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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
fo,' ;1
'.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
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