morphol03ical features

of the plaques of several CE viruses as a functi~n oE

initial pH differences in the overlays added to BHK cells

was examined as each virus strain was received. The

stability of the plaque characteristics of each vIrus on

BK{ cells was found to be influenced by the initial pH of

the overlay media. In contrast to chick embryo cells,

plaques were fO:lood on the baby hamster kidney cells by

Trivittatus, M'21ao, Ja:nestowi.1 Canyon and Snowshoe Hare

viruses. The optimum pH for plaque format. ion byth(~:3 e vIrus s

was between pH 7.0 a~d 7.2. Plaque formation by Ja~estown

Canyo~ and Melao viruses was observed to be greatly influenced

by slight differences in overlay pH: Plaques 'Nere only

observed When the overlay pH 'Ill as initially adjusted to 7.1.

The demonstratio~ of an apparent pH dependence of CE

virllses for pl<tqlll~ format.ion would hav\:.; add'2d an unrJ'2:3ire-­

c1blc var l.able requiring mani.pulation and intcrpretat_ion if

59

different overlay media had had to be used for each virus

stud ies were per focmor]. It was

decided to s tandarJ i Zt:; the plaque assay of these v iruses to

one overlay pH: The value selected was pH 7.1. This

allowed formation of plaqll(~S by every member stlHl:lec].

Tahyna virus, Which exhib d the highest pH optimum, pro-

duced smaller plaques at th~ lower pH (1-2 Inm 'J'erSU3 3-4 !TIm),

but. th,~ numbers of S \'Jer:e similar.

A'1 add it i')na 1 va r Ie Which influenced the m~rphology

of the CE virus plaques was the calf serum lsed in the QVer-

lay met]ium. In the course of these studi9s it was neces

to use several different lots and preparations of calf sera

and with each ~f th'3se the mo.:::-phology of th9 plaques pr-J­

duced by each of the viruses varied. The most apparent

differences were associated wi th the d iameters 'J~ the plaque:31

As noted abov2, sera inhibitory for

not used.

o~ the isola~es were

To summarize: Primary chick embryo cells and a strain

of hamster kidney cells both d replication of

the Californ encephalitis group viruses studied. Plaque

format on both types of cell was markedly dependent on

the init 1 pH of the overlay. The BHK cells supported

formation by a greater number of the CE viruses than

did the chick embryo cells: Every member of the group

stue] ied formed cas i ly detectab Ie plaques on the hamster

cell line.

II. PURIFICATION OF CALIFORNIA ENCEPHALITIS VIRUSES

The need for preparations of CE viruses from which

con-taminating non-viral matter had been removed was anti­

cipated and resulted in an attempt to devise a procedure

that would increase the specific activity, i.e., PFU:

protein ratio, of each virus preparation. Precipitation

60

of the infective virus present in crude mouse brain or

chick cell suspensions by buffered, sat~rated solutions of

ammonium sulfate was attempted but the recovery was low,

seldom exceeding 30 percent. Because the level of infec­

tivity remaining in the supernatant fluid did not total the

amount of unprecipitated virus, it was assumed that the

virus had been inactivated. The concentration of infective

particles that could be precipitated was not increased by

different pH values (6.3 to 8.1), by different levels of

salt saturation (35 to 65 percent), by the concentration of

protein present (3 to 11 percent), or by the method of

addition or mixture of the salt to the virus preparation.

Aggregation did not contribute sign icantly to the high

percentage of virus lost during precipitation: Filtration

of the resuspended precipitate by Millipore membranes with

pore diameters 2-4 times greater than the diameter of the

virus particles (Murphy et al., 1968) did not reduce the

levels of infectivity. Although precipitation of virus

with ammonium sulfate did decrease the level of contaminat­

inq protein by at least 80 percent, the accompanying loss

61

of infectivity precluded its usc.

In several instances virus preparations prccjpitated

with ammonium sulfate were further purified by centrifuga­

tion through 15-34 percent sucrose gradients. In every

experiment complete recovery of the input virus was noted.

Furthermore, the distribution of detectable protein in these

gradients was limited to the topmost fractions, indicating

that the nonviral material was unable to sediment into the

higher sucrose concentrations. This suggested the appl

bility of sucrose gradients to the purification of CE

viruses. Gradients could be composed of a low density

solution of sucrose capable of preventing sedimentation of

nonviral substances but permitting the sedimentation of

infectious particles. A high density sucrose solution could

serve as a cushion on which the virus particles would collect.

In practice the cushion consisted of 65 percent sucrose

while the low density sucrose solution contained 20 or 25

percent. When 25 percent. sucrose was used, a plot of the

distribution of infectivity indicated the presence of

significant concentrations of infectious particles throughout

the low density solution of sucrose (Figure 9). Although

the amount of contaminating protein excluded was greater

when this concentration was used, replacement by 20 percent

sucrose resulted in greLlter recovery of infectivity in the

frQctions constituting the sucrose-sucrose interface and

so the latter was employed throughout purification

62

9

4000

rl 7 ~ rl

" ~ ~ " ~1 3000 ~ ~ .~

I ill

() ~ rl 0 ~ < H 0 ~ .~

I 5 ~

~

1000 I

Fraction No.

FIGURE 9. DISTRIBUT ON VITY OL-LOWING C IFICAT ON OF SUCKLING ~OUSE

-PROPAGATED TRIV US VIRUS BY SEDI-ION 25% SUCRO ON~O A 65%

expc~r imcnt s . Fractions containing the highest conccntra-

tions of virus particles were collected, diluted 1:2 with

tris-buffered saline and layered onto gradients containing

25-65 percent sucrose. These were centrifuged for varying

lengths of time: Isopycnic banding was achieved by 3

63

houris centrifugation. When viruses were centrifuged onto

a cushion and then sedimented to density equilibrium under

these conditions, the highest concentrations of infectivity,

i.e., the peak, was spread over several fractions (Figure

10). In contrast, when virus prepared by sedimentation

into 25-45 percent gradients of sucrose (Figure llA) and

then resedimented - the second time to equilibrium - in

gradients of 25-65 percent sucrose (Figure lIB), the peak

of infectivity was much narrower, that is, was distributed

over fewer fractions. These results were interpreted as an

indication of particle size or density heterogeneity. By

either method the degree of purification was such that

protein could not be detected (less than 2 ug protein/ml)

by the Lowry procedure after the virus had been "partially

purified", that is clarified and sedimented to equilibrium

in sucrose gradients.

To summarize: California encephali t.is viruses Were

purified without substantial loss of infectivity by a two­

step procedure. Virus particles were first sedimented onto

a sucrose cushion; this resulted in removal of substantial

amounts of nonviral material. Virus particles thnL had

8

rl n

8 (

"'" ~ fI--t r.:y

0 rl

6 QO 0

H

5 10 20 30

Fraction no.

FIGURE 10. DISTRIBUTION OF SUCROSE CUSHIOf\-,CLAB.IFIED, MOGSE BRAIN­DERIVED TRIVITTATUS VIRUS INFECT­IVITY FOLLOWING SEDIMENTATION TO EQUILIBRIUNi IN A 25-65 PERCEN'I' SUCROSE GRADIENT.

64

65

8

7

6

$.-1 5 Q)

P.J

H Q)

..p ·rl -tJ

7 B 0 r1

QO 0

r-=!

6

5

10 20 30

Fraction No.

FIGURE . PARTI PUR IC ION MOUSE N-PROPAGATED SAN ANGELC VIRUS BY ION OF A CRUDE SUSPENSION 25 PERCENT SUCRD (A) GRADIENTS AND 1-FUGA11ION 110 EQU LIER UM (B) A PEAK FRACTION (II ) THROUGH 25-65

SUCHOSE.

66

sedimcntcll onto t~h(:! sucrose-sucrose interface were collected,

layered onto sucrose gradients consisting of 25-65 percent

sucrose in tris buffer, and then sedimented to density

equilibrium. Fractions of each gradient containing the

highest amount of infectivity were collected and used as

"partially purified" virus.

III. VIRUS-ANTIBODY INTERACTION STUDIES

The conditions permitting plaque formation by members

of the California encephalitis virus group were determined

in order that plaque techniques could be applied to the

serological investigation of the viruses' antigenic proper­

ties and intragroup relationships. Before this was attempted,

however, some basic features of the CE virus-antibody

interaction Were studied.

In early immunization trials, mice and rabbits were

inoculated intraperitoneally and subcutaneously, respect­

ively, at 7 day intervals for 35 days with suspensions of

BFS-283 and "30521 11 viruses propagated in suckling mice.

Ten days after the sixth injection, sera were collected

following exsanguination by cardiac puncture. The neutral­

izing capacity of each serum was determined by making

serial tenfold dilutions of the sera and viruses and

incubating mixtures of each combination for 60 minutes at

37 C. Assay for virus surviving the interaction with

antibody indicated that in no i.nstance did the decrease in

67

infectivity by neutralization exceed one log of the inoculum

present at zero time. Moreover, cross-reactions in the

heterologous combinations were complete, i.e., heterologous

virub was neutralized to the same extent as homologous

virus. Immune sera specific for these viruses were also

obtained from K. L. Smart at Dugway Proving Grounds, Utah

and studied. Similar results Were noted.

To examine the possibility that the animals had

responded poorly to the antigenic stimulus provided by the

mouse brain-derived viruses, two rabbits were injected with

chick cell-propagated San Angelo virus emulsified in com­

plete Freund's adjuvant. Both were inoculated in each foot

pad and hind quarter (intramuscular); three weeks later

subcutaneous injections were administered at three positions

along the back in addition to intramuscular injections in

both hind quarters. Two weeks following the second injec­

tion blood samples were removed and the sera collected.

The neutralizing capacity of each serum was tested

with homologous mouse brain-derived (MBdSA) and chick cell­

derived (CCdSA) San Angelo viruses (Figure l2). As was

previously observed for BFS-283 and the local isolate, the

neutralization of MBdSA did not exceed one log during 30

minutes' incubation. In contrast, the neutralization of

CCdSA was rapid and exceeded three logs of inactivation

within 10 minutes. These results suggested differences

betweon viruses propagated in the two types of cells,

100 Mouse brain-derived w virus ::5 $-~ -----------·M :> on 10 ~ \

'M \ :> 'M • :>

\ H ::5

1 \ w \

Ct--! 0

\ +' \ ~ Chick cell-derived (J) .. 0 0.1 virus H "-(])

tl.

0 10 20

Time (minutes)

FIGURE 12. INFLUENCE OF THE HO USED TO PROPAGATE

VIRUS ON NEUTRALI ION BY ANTI-SAN ANGELO VIRUS (C DERIVED) SERUM.

30

68

69

although the possibility could not be discounted that anti-

bodies to host cell substances had either interfered with

the inactivation of MBdV (in the earl r studies) or had

enhanced the neutralization of CCdV in this experiment. To

examine the possibility that anticellular antibodies had

influenced viral inactivation, two experiments were

initiated.

In the first experiment, dilutions of antiserum

obtained from a rabbi t in jected wi th ho'mogenized normal

chick embryo cells were mixed with CCdV and incubated;

fresh guinea pig serum was included in one reaction set

TABLE 10

NEUTRALIZATION OF SAN ANGELO VIRUS PROPAGATED IN MOUSE BRAIN (MBdSA) AND CHICK EMBRYO CELLS (CCdSA) BY RABBIT ANTISERA TO NORMAL CHICK CELLS (Ra-ANTI CC) AND CHICK CELL-PROPAGATED SAN ANGELO VIRUS (Ra-ANTI CCdSA).a

Serum Present Absent

MBdSA CCdSA MBdSA CCdSA ----------

Normal rabbit 1.4 x 106b 1.6 x 106 1.5 x 106 1.7 x 106

Ra-anti CCdSA 4.9 x 105 5.0 x 102 3.1 x 105 5.0 x 102

Ra-anti CC 1.6 x 106 2.4 x 104 1.8 x 106 1.9 x 106

--------a. Assay on chick embryo cells.

b. PF'U/ml

c. Fresh unheated or heated guinea pig serum present at 5 percent (vol/vol).

while heated guinea pig serum was present in another. 'rhe

data sumnorized in Table 10 indicate that anti-chick cell

antibodies did cause inactivation of virus but only when

unheated guinea pig serum was included. The effect WaS

specific for the chick cell-derived agent; MBdV was not

inactivated. Similar experiments with anti-normal mo~se

brain cell sera and mouse brain-derived viruses indicated

no neutralizing effects of anticellular sera. Moreover,

pre-incubation of either type virus with the corresponding

anticellular serum, followed by incubation with specific

antiviral serum, did not decrease the amount of virus

neutralized.

70

In the second experiment, antiserum obtained after

immunization of a rabbit with mouse brain-derived BFS-283

virus was examined for its ability to neutralize the homo­

logous mouse brain- and chick cell-derived viruses. The

data presented in Table 11 indicate that this antiserum did

not inactivate MBdV to the extent detected with CCdV. This

indicated that the host source of the virus used in the

immunization procedure did not affect the ability of the

antiserum to neutralize virus. In contrast, the host source

of the virus used in the neutralization test was important

in determining the amount of virus inactivation.

Numerous viruses, enveloped and non-enveloped, have

been shown to maintain substant 1 amounts of infectivity

following incubation with neutralizing antibodies. The

TABLE 11

NEu'rRALIZATION OF MOUSE BRAIN- AND CHICK EMBHYO CELL­DERIVED SAN ANGELO AND RFS-283 VIRUSES BY RABBIT AN11I­BFS-283 VIRUS (MOUSE BRAIN-DERIVED) SERUM.

San Angelo 283-LP Antiserum

MBdva CCdV MBdV

Ra-anti MBd- 8.7 x 106b 3.4 x 104 6.0 x BFS-283

RNS 3.0 x 10 7 2.6 x 10 6 5.8 x

a. MBdV mouse brain-derived virus; CCdV derived virus.

b. PFU/ml

CCdV

106 4 x 103

10 6 4 x 10 6

chick cell-

virus resisting neutralization has been referred to as the

persistent fraction, protected fraction or simply non-

71

neutral~zable virus and has been attributed to a variety of

causes. The discovery of two viruses - presumably of

identical genotype but distinct phenotype - that exhibited

such marked differences in neutralization by specific anti-

sera, presented an opportunity to examine the nature and,

perhaps, a cause of the persistent fraction. Besides the

contribution of the virus to the persistent fraction, the

possible influence of other components involved in the

plaque reduction technique was also stud d. These included

the assay cells and factors associated with serum such as

spccj fic antibodies, cornplcmc.:nt and other "accessory fact.ors I:

72

Components of complement can contribute to the neutrul­

ization of most viruses, especially when early, IgM-contain­

ing sera are used (Daniels et al., 1970: Yoshino and

Taniguchi, 1965). In addition, cytomegalovirus neutraliza­

tion by hyperimmune, IgG-containing sera has also been

shown to be enhanced when complement is present (Andersen,

1972). Other serum components have also been identified

which increase the amount of virus that can be neutralized

by antiserum. These "accessory factors'll are distinct from

complement, although they may constitute a complex of

factors in which one or more components of complement

participate (Way and Garwes, 1970). Earlier, data were

presented which indicated that the addition of fresh guinea

pig serum did not increase the amount of mouse brain-derived

virus neutralized (Table 10). In other experiments with

different CE viruses from the same cell source, the size of

the surviving fraction was not altered by the addition of

fresh guinea pig sera. Similarly, the addition of unheated

normal rabbit serum to mixtures of antiserum and viruses

prepared from infected mouse brains had no affect on the

neutralization of the viruses. As a comparison and an

indication of the activity of the guinea pig serum prepara­

tion, the latter was included in a mixture of BFS-283 and

homologous early immune serum (Figure 13). The rate of

jnactivation of this virus was considerably greater in the

presence of unheated guinea pig serum. It was concluded

10 U)

::i ~I

.,-j

?

ttO s::::

.,-j

? .,-j

? H ::i U)

ct-J 0

...j-.:>

s:::: Q)

0 0.1 H • Q)

P-t

0 20 o

Time (minutes

FIGURE 13. NEUTRALIZATION BFS-283 (LARGE PLAQUE VARIANT) VIRUS BY HOMOJ-lOGOUS "EARLY II'v1IV1UNE ff HAB­BIT SERUM COLLECTED 7 DAYS INTRAMUSCULA.R I I OF VIRUS: EFFECT OF FRESH (8) (0) GU G SERUM. SENT 95 PERC C E S.

73

74

that heat labile components of sera as well as other

"accessory factors" influenced the inactivation of CE viruses

only when sera that might be expected to contain 19 S,

IgM-type antibodies Were present.

The reduction or depletion of antibody concentration

was investigated as a possible cause of the persistent

fraction by Lafferty (1963a) and Lewenton-Kriss and Mandel

(1972), although neither were able to demonstrate sufficie~t

depletion to account for the surviving infective virus.

This possibility was also entertained with the CE viruses

and their antisera (Figure 14). Mouse brain- and chick

embryo cell-derived San Angelo viruses were both used. At

time zero o~e virus type was mixed with an equal volume of

homologous antiserum; when the surviving virus was limited

to that present in the persistent fraction (pre-determined

by examination of data from neutralization curves) enough

fresh virus (MBdV or CCdV) was added to return the level of

infectivity to the concentration present at zero time.

This resulted in dilution of the antiserum by a factor of

two. When MBdV was added to previously incubated mixtures

containing virus propagated in either cell type the neutral­

ization rate was similar to that of the initial mixture of

MBdV and antiserum. This was also found to be the case

when chick cell-derived virus was added to a previously

incubQtcd mixture containing virus from this sume cell

source. Because of the extremely high persistent frilction

75

100

1

0.1

m A :::::i 0.01 N

.r-l :> bJ) 100 ~

.r-l 10 :>

.r-l :> N 1 :::::i m

ct-I 0.,1 0

B 0.01 ----.-I

0 N Q)

100

~. flt

10 \ I

1 I

I

0.1 __ ..J

0.01 8 16 24

Time (minu s)

FIGURE 14. NEUTRALIZATION OF SAN ANGELO VIRUS TO PREVIOUSLY INCUBATED SAN VIRUS-HOMOLO-GOUS ANT MIXT SEC ADDIT ON OF VIRUS WAS MADE AT 12 MINUTES. 0, MOUSE BRAIN IVED VIRUS; ~t CHICK C IVED VIRUS.

76

of MBC]V, it was not possible to follow the inactivation of

CCdV added secondarily to a mixture of MBdV and antiserum.

Instead, heat-inactivated MBdV was incliliated with the anti­

serum before CCdV was added. The pattern of neutralization

(Figure 15) was similar to that observed in Figure 14B.

Because the effect of heating on the viruses' antigenic

structures was not known, the mouse brain-derived virus

used was heat-inactivated at 37 C and 56 C. As a control,

normal mouse brain material was treated the same as mouse

brain-derived virus, inactivated at 37 C, and then incubated

with antiserum prior to the second add ion of virus. Again,

the capacity of the antiserum to neutralize virus added

secondarily was not significantly diminished.

In precipitin reactions the presence of excess anti­

body gives se to a "soluble" complex characterized by

markedly reduced precipitation. An analogous state has

been described for some viruses (Lafferty, 1963b). High

antiserum concentrations reduce the amount of virus neutral­

ized, i.e., increase the size of the protected fraction.

By mixing MBc]V with increasing dilutions of antiserum, it

could be shown that the concentration of antiserum employed

in the experiments descr d to this point did not prevent

neutralization of MBdV (Table 12). Moreover, a 1:800

dilution of rabbit anti-San Angelo virus (chick cell-derived)

serum 'NdS found to contain enough antibody to neutralize

all dctecLLlble infectivity of the homologous chick cell-

w 100 ::> $..4

• .-1 :>

10 :>

-.-I :> $..4 :'J w

tt-; 1 0

-J-:> ~ OJ 0 $..4 0.1 OJ ~ 0 3 6 9

Time (minutes)

F 15- RALIZATION OF CHICK C PROPAGATED SAN ANGELO VIRUS BY ANTISERUM PRE-INCUBATED W HEAT INACTIVATED MOU 3RAIN-DE-

VED SAN ANGELO VIRUS OR MOUSE BRAIN MATERI . At 37 C HEATED MBdSA; s, CHEATED MBdSA; .t 37 C NMB.

77

TABLE 12

INFLUENCE OF ANTISERUM DILUTION ON THE NEUTRALIZATIO':'J OF MO'JSE BRAIN-PROPAGArr'ED SAN ANGELO VIRUS BY RABBIT ANTI-SAN ANGELO VIRUS (CHICK CELL-DERIVED) SERUM.

Expt. No. Rec procal of Incubat time Antiserum Di- (minutes) lution

0 30 60

1 0 1.7 x 106a NDb 2.0 x

BOO ND 3.0 x 105 2.4 x

1,200 ND 3. B x 105 2.4 x

1,600 ND 3.B x 105 2.B x

2,OOJ ND 4.4 x 105 2.B x

2 0 1.5 x 106

ND 1.7 x

2,00:) ND 1.6 x 105 1.4 x

4,OOJ ND 2.4 x 105 1.2 x

B,OOJ ND 7.5 x 105 2.B x

16,000 ND 1.2 x 106 7.0 x

78

106

105

105

105

105

106

105

105

105

105

--------------------a. PFU/ml

b. Not determined

79

derived virus (Figure 12), although only one log of the

corresponding mouse brain-derived virus was neutralized at

this dilution. Further dilution of the antiserum did not

decrease the persistent fraction of the latter virus. These

results suggested that the concentration of antiserum

utilized did not contribute significantly to the quantitative

level of the persistent fraction.

Mandel (1958) achieved secondary neutralization in a

poliovirus-rabbit antiserum mixture by the addition of

guinea pig anti-rabbit globulin serum. The increased

inactivation was attributed to antiglobulin-induced stabili­

z3tion of primary virus-antibody b~nds. Inactivation of

virus after antiglobulin serum has been added suggests that

viruses can survive the interaction with antiviral anti­

bodies, even when the latter are bound to the virus particle.

An attempt was made to reduce the level of the p2rsistent

fraction associated with mouse brain-derived viruses by

adding goat-anti-rabbit globulin serum to the previously

incubated mixture of virus and rabbit antiviral serum.

Although the experimsnt was performed twice with different

commercial preparations of goat-antiglobulin serum and

included a wide range of dilutions of the goat antiglobulin

and rabbit antiviral sera, secondary neutralization could

not be detected (Table 13). The activities of the anti­

globul in sera 'Nere not tested 7 however I the first lot was

biologically active against rabbit globulins in a

80

'rABLE 13

ENHANCEMENT OF NEUTRALIZAT ION OF MOUSE BRAIN-PROPAGATED SAN ANGELO VIRUS BY GOArr ANrr I-RABBIT GLOBULIN SERUM FOL­LOWING INCUBATION WITH RABBIT ANrI-SAN ANGELO VIRuS SEK;JM.

------~----,--------.------------

Initial incubation Subsequent incub- Titer (PFU/ml) of .l6.Q"I of virus wi: ation-L6QII) w/: surviving virus

NRS*

..

GARS (1:20)

IRS (1:8:J)

..

"

II

II

II

IRS (1:8:J0)

II

II

II

II

..

Diluent

Diluent

IRS

GARS (1:20)

GARS (1:100)

GARS (1:200)

Diluent

IRS

GARS (1:20)

GARS (1:100)

GARS (1: 200 )

3.6 x 10 7

1.2 x 10 7

3.0 x 10 7

2.5 x 106

1.2 x 10 6

1.2 x 106

1.4 x 106

1.5 x 106

1.3 x 106

5.8 x 10 6

3.0 x 10 6

1.8 x 106

3.0 x 10 6

t::

3.0 x 10 0

3.0 x 106

* NRS normal rabbit serum; IRS immune rabbit (anti-San Angelo) seru~. GARS serUl1.

goat anti-rabbit glob~lin

radioimmlnoprecipitin assay with polyoma virus (Dr. P.

Lombard i, personal communicat ion) •

81

Attention was next directed to the possible influence

of the cells employed to assay virus surviving neutraliza­

tion. Kjellen and Schlesinger (1959) found that "neutral­

ized" vesicular stomat is virus was characterized by a

lOOO~fold high2r persistent fraction when assayed on primary

chick embryo cells than on a continuous line leukemic

b::>ne marroN cells. Similarly, Lafferty (1963b) reported

similar a~~unts of neutralization of rabbitpJx virus whan

assayed In chorioallantoic ffit-=mbranes and in suckling mice

but substantially lower am::>unts of inactivation o~ th2

virus Wh211 the mixture was 3ssayed in rabbit skin. He

concluded th3t one of the causes of the resistant fraction

was th2 inability of certain species of cells to recognize

virus as being neutralized, even though illolecules of

"neutralizing" antib::>dy were firmly bound to the virus

particle.

When chick cells and baby h3:l1ster kidney cells -were

used to assay neutralized mouse brain-derived San Angelo

virus, significant differences in the level of surviving

virus could not be detected (Figure 16). When these same

cells Were used to assay neutralized chick cell-derived

virus, a detectable difference was observed, i.e., the

degree of neutralization ~f CCdSA appeared greater wh2n

assayed on BHK cells.

UJ 1 ::> ~

-" ?

QO A ~

0.1 -" ----.-J ? 3 6 9 -" ? ~ ::> UJ

Y--l 0

+' ~ (l)

0 ~

10 (l)

Ili

1

a . 1 L _____ .--'--_____ -'-·-___ ·_··· ___ -'

3 6 9

Time (minutes)

FIGURE 16. COMPARISON OF NEUTRALI­ZATIO~ OF MOUSE-BRAIN (A)-AND CHICK CELL(B)-DERIVED SAN ANGELO VIRUSES BY RABBIT ANTI-SAN ANGELO VIRUS ( DR N-DERIVED) t ASSAY-ED ON PRIMARY CHICK (~) AND MODI BABY HA[V1S reER KIDNEY (.) CELLS. BARS INDICATE 95 PERCENT CON­FIDEr'{CE LIMIrfS.

82

The contribution of the virus component to the high

surviving fraction, as well as the material with which it

was mixed as a pool, was also studied. The experiments

included a determination of the presence of aggregates

within m~use brain-derived virus samples and the existence

of non-infectious virus-specific antigens that might have

possessed serum blocking power. Both have previously bee~

advanced as underlying causes of the persiste~t fraction

(Wallis and Melnick, 1967; Cardiff et al., 1971).

Wallis and Melnick (1967) were able to eliminate the

persistent fraction of several viruses by preliminary fil­

tration of samples through me~branes with porosities not

exceeding twice the reported diam2ter of the individual

virus particles. Their results suggested that virus

sequestered within aggregates did not come into contact

with antibody and, hence, escaped neutralization. Similar

experiments were applied to mouse brain-derived CE groi.lp

viruses, using 220 nm Millipore m2:nbrane filters. An

attempt was made to differentiate between viral aggregates

present before interaction with antibody and immJne a:Jgre­

gates that might be present after reaction with antibody.

However, filtration did not alter the neutralization rate

significantly or decrease the extent of the persistent

fraction (Figure 17).

An Qlternate approach was attempted which provided

information concerning the possible interference of

83

100~----~-_________________ Q

10 ~

bD ~ A .,j

~ 1 .,j

~ H ::> 100 '. UJ

" ct-t '0, 0

+' "0 - _ _ Q_

~ (l) (J\ 10 0 H (l) ~-

....... -----0...

B '0- _ $

-. 1 ~ __ --'--___ I'-----__ L-_____ J

40 80 0

Time (minutes)

FIGURE 17. NEUTRALIZ RIVED LACRO VIRUS RABB V S ( BRAIN-DERIVED) REMOVAL VIRAL(A) AND

FI ION THR 11 220 nm AFT 60 MIN AT

LINES: REACT OF V WITH DASHED LINES: REACTION OF V SERUM.

84

85

neutralization by non-infectious, virus-specific, antigenic

comp~nents. Cardiff et ale (1971) were able to separate two

mouse brain-derived Dengue virus hemagglut ting ant

by sedimentation through a sucrose grad nt. The "rapidly

sedimcnting h2mag'j"lutinin" consisted c>f whole, infectioi.l:3

virions, while the "slowly sedim2nting hemagglutinin" Was

regarded as incomplete virions containing unique anti ic

cO!1formations and protein strllctures. The presence of the

latter in a neutralization mixture was suffic to block

inactivat of the ctious particles by specific anti-

bodies. An effort was made to separate infectious CE virus

particles from nO!1-infect viral components that might

have served to block neutralization.

Mouse brain-derived viruses were fuged thro'..1gh

solutions of 20 percent sucrose onto 65 percent sucrose

cushions and then sedim3nted into 25-65 percent gradients

of sucrose; a fractio~ of each gradient containing a part

of the infectivity peak (F ure 11) was mixed w h~mc>lo-

gous antiserum and incubated. In every case th'3 frac t ion ''Jf

virus surviving neutralization was similar to that measured

when crude infected mouse brain susp'2nsions were used.

The experiments described above ~ere each performed

in an attempt to identify conditions that might contrib~te

information relevant to the existence and nature of th(~

fraction of CE virus resistant to neutralization by specific

antisera. None were fruitful in terms of a definition ~f

Ei6

the factors that wO~lld gi ve rise to this stilte. p." 'xami na-

tion of th2 observation regarding the inactivatin'

genetically identical viruses propagated in diffet It cell

species suggested that phenotypic differences could account

for the distinct patterns of neutralization. California

encephalitis viruses presumably mature when an envelope is

acquired as a result of passage through the hast cell mem­

brane. Differences in me:nbrane compositions between cell

species would be reflected in the surface properties of

the viruses. Three experiments were designed to investi-

gate the sibility that viral envelope differences had

determined the reactivity of virus with antibody and, h~nce,

the size of the persistent fraction of CE viruses. In

each Jf these, the viruses were prepared by sedimentatio~

onto sucrose cushions and then centrifuged into 25-65

percent sucrose gradients. Purified preparations were

mixed with enzymes that might be expected to alter the

antigenic properties of the viruses - if their specific

substrates were present at the virion surface. For each

enzyme, a range of concentrations was studied; at. different

tim::s the enzymatic activity was arrested and homolo:l:)'Js

viral antiserum was added to the mixture. The effect of

each enzyme on viral infectivity W3S determined as well

as its influence on the' ability of specific antisr3rum to

neutralize the treated virus.

rrho first enzyme studied was trypsin. Hannoun (19G8)

reported that trypsin trcatm3nt of sucrose-acetone

87

preparations of CE virus hemagglutinating antigens caused

a significant increase in the hemagglutination ti tors. rfhis

is contrasted by enzymatic treatment of Gro~p B Arbovirus

antigen preparations, which exhibit loss of hemagglutinating

activity after trypsin treatm3nt (Cheng, 1958). A comparison

was made between the trypsin s~nsitivities of the infective

particles of m~use brain- and baby hamster kidney cell

propagated LaCrosse viruses (Table 14).

Trypsin (Nutritional Biochemical Corp., Cleveland,

Ohio) was prepared as a 400 ug/ml solution in tris-b~ffered

sa line, pH 7.5, and was sterili zed by fi 1 trat ion thrOi.lrJh a

220 nm Millipore membrane before use. Tenfold dilutions of

the stock solution were IMde prior to each experiment.

Equal volu~es of 37 C equilibrated trypsin and virus

preparatio~s were mixed and incubated. At designated times

the activity of the trypsin was arrested by adding 0.1 m1

heat-treated calf seru~ to each ~l of the mixture. When

neutralization of trypsin-treated viruses ~as stujied,

appropriate seru~ dilutions were added to each of these

mixtures. At the lowest trypsin concentration added

(2 ug/ml), 9J percent of the BHK-derived virus 'Nas inacti­

vated. In co~trast, at the highest trypsin concentration

used (20,] ug/ml), less than 10 percent of the mouse bra in-­

propagated virus was inactivated. Trypsin treatment of

the latter virus had no effect on the level of non-nQ~tral­

izec] virus following reaction with antiserum. ThGS(~

TABLE 14

EFFECT OF TRYPSIN TREATMENT O~ THE INFECTIVITY OF PARTIALLY PURIFIED LACROSSE VIRUS PROPAGATED IN BHK CELLS AND THE SUCKLING MO~SE.a

Trypsin CO!1cn. Time (minutes)

(ug/ml) MBdLaXb BHKdLaXb

0 10 20 40 0 10 20 40

0 S.Oxl05c ND ND 6 6 1.OxlO 1.2xlO ND ND 1.6xlO 6

2 ND 1.2xlO6 1.lxl06 1.2xl06 ND 1.lx106 7.2xl05 1.6x105

20 ND 1.3xlO 6 1.2xl06 1.2xlO6 ND S.Oxl04 4.0xl04 7.0xlO3

200 ND 1.lxl06 1.Ox106 9.0xl05 ND 333 3.0xlO 3.0xlO 3.0xlO

a. Virus pools clarified by sedimentation onto sucrose cus.hiJn, f~llovled by purification by centrifugation through a sucrose gradient.

b. M3dLaX mouse brain-derived La.Crosse; BHKdLaX = BHK cell-derived LaCrosse virus.

c. PFU/m1.

d. Not determined.

00 00

findin~fS W'2rc also noted when Trivittatlls virus was used.

The results indiCdted that susceptible peptide bonds

associated with BHK-propagated virus were n~t exp0sed on

the mOUSe brain-derived virus.

LaCrosse virus propagated in the brain tissues of

suckling mice Was treated with a preparation of phospho-

lipase C in an attem?t to select ly remove phospholipid

89

compone.:.1ts of the viral envelope that could contrib:.r':e to

steric hindrance of antibody rn~lecules. The enzyme is

kn~::r.rJn to cleave the ph~sph~diester linka h3twee.:.1 ph'~s­

phatidic acid and the nitrog(~nous base of phospholipids

(Mahler and Cordes, 1971). Friedman and Pastan (1969) were

able to remove 60 p'::!rcen t of th'2 phosph'Jlipid of Semliki

Forest virus withaut causing loss of infectivity. In con­

trast, the infectivity of influenza virus (WSN strain) was

greatly decreased a a short exp0su~e to the enzyme

(Simpson and Hauser, 1965). LaCrosse virus was mixed with

increasing cO.:.1centratio~s phosph'Jlipase C (Worthingto:)

Biochemical Corporation, Freehold, New Jersey) and the pre­

equilibrated (37 C) mixtures were incubated for variou3

tim~s (Table 15). The dilu'2nt resembled tr -b'..1£fered

sal b'.lt contained .OJI M CaC12 and n'~ EDTA. Because th2

enzym2 requires calcium ions (Friedman and Pastan, 1969),

its activity could be arrested by adding 0.2 ml of 0.01 M

EDTA to each ~l of th8 mixture. Subsequ'2nt neutra 1 iZdt. io()

expcrilIv~ots 'w'2re per for me.: c1 in a manner similar to the

90

tryps in studies. Exp0sure of the virus to 10'J u'J of phos-

ph()lipase C resulted in an increase in the infectivity titer

of the virus preparation 'tlhich "was rnaintuined for at least

15 :ninutes. In the presence of 1 mt] of th(~ enzyme increasec1

infectivity was noted after 5 minutes but by 15 minutes a

considerable loss was apparent. The size of this persistent

fraction of th0 phospholipase C-treated virus susl?ension

was similar to that of the control suspension that had not

been incubated with the enzyme.

TABLE 15

EFFECT OF PHJSPHOLIPASE C TRE..l\'TMENT ON THE INFECr­IVITY OF PAR"fIALLY P'JRIFIED M'J~JSE B!l..l\IN-DERIV£.iJ LACROSSE VIRJS.

PhJspholipase C Con2entrat on Time

(minutes) ____ . __________ .LlJ9.LIIl!:.t: _________ ~ _____ _

o 10 108 100J -- -.-----.---~.-~------ -_._-,------_.- --.--.--

0 4.2 x 107b NDc

ND ND

5 ND 4.8 x 10 7 1.0 x 108 9.9 x 10 7

15 3.8 x 10 7 4.0 x 107

8.6 x 107 2.2 x: 1J 7

Cl. Worthin3ton Biochemical Corp., 1.5 u~its/mg.

b. PFU/m1.

c. Not determined.

Expcri~ents were also conducted with a preparati~n J£

ne',JraminidZlSC (Sigma ChemicQ1 Company, St. Louis, MissQuri).

rrho rat ionale behi nd t.h:! usc of this enzylw:: was simi lar to

that of phJsp!l,)lipase C: RCITl:Jval of negatively-churgcd

molecules of neuraminic acid present on viral glycolipids

and glycoproteins (Klenk and Choppin, 1970) might alter

the surface of the virus in such a way that molecules of

91

antibody would no longer be sterically hindered. 'rhe enzyine

was used at a concentration of 0.2 units/ml~ its activity

was inhibited by the addition of 0.01 M N-acetylneu~aminic

acid (0.2 ml per ml of the incubating mixture). After 3

hours of incubation the mouse brain-der~ved LaCrosse virus

titer decreased from 2.2 x 10 7 PFO/ml to 1.2 x 10 7 PFO/ml.

When specific antiserum '/'Jas added 30, 60 or 120 min'ltes

after neuraminidase had been, the amount of virus neutral-

ized wa.s similar to that observed for untreated controls.

To summarize: The use of California encephalitis

viruses (propaga ted in th l3 brains of suc::kling :nice) in

plaque reduction studies resulted in the presence of an

extremely high level of non-neutralizeable virus. Neither

the source of the antiseru~ nor the source of the virus

used to proj~ce the antiserum influenced th9 amou~t of

surviving virus. In contrast, the h~st cell in which the

virus used in the neutralization mixture was propagated

determined th2 degree of virus inact.ivation, and h'2nce,

the level of the persistent fracti~n. Neutralization of

mouse brain-df~Livcd virus was not blocked bi virus­

specific, non nfectious material present in infected

iTI,)use brain s~..lspensions and filtration to remove viral

ag'3rcgatcs did not inccease th·; amount of neutral iZdt ion.

92

The add ion of antiglobulin t.O virus-antibody mixtures did

not result in increased loss of infectivity. Indirect

evidence was obtained sllggesting that surface differences

between viruses propagated in the two cell types lTIay have

been responsible for the marked differences in degree of

neutralization.

IV. NEIJTRl1.LIZl\TION STUDIES

Comparis0n of the antigenic relationshi among

several California enc litis viruses was attempted by

applying the plaque reduction techniques desccibed by

Dulbecco at ale (1956), and used by M=Br e (1959) in the

study of antigen lly related viruses. B2 the

riments W(2re performed, the influence of several

potential variables was investigated.

The first variable considered was that of initial

virus concentration and its effect on the resulting rate of

viral inactivati:Jn by a!1t.isex-um .. It was found that varying

the concentrati8n of virus present in the neutralization

'nix"tul-e at time zero over a hundred fold d ilut.iol1. ran'3t:; did

not affect the rate of neutralization gure 18). This

permitted at ast a tenEold dilutian away from the

undiluted v pools. Subsequt=ntly, virus stock prepara-

tiol1S were diluted at the time Df each experiment to con-c:.

tCl in 1-5 x 10° P';:"U/ml.

100

en :::; ~l

.r-j

:> QD s:::!

.r-j

:> .r-j

:> H :5 en

4-l 0 1

...j-:>

s:::! (J)

0 H (J)

P-t

0.1 0 3

Time (minutes)

FIGURE 18. EFFECT OF JNITIAL V TRATION RATE NEUTRALIZATI CELL-PROPAGATED SAN ANGELO V S HYPERlMMU RABB ISERUM. ~, 10 · e, 10 ·5 PFU/ml; A, 105.5 PFU/ml.

a o ...

9

CONC CHICK

OUS PFU/ml;

93

The nurnber of virus particles in a virus-antiserum

mixture inastivated by neutralizing antibodies have been

shown to be influenced by the type of cells on which the

mixture is assayed (Kjellen and Schlesinger, 1959) and

als'J by the h·")st cells in which the test virus has been

propa3ated (Lewenton-Kriss and Mandel, 1972). An atte:npt

was made to detect any contr ib'.1tion ei th'er of these might

make to the CE virus-rabbit antiserum system ·-1nder study .

94

. Chick e:nbryo and baby hamster kidney cells were used to

propagate as well as to assay the neutralization of San

Angelo virus. The results of this experiment are pres r3nted

in Figure 19. Chick cell- or BHK cell-derived San Angelo

virus \!vas mixed wi th an equa 1 volume of appropr ia te ly

diluted rabbit antiserum to m~)llSe br.-ain-deri vee] San Angel,")

virus. Each component of thf3 mixt.ure '.-las equilibrated to

37 C before admixture and both virus preparati8ns were ~3ed

at similar concentrations. Infec ti vi ty sample:3 '·rlere

removed from -:'h '3 incubatin9 m=.x·tures at on·3 miOtlte intervals

between ze r-o tim(:;! and 6 minu tes. Previously, it had been

nJted that the curves d:cawn to represe.::1t th r3 k Lnet of

CCdV neutralization frequen·tly exhibited an in it 1 la3

that preceded exponential loss of infectivity. This lag

(sh0ulder) was evident in the experimental results pre­

sented in Figure 19Jn both typ:=s of assa.y cells. Th2

deviation [rom linea.rity of the plot of B:-iKdSA in3.ctivc1ti')n

W~1S not as prot1:Junced ()n ei thor tYP'2 of co 11. Th0 rcsl11ts

100

10

1

A o • l _________ L ____ --.-L ___ 1

o 2 4 6

100

10

1

0.1 t

o

B

2

Time (minutes)

FIGURE 19. NEUTRALIZATION OF BABY HAIVlSrrER KIDNEY (,,) -AND CHICK EMBRYO CELL (A)-DERIVED SAN ANGELO VIRUSES BY RABBIT AN~I(MOUSE BRAIN-DERIVED) SAN ANGELO VIRUS SERUM: ASSAY ON PRIMARY CHICK ElVlBRYO (A) AND MODI­FIED BAP), HAIVi~;ITER KIDNEY (B) CELLS. BARS INDICATE 95 PERCENT CONFIDENCE LIMrrS.

95

96

also indicated that the two cell types Were similar in

their discrimination between neutralized and infectious

viruses.

Whi le c neutralization rate constants have been

used by several inve~tigators to indicate the ant lC

relatedness of viruses (McBride, 1959; Ozaki and

1967), th·~ ob3erved deviations from linearity precluded

th,~ir appl ication to the campa rison of CE v l.rLlses .

Simi larly, oth';::r met.hods that have been us to compare f

in quantitat terms, the antigenic relat . c lpS 0_

viruses ware nat used beca~se, generally, they have nat

taken into acco~nt the possible ex tence of an initial lag

in neutralization or have overlooked the sen::::::e of nO(1-

neutralized virus. This necess i tated ·th~ deve lopmen t of

an a ~~ans of presenting a meanin 1 comparison of

the antigenic celationships of the CE viruses.

Num~rous investiga.to:-s have pointed out thl~ import.an~e

of lo\ving th.;:: interaction of v cles and 3.nti-

sera kinetically, particularly When se'leral viruses are

studied that demonstrate a high de e cross-rea~tivity.

Bes 2S nl~utralization tests, kinet method3 have been

d to complement fixation campa sons of viruses

(Hatgi and Sweet, 1971) and hemagglutination inhibiti0n

stul]ies of related viru3 isolates (Casa.ls I 19(4). In each

rate th~n any heterologous reilcti~n. Th~jS, kin.~t_ ic techn iqUC:3

97

pennit easy identification of homolo90us viruses and in some

instances can be used to indicate relative relationships

among heterologous viruses. Conversely, Hashi~oto and

Prince (1963) were able to detect virus-antibody dissocia­

tion by followin9 the neutralization of Japanese encephalitis

virus kinetically. With these observations in mind, an

effort was made to determine a dilution for each antiserum

that would result in a similar rate of inactivation of the

respective hom~)logous viruses. Within the limitations

inheren·t to the plaque assay, comparis:)l1s of the neutral iza·­

tion of any virus by different antisera would then be

possible. The assumption was made that antisera giving

rise to similar virus inactivation rates with their respec­

tive h()lllOlo90US viruses would contain similar numbe::::-s of

neutra l.izing antibody molecules. It was found I experimen·-

tally, that dilutions of each serum could be determined

that would leave 20-25 percent, 1.7-5.0 percent, a~d 0.3-1.0

:percent of the oJ.:-iginal infectivity at 3, 6 and 9 minute:3,

respectively. The semi-logarithmic plot of these values

W:.lS a straight line. In practice the fina 1 dlletti ')ns for

the different s~ra tested varied between 1:80 and 1:1400.

Tne va 1 id ity of the assu;nptions mentioned above was

tested by comparing the neutralization of BFS-233 and th~ee

heterologous viruses by two different preparations of

antiserum obtainQc] by inoculation of mice with BPS-23J Cl.!1d

foll~)\v'ed by collecti:)l1 of the immune ascitic fluids

(Figures 20 and 21). These sera were prepared at the

National Institutes of Health in 1963 and 1956 and '/J1;!re

distribu.ted as reference antisera. Th,= optimum d i llltions

Were determined to be 1:1100 ior the earlier preparation

and 1:408 for the fluid prepared in 1966. Experimentally,

the reactions of the heterologous viruses with each an-ti­

serum were unique but were similar between antiserum

pre ra tio!1s. Reprodu::::ibi li ty between sera pared in

98

rabbits was also noted. It was observed that the rabbit

sera t=xhibited a degree of variation that might be expected

between similar animals gure 22). In every instance the

patterns ''Jbserved b'2tween th'2 pa o~ rabbit sera Were

similar with respect to th9 neutralization of heterologo~s

viruse:3.

The possibility that neutralizing antibodies continued

to inactivate virus comprising the surviving fra~tion after

infect ity sa~ples had been removed from the incuDatin9

mixtures was also considered. In neutralization experi-

ments, samples removed for assay of residual infectivity

were routinely diluted ol1e hundredfold in chilled buffer

as quickly as ~ossible to minimize further neutralization.

An indication of the neutralizing activity contained in

sera diluted this far beyond the described optimum dilution

was ascertained by incubating sera at the h

and hOl11o viruses for 3 hours (Table 16).

dilutions

In no

instance di(l the amount of hOlTIt)logolls virus inactivatccl

exceed on(~ ll')g.

100

10

1

0.1

100 ---o-e

10

1 LaCrosse

\

San Angelo ----1.------1._-.1--

,

Trivittatus

\

\

, '0

0.1 --L_--,-I _--'-_--'

a J 6 9 60 a J 6 9

Time (minutes)

FIGURE 20. NEUTRAIIZATION OF CALIFORNIA ALITIS VIRUSES BY MOUSE I-BFS­ASCITIC FLUID (NIH REFERENCE ANTI PARED 6-66.) ANTISERUM DILUTED 1:400.

100 rn ::5

10 ~ or-! :> bD 1

0 s::: or-! :> 0.1 or-! :> ~ ::5 100 rn

~

----4t-----.--------, ct-1

, -~~~ 0 10

, • .p

s::: (J) 1 0 ~

Trivittatus Q) LaCrosse P-i O.l I I I

0 ') 6 9 60 0 J

Time (minutes)

60

\

\

" '0

0

FIGURE'; 21. NEU fllRALIZ ION OF CALIFORNIA EPH-ALITIS V SES MOUSE ANTI S-28J VIRUS ASCIr:l'IC 1D (NIH REFERENCE ISERUUl, PAHED 3-26 3.) AN~iISERUM DILU11 1: 1100.

99

100

10

1

0.1

100

10

1

0.1 o

-'- -- ....• -.---.-.- ....... -.--.---- ~-'. ---0

----4~ '- .... '- " ..

A

\ \

\ \

\

_-J.. ___ ----L ____ ---lI ____ -'

--l-.---JI"-~---Ar- - - _ _ •

'-

B

----~------~I------~----~ ) 6 9 60

Time (minutes)

FIGURE 22. TRALIZATI OF CALIFORNIA EPHALrrIS VIRU BY ANTISERA COLI,ECTED

FROM TWO RAEB S SUB~ITTED TO S ILAR SCHEDULES OF I ION WI1'H BFS-28) VIRUS. IT A SERUM DILUT 1:600; RAB­BIT B SERUM DILUTED 1: 00. e, TRIVITTATUS VIRUS; A, JERRY SLOUGH VIRUS; 0, BFS-2o) VIRUS.

100

TABLE 16

RESIDUAL ANTIVIRAL ANTIBODY ACTIVITY OF RABBIT SERA AFTER lOO-FOLD DILUTION BEYOND PRE-DE'rERM[N8D OPT I'1AfJ DILUTION FOR NEUTRALIZF\TION OF HOMOLOGOUS VIRUS.

Rabbit hyperimmune, Time (minutes)

antiviral serum 9irected against: 0 60 180 13:-la

Jerry Slough 1.9 x 106c 1.4 x 106 8.5 x 105 1.4 x

Trivittatus 1.4 x 106 1.4 x 1116 1.4 x 106 1.2 x

San Angelo 2.6 x 106 2.1 x 106 1.6 x 106 2.2 x

San Angelo b 2.3 x 106 2.3 x 106 1.6 x 106 2.0 x

Tahyna 2.4 x 106 2.6 x 106 2.3 x 106 2.8 x

B8'S -283 2.0 x 106 1.6 x 106 1.5 x 10 6 1.4 x

L.:.Cross8 8.8 x 105 5.5 x 105 3.0 x 105 NDd

a. Normal rabbit serum controls.

b. Rabbit anti-S:::.n .l\ngelo virus (chick cell-propagated) serum. All other sera are rabbit anti(mouse brain propagated) virus sera.

c. PFtJ/ml

d. Not determined.

101

106

106

106

106

106

106

Another variable considered was the influence of anti-

serum concentration on the neutralization of heterologous

vIruses. The two viruses selected, Jerry Slough and James-

tow~ Canyon, have been classified as variants of one virus

(Sudia et al., 1971). When mixed with the optimal dilution

of LaCrosse antis(.jrum (Figure 23B), inactivation of lJames-

town Cunyon v irus could not: be detected. However, when

rn 1 ::::5 ~

.r-! :> b.O A ~

.r-! 0.1 :>

.r-! :> ~ ::5 rn

ct--l 100 0

-j-J

~ Q)

0 H Q) 10

Pi

1

0.1 ~-------~----~----__ ~ o 3 6 9

lJ.lime (minutes)

FIGURE 23. INFLUENCE OF ANTISERUM CONCENTRAlJ.1 ION ON THE KINE'I'ICS OF' HOMOLOGOUS AND HETEROLOGOUS CALI­FORNIA ENCEPHALITIS VIRLS NEUTRAL­IZATION. At RABB ANTISERUM TO OCOUSE BRAIN-DERIVED LACROSSE VIRUS DILUTED 1:100; B, 1:]00 ("OF:[1IMAL") e, LACROSSE VIRUS; A, JERRY SLOUGH VIRUS; 6, lTAlVlESTOWN CANYON VIRUS.

102

103

the antiserum concentration was increased threefold (Figure

23A), neutralization was apparent. Interestingly, the

neutralization of Jerry Slough virus was not affected by

these antiserum changes .

. It was concluded that the use of rabbit antisera at

the dilutions effecting the described rate of neutraliza­

tion would provide an acceptable basis for the comparison

of CE viruses by homologous and hetero19gous antisera.

Having considered the influence of assdY cell, virus source

and concentration, antiserum ~oncentration and biological

differences between irnmllnized animals, several experiments

were performed to determine the degree of cross-reactivity

detectable when heterologous viruses "'Iere mi.xed with each

of several different antisera. For these experimen~BFS-283

(large plaque), LaCrosse, San Angelo, Trivittatus, Tahyna,

Jerry Slough and Jamestown Canyon viruses were each inocu­

lated into +:.wo rabbits which wece then immunized according

to the schedule outlined in Table 2. When the hyperimmune

sera of these rabbits were stud d, was noted that each

serum except those specific rqr Jerry Slough and Jamestown

Canyon viruses could be used in neutralization experime~ts

at dilutions greater than 1:100. Jerry Slough antiserum

was used at a 1:80 dilution; neither serum frOILl 1:..11e rabbits

injected with Jamestown Canyon virus was able to neutralize

homologous virus at dilutions as low as 1:40 and so they

Were not used. The rabbits Were inoculated with viruses

104

propagated in mouse brain tissue whih.~ the vi.rI.U:)(~~; used in

plaque reduction studies were propagated in BHK cells: This

maximized the likelihood that antibody would react with

virus ific antigenic components and that anti-host cell

antibod s would not interfere with or contribute to neutral­

ization. Infectivity samples were removed from the incubating

mixtures at 3 minute intervals for 9 minutes; the next

. samples were rem:)ved at 60 and 180 minutes. Normal serum

was used in zero time and 18J minute control tubes: After

3 hours' incubation, s()me loss of ctivity was noted,

although this seldom exceeded 0.5 log (32 percent). In

each of the llo\"ing F l gures the neut La 1 i za ti on of the

homologous viruses is plotted in the lefthand graph.

The neutralization of viruses that did not cross-react to

a significant degree, i.e., lose more than 50 percent of the

zero ti me infec vi ty by 60 rninutes, has bec?n reported

separately as the reduction of infectivity at 180 minutes.

The advanta of kinetic campa sons became evident

when the results of inactivation of se~/eral CE viruses by

BFS-283 antiserum (E'igure 24) Were compared with data ob­

tained by the immunodiffusion studies of Calisher and

Maness (1970). In the neutralization stud s significant

sh;')ulders were evident in the plots in Which no virus hao

been inactivated. These were apparent for each the

heterologous viruses batween zero and 6 or 9 minutes. The

homologous virus was eusily distin9uished from all o:'her

W :J H

•. -1 :>

:> .r-! :> H ::::> w

'H o

+..:> c C> u H

100

10

1 3-283

0.1

, 100 '6

10

1 Janie s town Canyon

0.1

100 ~-----w Q ,.

" " 10 Q

1 Keys

--.------. \

San 10

Trivittatus

I) I) ~

\

\ \

\ •

'\.

'0

\ \

\ Tahyna \

I loooV o 3 6 9 v Q 60

(minutes)

.--. . . -. l

LaCrosse

~ " .........

Jerry Slough I" "" " I

f t determined

[ Me~ao o 3

URE 24. NElJTRA1IZATION CALIFORNIA IS VIRUSES BY MOUSE BRAIN IVED BFS- VIRUS. 1-

1:800. r-' o U1

106

viruses studied; the heterologous viruses could be grouped

according to their patterns of inactivation. Thus, San

Angelo and Tahyna viruses were easily distinguished from

Jamestown Canyon, Trivittatus and Keystone viruses. La­

Crosse virus was distinct in that it did not cross-react

with anti-BFS-283 serum during 60 minutes' incubation.

After 180 minutes less than one log (0.7 log) of this virus

had been neutralized. In the experiments of Calisher and

Maness (l970), LaCrosse, San Angelo and Jamestovln Canyon

viruses exhibited "3+" precipitin reactions against BFS-283

antiserum while the homologous virus was characterized by

a "4+" reaction.

The results of experiments on neutralization of several

California encephalitis viruses by laCrosse hyper-immune

serum have been summarized in the graphs of Figure 25.

The time course of neutralization of Jerry Slough virus

was similar to that of the homologous LaCrosse virus,

although an initial lag was evident and after 9 minutes

the rate of neutralization decreased considerably.

As has be~n noted previously, Jamestown Canyon and

Jerry Slough viruses are regarded by some investigators as

variants of one virus (Sudia et al., 1971). It was inter­

esting to note the unique patterns of neutralization of

these viruses when mixed with anti-LaCrosse serum. For

the other viruses the degree of cross-reactivity was

limitcc]. The extent of neutralization of Trivittatus,

W :::5 H

.r-j

~

> H :::5 tn

<+-1 o

o H C)

P-t

100 0 i " ,"'-i " 10 L 0, i "

1 L "~ O

I La.,C"Y'o("'s 1

I . ~ c e • ~I ! _ I,.., D. D I

U 0 v

100

\ 10 \

\

1

0.1

~ 100 r Q ~ I

10 L

1

0.1 0 o

• II

l .. .. - ..

[ sa~ An~elO IOV>,A,I

roo 0--0

I LTr\Vi tt1a tU~"AA.1

--....

()

Tahyna

TimE~ (minu S)

283

, , '0

I 100"\A,1

, , Jerry S10u.gh '.It.

1

,.---.y I Y--y

I-I

[Me~~o 1 1--.1

o 3 6 9 60

ZATICN CALIFORK ENCEPHALITIS VIRU TO SE BRAIN-DERIVED LACROSSE VIRUS. ANTI-

1:300. t--' o -..J

108

Melao and San Angelo viruses after 180 minut.es lS in(lif~at!:!ll

in Table 17. The neutralization of each of these viruses

did not exceed one log after 3 hours.

TABLE 17

EFFECT OF E>ITEND2:D TIME ON THE NETJrRALIZATION OF SE­LECrrED CALIFO::<!.JIA E~\IC£PHALITIS VIRUSES BY RABBIT ANT 1-MOUSE BRAIN-DERIVED LACROSSE VIRUS SERUM. a

Virus Time (minutes) -~--,-~--

Strain Ob 60 180 180b

,.. 105 105 TVT 1.4 x 10° 7.0 x 5.0 x 1.0 x 10 6

MEL 1.8 x 106 9.0 x 105 1.6 x 105 1.0 x 106

SA 1.7 x 106c 1.1 x 106

2.0 x 105 1.6 x 106

a. Antiserum diluted 1:300.

b. Normal rabbit serum controls.

c. PFU/ml.

When Tahyna virus antiserum was used to inactivate CE

viruses only Su.:! Angelo and BPS -283 'were neutralized to a

significant extent (Figur2 26). As was noted with the

other antisera, inactivation of these viruses was preceded

by a lag. LaCrosse, Trivittatus, Jerry Slough, Keystone

and M21ao viruses were indis nguishabl'2 whC:.:tl their neu-

tral tion curves Were compared. Extension of the reac-

tion time of four of the isolates to 180 minlltes die] not

Eaei 1. i tat(~ their serological separation by th is antiserum

(TaLle IFn.

{;)

:::s ~

.r-l

>

100 (Z

10 I,,(I~ 1 [- ()

o LTahyna . 1 . ____ --'__ I ~ _ or.r.1 ~~\J

100 43---<bl!.~---_ 6

> .,-1 10 > H

c75 1 'H o +-" C (])

o H Q)

P-i

Jamestown Canyon o • 1 I I ('\vo"c\v t

o _'-11---------,...-..

" ~

10

1

II III I \ I

\

San Angelc

\

\ \ IZI

I 1 I" r. A I

Trivittatus

-Q

.......

\

\

"D

\

Keystone BFS-28J \ o Ir. r. "" I

o 0 J 6 9 v v6vo

Time (minutes)

.-..

LaCrosse

~ A,

Jerry Slough

Melao I

-.

'.

" y

I I I I" r... '" I o J 6 9 v~60

26. NEUTRALIZATION OF CALIFORNIA ENCEPHALITIS VIRUSES BY ANTISERUM ~O MOUSE BRAIN-DERIVED TAHYNA VIRUS. ANTISERUM

DI l:JOO. I--' o \.0

TABLE 18

Nf:UTRALIZATION OF CALIFORNIA ENCEPHALITIS VIRUSES BY RABBIT ANTI-TAHYNA (MOUSE BRAIN-DERIVED) VIRUS SERUM. a

Virus Time (minutes)

Strain Ob 180

b 60 180

JS 5.0 x 105 8.0 x 104

2.0 x 104 4.8 x 105

LaX 1.7 x 106 7.0 x 105 1.1 x 105 1.7 x 106

TVT 1.1 x 106 2.0 x 105 1.0 x 105 1.0 x 106

KEY 5.0 x 105 1.0 x 105 4.0 x 104 4.0 x 105

a. Antiserum diluted 1:300.

b. Normal rabbit serum controls.

The antiserum s ic for Jerry Slough virus was the

most specific of the sera used: Fewer cross-reactions were

noted with this antiserum than with any the ot.heJ:-s

(Figure 27). After 180 minutes the amount of each virus

that had been vated had not increased from the degree

of neutralizat noted at 60 minutes (Table 19). A four-

f·J1d i·1.crea.s(~ in the antiserum concentrat did not

significantly increase the amount of virus inact

(Table 19).

110

Keystone, Melao, Tahyna and San Angelo viruses cxhib d

significant cross-reacti\'it.if~s when mixed with Trivittutus

virus antiserum (Figure 28). The neutralization of the

other viruses was not eXi:lmined at extended times.

W :3 H .~

:->

:-> .~

:-> H :3 tf)

ct-l o

+' ~ Q)

o H Q)

P-t

100 A I >-,

l:r~, ~Je Slough

o • 1 l I I 10 0 ') I

100

t termined 10

1 Jamestown Canyon

0.1

100 r ~,

"'-• 10 I

11 [Keylstone

0.1 I 10 ~ 0 I v v v

0 3 6 9 60

I q a a -6

I San Angelo h' "I

o

Trivittatus

r () () ()- -()

I Tahyna 10 "0 I I j 9 v v 60 0 3 6

Time (minutes)

[] 0 O--EJ - .....0 , I

I

l I BFS-283 I, , , 1

vvv

---.-. 0 0- -e I

L

1- La~ros~e Ip ~ I oe~

I-V,

"-• Melao

FIGURE 27. NEUTRALIZATION OF CALIFORNIA EPHALITIS VIRUSES BY RA5B ANTISERUM TO MOU BRAIN IV JERRY SLOUGH VIRUS. 1-SE? DI 1:80.

t-' I-' I-'

ill ::::s H

.r-!

>

.r-!

> .r-!

> H ::::s ill

!f-i o ~

>:: OJ u H OJ

P-t

100 ~, !~

l~ ~O I Trivi ttatus 0.1 I ! I 10 "'> A I

lOC! Ls n 6- -6

10

1 Jamestown Canyon

o • 1 '00 ° 0 "0'

100~ 10 L

I

1 Keystone

0.1 o

" '~

FIGURE 28. NEUTRALIZAT RA3B ANTISERUM SERUM DI 1:900.

o

San Angelo

\

\ \

• If) f) D I v v

........

LaCrosse

~ \

\

........

\

Tahyna \ Lr" r.. A'rt

3 6 9 v V6V o

Time (minutes)

GJ 0 i ""-

i r t BFS;-283

IL, ,~,J

A: .A

o

A

Jerry Slough

lao

10 " I'. I v <po

" '"

--'...c,"-~

3 6 9vv

60

OF CALIFOR~IA EPHALITIS VIRUSES BY BRAIN-DERIVED TRIVITTATUS VIRUS. ANTI-

f-' t-' tv

113

TABLE 19

INFLUENCE OF Kl(TENDED T IMg AND [I..J"~~REASED CONCEN­TRATION OF' ANfrISERrJM 0::1 Ti-IE NEUTRi\LIZATION OF CALIFORNIA ENCEPa~LITIS VIRUSES BY RABBIT ANTISERUM TO JERRY SLOUGH -vIRUS PROPAGATED IN SUCKLING MICE.

Virus Time (minutes)

Strain 180b Oc 180a 180c

JS 1.4 x 106d ND ND 1.3 x 106

rrAH 1.4 x 106 1.7 x 106

1.4 x. 106 1.6 x 106

LaX 1.2 x 106 1.2 x 106 7.7 x 105 1.2 x 10 6

106 106 106 r

SA 2.2 x 1.8 x 1.6 x 1.9 x 10°

106 106 105

r

Tv'r 1.6 x 1.2 x 7.7 x 2.2 x 10()

283 1.8 x 106 1.8 x 106 1.2 x 106 1.6 x 106

KEY 2.0 x 106 1.5 x lOS 4.2 x 104 1.2 x 106

MEL 5.0 x 105 1.4 x 104 3.0 x 103 5.0 x 105

a. Antiserum diluted 1:80.

b. Antiserum diluted 1:20.

c. Normal rabbit serum controls.

d. PFU/ml.

e. Not determined.

When San Angelo virus antiserum was used to neutralize

other CE viruses, cross-reactivities were noted with B?S-283,

Jamestown Canyon, Trivittatus, Jerry Slough, Keystone and

Tahyna viruses; only LaCrosse virus was not neutralized by

the serum (Figure 29). Melao virus was not studied. San

Angelo virus, significantly, could not be distinguished

w ::s H

'M :> cD >=:

'M :>

'M :> H ::s m

ct-1 o

+> >=: (!)

o H (!)

P-i

100~

10 1_ '1II~ I

1 L San 10

o • 1 I __ L... __ 1. I ov"',.p,)

100i~ 10 L "

1

0.1

100 ';I \iii n

10

1

0.1

, '~

Keystone l----kv-V'VJ

o 3 6 9 60

FIGURE 29. NEUTRALIZATION RP..B3IT ANTISERUM TO MOUSE SERUM LUTED 1: O.

- -.

LaCrosse 1"\,0.,,1'\. ,

....... .... 0

Trivittatus

Tahyna

o 3 6

\ , , \

\

\ lov'vl'.,f)

9 60

'Time (minutes)

~ \

\

5-283 \ b., ,,b

U 0

, ' ...

Jerry Slough

~ Not determined

I Melao 1. I I ...... 0 I v v v

o 3 6 9 60

IS VIRUSES ANGELO V • ANTI-

f-' f-' ~

115

from Trivittatus virus by the anti-San Angelo virus serum

during the first 9 minutes of reaction. Significant,

immediate inactivation was also apparent with the neutral­

ization of BFS-283; however, the rate was lower than that

of the homologous reaction.

DISCUSSION

The experiments described herein Were conducted in

an attempt to further the understanding of the complex anti­

genic relationships which characteri~e the California

encephalitis group of viruses. While the intention was to

co~pare the viruses by plaque reduction techniques, the

studies found necessary before this could be achieved c~~­

prised a significant proportion of the experiments. For

purposes of discussion the data can be separated into four

topics: pH and plaque formation; the persistent fraction

in neutralization reactions; the kinetic lag noted in homo­

logous and heterologous neutralization reactions; and the

cross-reactivities observed between members of the CE virus

group.

The behavior of the two types of cells used to investi­

gate plaque formation by CE viruses was similar in most

mensurable respects. One obvious distinction was the

ability of the line of hamster cells to support formation

of plaques by certain members of the virus group that did

not form countable plaques on chick embryo cells, at least

not under the conditioI)s studied. St~veral experilOont.3

indicated an extraordinary sensitivity of most of the

viruses to the conditions prevailing during incubation of

117

infected cells under overlay media containing agarosc.

That this gelling agent may have influenced plaque

formation Was suggested by several experiments. For

example, the size and number of plaques Viere influenced by

the medium employed in the overlay, while the patterns of

replication - in fluid cultures - in terms of the kinetics

of appearance of viruses and yield of particles - were not

affected by the medium used (Figure 6). The pH of the

overlay ~9dium at the ti~e it was added to infected cells

also influenced plaque formation. This affect was also

influenced by the presence of agarose: A virus that pro­

duced plaques at pH 7.0 but not at pH 7.3 replicated in

fluid cultures equally well at pH 7.0, 7.3 or 7.6. The

observation that plaque sizes were increased when DEAE­

Dextran was included in the overlay medium suggested the

presence of contaminating ani~nic substances i, the

agarose preparations that were capable of inhibi ting th,e

virusGs. The noted increase in plaque sizes and numbers

when the concentration of the agar-)sc; ',Jas decreased also

seemed to indicate the presence of inhibitory substances,

although the decrease in concentration that effected this

improvcmc~nt was not great. Indeed, the slight change in

concentration could have had a greater affect on the

diffusibility of nutrients or metabolic products through

the gf~l.

118

Plaque formation of several viruses is known to be

affected by the conditions generated by the use of solidi­

fying substances (Ventura, 1968; Colter et al., 1964).

The improvement in plaque sizes and numbers observed when

agar is replaced by agarose has been interpreted to mean

that the presence of contaminating sulfated polysaccharides

is responsible for virus inhibi on (Takemoto and Liebhaber,

1961a). Furthermore, when polycations are added to agar-

containing overlay med I plaque sizes and, sometimes,

numbers increase; this has also been interpreted as an

indication that polyanions interfere with infection of cells

by viruses (Liebhaber and Takemoto, 1961b). Restriction of

diffusion has also been advanced as the explanation of

virus inhibition by gelling substances. Wallis and Melnick

(1968) noted that polio and herpes viruses can be inhibited

by dextran sulfate, a sulfated polyglucose molecule, while

natural agar polysaccharides have no effect. In contrast,

Campbell and Colter (1965) reported that the plaques of

mengo-virus were enhanced by dextran sulfate. These

results suggested to Wallis and Melnick (1968) that poly­

anions present in the gels interfered with the diffusion

of viruses through semi-solid med but did not bind,

per to the particles. These investigators also

indicated that the inhibitory affect could be eliminated

by decreusing the gel concentration used.

119

Reports of viruses sensitive to the overlay medium pH

have indicated that, where sensitivities can be demon­

strated, they are due to prevailing acidic cond ions.

Vogt et ale (1957) showed that the tld" mutants of polio­

virus produced significantly fewer plaques when the pH,

established by changing the bicarbonate concentration, was

below 7.1. Similarly, small plaque-producing strains of

rubella virus were inhibited by acid pH overlay conditions

(Fogel and Plotkins, 1969). The plaques of foot-and-

mouth disease virus variants were also inhibited under agar

media when the initial pH was 7.0-7.2 (Martinsen, 1970). In

each of these examples, the viruses produced plaques at

higher pH values, even at pH 8.6. The observations

reported for the CE viruses were markedly different: The

majority of the variants studied did not produce plaques

above pH 7.5 and produced plaques of greatest size, number

and clarity between pH 7.0 and 7.2.

Results of the experiments involving plaque formation

by Californ encephalitis viruses d not favor either of

the above-mentioned proposed mechanisms of inhibition. The

addition of a polycation such as DEAE-Dextran would have

contributed to an increase in the net positive charge of

the agarose gel. This might have been expected to enhance

the ability of the virus particles to diffuse through the

overlay meclium beCCluse of charge differences or becuusc

anioni.c inhibitors had been sequestered by cationic

120

components. Similarly, the increased plaque sizes which

accompanied the decrease in agarose concentration could

have been due to enhancement of diffusibility by reducing

the viscosity or density of the gel or by reducing the

concentration of the inhibitor. Wallis and Melnick (1968)

suggested that reports of enhanced plaque formation under

agarose Were probably not attrib~table to the absence of

sulfated polysaccharides but, instead, to the higher pH

which characterizes preparations of agarose. The results

of the CE virus studies do not support this explanati~n.

Still to be explained is the extraordinary affect of small

initial pH jifferences on the ability of CE virus2s to

produce plaques on the two cell species studi9d.

The determination of condi t.ions tha t ""QuId pr J!fiot'::!

formation of plaques by CE viruses permitted application

of plaque re~uction techniques to the determinati~n of

the i':ltragrollp antigenic relationshi~)s sugges ted by other

serol~gical methods. I:1 preli~inary expe meni:s was

noted that the host cell in which the virus to be used in

ne~li::..ra 1 izd. ti ~n experiments \vas prepared de-!:::.erminedt.h(~

outcome of virus-dntibody interactions, while the virus

us~~d to immll1,ize animals had no influence. This was

represented gr3phically by the presence of a significant

persistent fraction of surviving virus following neutral­

ization when the virus used had been propagated in suckling

mouse brain tissues. When viruses propagated in chick

121

embryo cells or baby hamster kidney cells were studied,

the surviving fractions were much smaller or could not be

demonstrated. Other investigators have also noted diffi­

culties associated with the use of mouse brain-derived

viruses. Hashimoto and Pr ince (1963) concl u(l,:;:!:l I:hat neu­

tralization kinetics were not applicable to the study of

Japanese encephalitis virus: The virus used was derived

from preparations of infected mouse brains. Cardiff et al.

(1971) demonstrated the presence of virus-specific, non­

infectious substances in mouse brain preparations of virus

that were able to interfere with the neutralization of

infectious particles. Removal of these by centrifugation

reduced the persistent fraction considerably but did not

eliminate it. The significance of the virus source to be

used in neutralizati·-:>n stud s has ,:llso been suggest:.ed by

others .. Le~vent;:>n~-K.r-iss and Mandel (1972) found that the

persistent fraction of poliovirus propagated in HeLa cells

was hi0her than the surviving fraction of the same virus

prepared in monkey kidney cells. These differences suggest

the involvement of host cell-determined variations which

could also be used to explain the above-mentioned differ­

ences between CE viruses of one strain propagated in

dissimilar host cells. Cells are known to contribute to

phen~typic differences between otherwise similar viruses.

For example, a single passage of Venezuelan equine

encephulitis virus into a host or host cell unrelated to

122

that in which the virus was first propagated, resulted in

detectable differences in the lipid composition of the

viral envelope (Heydrick et al., 1966). Similarly, the

lipid composition of SVS virus varied with the host cell

in which it was propagated and resembled the lipid com-

position of the plasma m3mb.r.anes the respective cells

(Klenk and Choppin, 1969). Density diffecences haV8 ~lsa

been demonstrated when one v has been propagated

different host cells. Stenback and Durand (lg63) showed

tha t N'2wcastle disease v gruwn i11 ce lIs of avian Qt- i ~

was characterized by lower dens s than the same virus

g~ted in mammalian cells. In an experiment that has

not been presented as a part of this thesis, the rate of

sedimentation of mouse bra -derived virus was found to be

Sll)vlei:- than that of the same virus propagated in baby

hamster kidney cells. This suggested different densit s

of the two virus types and prompted the attempts to demon-

strate dissimilarit s their envelopes. When purified

preparations of each type of virus were treated with

trypsin, distinct d rences between their properties

became evident. The sensitivity of hamster cell-derived

viruses at low concentrations of the enzyme was contrasted

sharply with the resistance of mouse brain-de ved viruses,

even at high enzyme concentrations. Trypsin is an

cnclopcptidase Which exhibits specificity for pept bonds

contuining the carboxyl groups of arginine or lysine

123

(Mahler and Cordes, 1971). The susceptibility of the one

virus to this enzyme indicated that the determinants

associated with infectivity contained at least one of these

amino acids. Conversely, the resistance of the other virus

suggested that determinants had been protected from the

enzyme, ther by proteins that were not affected by trypsin

or by non-prote

have been invo

substances. That the latter may

was suggested by the results of an ri-

ment in which mouse brain-propagated virus was treated with

phospholipase C. This enzyme has been used by others to

release phospholipids and cholesterol from the envelopes

of viruses (Friedman and Pastan, 1969; Cartwright et al.,

1969). As has been mentioned, the effect of phospholipase

C on infectivity var In the

strain used, an increase In

While it would provide support

stance of the CE virus

ct ity was observed.

evidence to interpret

this finding as an indication that the enzyme cleaved

material from the virus in such a way as to render inactive

particles infectious by exposing cr ical sites, the data

could also be interpreted to indicate that the enzyme had

dispersed aggregates of virus. The ability neutralizing

antibodies to inactivate mouse brain-der viruses was

not enhanced by treatment of the viruses with either trypsin

or phospholipase C.

The apfBrent interference with neutralization by sur-

face st.ructures associated with viruses grown mouse

124

brain ssues seemed to suggest that, while antibod ios may

have been able to attach to these viruses, they may not

have been able to assume stable configurations or bonds

that might have resulted in inactivation. Lafferty (1963b)

hypothesized that the initial interaction between an in­

fectious v ion and a molecule of antibody is reversible

and involves one viral determinant and only one of the two

combining sites of the antibody molecule. This inter­

action, which could involve more than one determinant-

combining site pa , was referred to as "sensitization ll

and by definition preceded neutralization of the particle.

Neutralization sensitized particles would follow the

reaction of a second combining site with a second viral

determinant on the same virus particle. When th stabili­

zation does not occur the sensitized viruses remain infec­

tious and give rise to the persistent fraction. Mandel

(1958) has shown that some preparations of poliovirus

retained infectivity in the presence of neutralizing anti­

bod s which were associated with the virus particles:

Addition of anti-globulin serum to a "neutralized"mixture

decreased the amount of surviving virus.

The persistent fraction associated with CE viruses

prepared from infected moUSe brain tissues can be explained

in terms of Lafferty's hypothes The resistance of mouse

brain-derived viruses to trypsin, contrasted by the sensi­

tivity of hamster cell-derived viruses, suggested the

125

presence of non-protein muterial which, in effect, protected

the antigenic determinants from the action of the enzyme.

This masking e could also have prevented antibody

molecules from assuming stable configurations with antigenic

determinant pa either by ste lly hindering the anti-

body molecule or by making the effective distance between

available determinants too

When the results of the neutralization of chick cell-

derived CE v s were plotted semi-logarithm lly, a

second departure from linear was detected. For a limited

time after virus and hyperimmune serum had been mixed, the

rate of inact ion was slower than was noted at subsequent

times (Figure 19). This lag was also evident when early

immune sera were studied (Figure 13). The application of

specific rate constants to the comparison of neutra ion

of related viruses depends, in part, on the assumption that

a single molecule of antibody is sufficient to inactivate

a virus icle. If the additional assumption is made

that the change in the concentration of antibody molecules

after reaction is negligible, the equations of first order

or monomolecular reaction kinetics may be applied. A semi­

logarithmic plot representing this kind of reaction is a

straight line, while departure from linearity, i.e., a lag,

indicates a reaction greater complexity (Svehag, 1968).

The demonstration of a lag preceding the exponential loss

of CE virus infectivity in at least two situations seemed

l26

to preclude the use of these mathematical techniques. In­

stead, a method was developecJ which permitted visualization

of antigenic similarities and dissimilarities of the viruses

by determining and plotting the kinetics of inactivation of

each strain. Experimentally, the homologous reactions Were

similar: This led to the assumption that, at the dilutions

used, the concentration of neutralizing antibody molecules

in each Serum was approximately the same. In the heterolo­

gous combinations, three types of cross-reaction were

apparent: There was either no detectable reaction, cross­

reactivity in which the curves of inactiva on were charac­

terized by significant lags, or reactions in which no lag

was demonstrable and the rate of inactivation resembled the

rate of neutra zation of the homologous virus. These

distinct patterns provided the basis of an attempt to

explain the cross-reactions and, hence, relationships within

the CE virus group.

The presence of a kinetic lag in neutralization experi­

ments suggests that more than one event must transpire or

that the critical event leading to inactivation proceeds

slowly. The rapidity of primary antigen-antibody reactions

(Haurowitz, 1968) favors the first possibility. If only

one antibody molecule is required for neutralization, a

lag would not be expected, even in heterologous reactions.

Rather, a linear neutralization curve of decreased rate

would be noted. If, on the other hand, more than one

127

antibody molecule is necessary to bring about hct{~r.\~)lol}()uS

virus neutralization, a lag should be demonstrable. Further­

more, the extent of the lag might be expected to be influ­

enced by the specificity, avidity and heterogeneity of the

antibod s part ipating in the neutralization reaction.

In this context, spe ficity is defined as the degree of

cross-reactivity an antiserum exhibits while avidity is

viewed as the tendency of antigen-antibody complexes to

dis soc spontaneously or upon dilution. Webster (1968)

measured the avidity of antisera reacting with influenza

viruses by determining the equilibrium constants of the

reactions. His data indicated that antisera of low avidity

reacted only with the homologous antigens and were, there­

fore, highly spe fie. Similarly, antisera of high avidity

were found capable of reacting with heterologous antigens;

these were considered to be low specificity. These

observations can be extended to the interpretation of the

CE virus neutralization results but do not account for all

the observations.

advanced.

For these, other explanations must be

That little is known about the mechanism of viral cross-

reactions is evidenced by the controversy between proponents

of the single determinant and mosaic hypotheses regarding

the antigens involved in neutralization (Rappaport, lr)~·)L).

A virus exemplifying the single determinant hypothesis con-

tains repeating units of a sing antigen: a cross-reacting

128

virus would be characterized by a structurally simi

antigen present as a repeating unit. Adherents of the mosaic

hypothesis propose the presence of more than one antigen,

each capable of interacting with neutralizing antibodies.

A cross-reactive virus would possess at least one of these

anti and it would be structurally identical to the

ant present on the other virus. Support for either

hypothesis is limited and in most instances indirect.

Serologically, a lag would not be expected if the relation­

ships between antigenically similar viruses were attribu-

Ie to common, identical antigenic determinants and if

only one molecule of antibody was necessary to inactivate

the virus. Among luenza viruses, the peptide maps of

hemagglutinating gens isolated fro~ closely related

strains (determined by cross-reactions) have been found to

be very similar. In contrast, similar antigens isolated

from viruses before and after a major antigenic shift were

observed to be s ificantly different (Webster and Laver,

1972). These observations suggest ions in single

determinants rather than the presence of mUltiple discrete

antigens. Evidence from the study of other enveloped

viruses also s sts the presence single determinants.

For example, Kennedy and Burke (1972) isolated a single

envelope prate from vesicular stomatitis virus. Neutra1-

izing antibody specific for this polypeptide inact.ivated

the homologous virus only (Wagner et al., 1969). Whi

129

several investigators have reported the presence of single

polypeptides in the envelopes of arboviruses (Strauss et al.,

1969; Shapiro et al., 1971), Sehlesinger et ale (1972)

identified a second glycoprotein in Sindbis virus that was

found to co-migrate in polyacrylamide gels with the pre­

viously identified envelope glycoprotein. Three LaCrosse

virus polypeptides have been separated by polyacrylamide

gel electrophoresis, although their biologIcal function or

position in the virus was not reported (McLerran and

Ar 1 , 1973).

Two d tinct patterns of heterologous CE virus neutral­

ization were apparent in the studies of CE virus cross-

reactions. In the one, neutralization was , lacked a

signif lag, and in some

a rate of inactivation simi

nces was characte zed by

to that of the homologous

reaction, sting a high degree of structural simi rity

between the antigenic determinants of the viruses volved

(cf. Figures 25 and 29). That these viruses were not

identical, however, was indicated by the unidirectional

nature of their identities and by the dissimilarit s in

their reactions with immune sera corresponding to other

CE viruses.

The second type of heterologous virus neutralizat

was characterized by an absence of detectable inactivation,

lasting varying periods of time following admixture. The

lengthy lags might be (]scribec1 to the presence - in hi9h

130

concentrations - of antibodies of low specificity as well as

low avidity. Initial interaction between virus particles

and these antibodies would prevent reaction with antibodies

of greater avidity. However, dissociation of the form8r

would give the more avid antibody molecules the opportunity

to react with the virions. As the number of these inter­

actions increased neutralization would become measureably

evident. Inherent to this interpretation is the assumption

that neutralization requires more than o.ne molecule of

antibody to be affected.

The detailed comparisons presented herein for selected

CE virus isolates and their specific hyper immune sera sup­

ported, in some cases, the classificatory proposals advanced

by other investigators using less sensitive as well as less

specific serological techniques. In other instances,

however, significant differences were noted. With these

observations in mind the value of extensive studies with

each CE virus isolate seemed questionable without providing

parallel data using previously applied techniques such as

complement fixation and hemagglutination inhibition and

without examining larger numbers of immune sera represent­

ing different immunization routes, schedules, etc. The

most significant observation made, with respect to the

antigenic relationships of the CE viruses, was that the

employment of kinetic techniques permit much greater

discrimination in determining the nature of the relation­

ships of CE viruses that could not be differentiated by other

serological techniques.

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Name

Birthplace

High- School

University 1963-1968

Degree 1968

Professional Organizations

VITA

Joseph A:1thony Crookston

San Francisco, California

Highland High S::::-hool Salt Lake City, Utah

University of Utah Salt L3ke City, Utah

B.S., University of Utah Salt Lake City, Utah

A~erican Society for Microbiology