J. gen. Virol. (1989), 70, 37-43. Printedin Great Britain
37
Key words:flaviviruses/neutralization/antigenic relationships
Antigenic Relationships between Flaviviruses as Determined by
Cross-neutralization Tests with Polyclonal Antisera
By C H A R L E S H. C A L I S H E R , 1. N I C K K A R A B A T S O S , 1 J O E L M.
D A L R Y M P L E , z R O B E R T E. S H O P E , 3 J A M E S S. P O R T E R F I E L D , 4
E D W I N G. W E S T A W A Y s AND W A L T E R E. B R A N D T 6
1 Division of Vector-Borne Viral Diseases, Center for Infectious Diseases, Centers for Disease
Control Public Health Service, U.S. Department of Health and Human Services, P.O. Box 2087,
Fort Collins, Colorado 80522-2087, 2 U.S. Army Medical Research Institute for Infectious
Diseases, Fort Detrick, Frederick, Maryland 21701-5011, 3 Yale Arbovirus Research Unit,
Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College
Street, New Haven, Connecticut 06510, U.S.A., 4 Sir William Dunn School of Pathology,
University of Oxford, South Parks Road, Oxford OX1 3RE, U.K., 5 Department of Microbiology,
Monash University Medical School Clayton, Victoria 3168, Australia and 6 U.S. Army Medical
Material Development Activity, Frederick, Maryland 21701-5009, U.S.A.
(Accepted 4 October 1988)
SUMMARY
The recently established virus family Flaviviridae contains at least 68 recognized
members. Sixty-six of these viruses were tested by cross-neutralization in cell cultures.
Flaviviruses were separated into eight complexes [tick-borne encephalitis (12 viruses),
Rio Bravo (six), Japanese encephalitis (10), Tyuleniy (three), Ntaya (five), Uganda S
(four), dengue (four) and Modoc (five)] containing 49 viruses; 17 other viruses were not
sufficiently related to warrant inclusion in any of these complexes.
INTRODUCTION
The Subcommittee on InterRelationships among Catalogued Arboviruses (SIRACA) of the
American Committee on Arthropod-Borne Viruses periodically reviews published results of
tests of antigenic relationships of registered arboviruses. Such reviews provide guidelines for
defining the antigenic classifications of these viruses. Where possible, arboviruses are
incorporated into the general scheme of classification defined by the International Committee
for the Taxonomy of Viruses (ICTV) (Matthews, 1982). The rationale and scheme for classifying
viruses has been presented for the family Togaviridae, genus Alphavirus (Calisher et al., 1980).
Classification was proposed for the flaviviruses (formerly the group B arboviruses), which were
at that time included in the family Togaviridae (Porterfield et al., 1978). Westaway et al. (1985),
summarizing replication strategies and morphological, morphogenetic and biochemical
characteristics of the alphaviruses and flaviviruses, suggested that the differences between them
warrant creation of a separate family, Flaviviridae, for which the type species is yellow fever
virus; this change has been approved by the ICTV. While these viruses can be classified based
on available data, the most recent comprehensive study of the cross-reactivities of the
flaviviruses was in 1974 by de Madrid & Porterfield, who used cross-neutralization tests with 42
group B arboviruses, and were able to confirm the existence of antigenic subgroups (antigenic
complexes) within the genus (Theiler & Downs, 1973).
Since 1974, 24 additional viruses related antigenically to the original 42 have been registered
in the International Catalogue of Arboviruses (American Committee on Arthropod-Borne
Viruses, 1985) or have been isolated but not yet registered. Our work was carried out to update
that of de Madrid & Porterfield (1974), The results essentially confirm their conclusions and
extend them, suggesting that the flaviviruses may be separated into at least eight antigenic
0000-8526
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 22:23:38
38
c.H.
CALISHER AND OTHERS
complexes, including 49 viruses, and t h a t 17 o t h e r viruses are not related closely e n o u g h to
w a r r a n t inclusion in any of these c o m p l e x e s ; these are relegated to an ' u n a s s i g n e d ' category.
METHODS
Viruses. Sixty-two viruses registered in the International Catalogue of Arboviruses (American Committee
on Arthropod-Borne Viruses, 1985) and four unregistered viruses and their respective polyclonal antibodies,
prepared as hyperimmune mouse ascitic fluids (Tikasingh et al., 1966), were cross-tested. These 66 viruses are
considered members of the same serogroup, family and genus because they are serologically interrelated, as shown
by one or more tests such as haemagglutination inhibition, complement fixation, or neutralization (N). Not all
viruses of the family cross-react with all others; however, cross-reactivity of each virus with at least one other
member of the family has been documented. Serological cross-reactivity of viruses implies the sharing of closely
related antigenic sites or epitopes; cross-reactivity in N tests depends on epitopes present at the surface of the
virion on the envelope glycoprotein (Trent, 1977).
Absettarov, Hanzalova, Hypr and Kumlinge viruses were not included in this study because Clarke (1962, 1964)
and, more recently, Heinz et al. (Heinz & K unz, 1981 ; Heinz et al., 1983) and Stephenson et al. (1984) have shown
that these viruses have the same antigenic characteristics as the Stillerova strain, the suggested prototype for
Central European encephalitis virus strains (Clarke, 1962), which we used. Kedougou and Yaounde viruses,
although registered in the Catalogue of Arboviruses (American Committee on Arthropod-Borne Viruses, 1985),
were not available at the time of the complete cross-tests. Mention of the unregistered Yokose virus is not intended
to constitute priority of publication. Wesselsbron virus is restricted by the U.S. Department of Agriculture and
could not be included in this study, but a potent antiserum for this virus was included. Conversely, Central
European encephalitis virus was included for testing, but a potent homologous antibody preparation was not
available. Two possible members of the family Flaviviridae, cell-fusing agent and simian haemorrhagic fever
virus (Westaway et al., 1985) were not included in these cross-N tests.
Virus titrations and assays. Viruses were titrated by plaque assay in serially propagated Vero, LLC-MK2 and PS
cell cultures and in primary Pekin duck embryo cell cultures. Optimal systems, based on titre and size and clarity of
plaques, were chosen for N tests. Vero cells were used for Alfuy, Apoi, Bagaza, Banzi, Cacipacore, Bouboui,
Bussuquara, Gadgets Gully, Edge Hill, Entebbe bat, Israel turkey meningoencephalitis, Karshi, Koutango,
Kunjin, Kyasanur Forest disease, Langat, louping ill, Murray Valley encephalitis, Naranjal, Negishi, Omsk
haemorrhagic fever, Rio Bravo, Rocio, Russian spring-summer encephalitis, Saboya, Spondweni, Tembusu,
Tyuleniy, Uganda S and Zika viruses. LLC-MK2 cells were used for Aroa, Bukalasa bat, Carey Island, Dakar bat,
dengue 1, dengue 2, dengue 3, dengue 4, Ilheus, Japanese encephalitis, Jutiapa, Modoc, Montana Myotis
leukoencephalitis, Phnom Penh bat, Sal Vieja, San Perlita, Saumarez Reef, Sepik, Sokuluk, St Louis encephalitis,
Tamana bat, Usutu, West Nile and yellow fever viruses. PS cells were used for Cowbone Ridge, Jugra, Kadam,
Kokobera, Meaban, Powassan, Stratford and Yokose viruses. Duck embryo cells were used for Ntaya and Royal
Farm viruses. Homologous serum dilution 90~ plaque reduction N titres were determined for each immune
reagent (Lindsey et al., 1976). Homologous N titres also were determined in sub-optimal (lower titre or less distinct
plaques) systems for 25 of these viruses to detect differences due to the cell system used; none was noted. Mouse
hyperimmune ascitic fluids were screened for heterologous reactivity at a 1 : 20 dilution to detect minimal crossreactivity. Endpoint titres were subsequently determined for virus-antibody pairs reacting at the screening
dilution. Antibody was allowed to remain in contact with the cells throughout the assay, i.e. infected cells were not
washed before addition of the agar overlay (Westaway, 1966). Each virus was tested simultaneously with all
immune reagents, minimizing variation in virus dose (approx. 100 p.f.u.) and being convenient for subsequent
additions of second overlay containing neutral red stain. Considerable difficulty was encountered with Meaban
virus. Initially, it formed plaques in LLC-MK2 cells, but subsequent passages of these cells were not suitable for
plaque assays with this virus. Ultimately, plaque assays and N tests with Meaban virus were carried out using PS
cells overlaid with an enriched agar medium containing Eagle's MEM with Eade's salts, 10~ heat-inactivated
foetal bovine serum, 1~ non-essential amino acids, and 4 mM-L-glutamine per mL
RESULTS
F o r classification purposes, we use a previously p u b l i s h e d definition of serogroup (Calisher e t
al., 1980): two or m o r e viruses, distinct f r o m e a c h o t h e r by q u a n t i t a t i v e serological criteria (fourfold or greater differences b e t w e e n h o m o l o g o u s and heterologous titres o f b o t h s e r u m samples)
in one or m o r e tests, but related to e a c h o t h e r or to o t h e r viruses by s o m e (any) serological
method. Viruses closely related w i t h i n a serogroup but distinct f r o m e a c h o t h e r are c o n s i d e r e d to
constitute an a n t i g e n i c complex.
T a b l e s 1 to 8 present s u m m a r i e s o f N tests b e t w e e n 49 viruses and 48 a n t i b o d y p r e p a r a t i o n s
neutralizing one or m o r e flaviviruses. Cross-reactivities j u d g e d to be m i n o r , usually m o r e t h a n
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 22:23:38
Antigenic classification of flaviviruses
T a b l e 1.
39
Cross-neutralization of flaviviruses in the tick-borne encephalitis antigenic complex
Titre* of antibody to
Virust
RSSE OMSK
RSSE
OMSK
CEE
LI
KFD
LGT
NEG
POW
KSI
RF
CI
PPB
7
11
11
7
7
7
3
3
3
1
5
11
7
5
3
7
2
2
LI
KFD
LGT
1
7
9
7
1
3
1
1
1
5
5
7
5
9
7
2
2
5
9
7
5
3
9
2
2
1
1
NEG
POW
KSI
7
7
9
1
1
RF
CI
PPB
3
5
6
3
1
1
8
3
3
5
5
4
2
* Titre expressed as the number of dilutions with neutralizing antibody, i.e. 1 : 20, 1 : 4 0 . . . 1 : 1280 are shown as
1, 2 . . . 7; a blank space signifies < 1:20. Homologous reactions are in bold type.
t Viruses are designated RSSE, Russian spring-summer encephalitis; OMSK, Omsk haemorrhagic fever;
CEE, Central European encephalitis; LI, louping ill; KFD, Kyasanur Forest disease; LGT, Langat; NEG,
Negishi; POW, Powassan; KSI, Karshi; RF, Royal Farm; CI, Carey Island; PPB, Phnom Penh bat.
T a b l e 2.
Cross-neutralization offlaviviruses in the Rio Bravo antigenic complex
Titre* of antibody to
f
Virust
RB
ENT
RB
ENT
DB
BUK
SAB
APOI
5
2
5
DB
BUK
SAB
APO'I
1
1
9
1
5
3
3
3
5
2
2
1
9
* See Table 1.
t Viruses are designated RB, Rio Bravo; ENT, Entebhe bat; DB, Dakar bat; BUK, Bukalasa bat;
Saboya; APOI, Apoi.
T a b l e 3.
SAB,
Cross-neutralization offlaviviruses in the Japanese encephalitis antigenic complex
Titre* of antibody to
A
t
Virust
JE
JE
MVE
KOK
ALF
STR
SLE
USU
WN
KUN
KOU
7
5
4
4
1
1
1
MVE
KOK
8
4
5
5
1
1
1
ALF
STR
5
7
SLE
1
6
WN
KUN
KOU
3
5
3
5
2
3
3
1
2
1
4
2
1
1
1
7
2
1
l
2
9
6
1
1
6
9
1
4
4
6
6
10
9
4
USU
3
5
1
1
* See Table 1.
t Viruses are designated JE, Japanese encephalitis; MVE, Murray Valley encephalitis; KOK, Kokobera; ALF,
Alfuy; STR, Stratford; SLE, St Louis encephalitis; USU, Usutu; WN, West Nile; KUN, Kunjin; KOU,
Koutango.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 22:23:38
40
C. H. CALISHER AND OTHERS
T a b l e 4.
Cross-neutralization of flaviviruses in the Tyuleniy antigenic complex*
Titre~ of antibody to
~k
Virus:~
TYU
TYU
SRE
MEA
4
SRE
MEA
5
5
* Tyuleniy, Saumarez Reef and Meaban viruses were placed in a separate antigenic group because of previous
complement fixation test results.
t See Table 1.
Viruses are designated TYU, Tyu|eniy; SRE, Saumarez Reef; MEA, Meaban.
Cross-neutralization of flaviviruses in the Ntaya antigenic complex
T a b l e 5.
Titre* of antibody to
¢
Virust
NTA
IT
BAG
NTA
IT
BAG
YOK
KOK
TMU
4
2
1
7
5
2
5
YOK
4
KOK
TMIJ
1
3
1
8
4
3
3
7
4
5
* See Table 1.
t Viruses are designated NTA, Ntaya; IT, Israel turkey meningoencephalitis, BAG, Bagaza; YOK, Yokose;
KOK, Kokobera; TMU, Tembusu.
T a b l e 6.
Cross-neutralization tests with flaviviruses in the Uganda S antigenic complex
Titre* of antibody to
A
Virus:~
UGS
BAN
BOU
5
3
1
9
4
3
UGS
BAN
BOU
EH
EH
1
* See Table 1.
t Viruses are designated UGS, Uganda S; BAN, Banzi; BOU, Bouboui, EH, Edge Hill.
T a b l e 7.
Cross-neutralization tests with flaviviruses in the dengue antigenic complex
Titre* of antibody to
A
r
Virus'~
DEN-1 t
DEN-2
DEN-3
DEN-4
DEN-1
6
1
DEN-2
DEN-3
DEN-4
6
1
1
7
1
1
$
* See Table 1.
~fDEN-l, dengue 1 virus etc.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 22:23:38
41
Antigenic classification of flaviviruses
T a b l e 8. Cross-neutralization tests with flaviviruses in the Modoc antigenic complex
Titre* of antibody to
t
Virus]"
MOD
SV
CR
JUT
SI~
MOD
SV
CR
JUT
SP
9
4
2
3
3
4
9
3
4
1
3
2
5
2
3
2
2
1
2
4
10
9
6
* See Table 1.
i" Viruses are designated MOD, Modoc; SV, Sal Vieja; CR, Cowbone Ridge; JUT, Jutiapa; SP, San Perlita.
T a b l e 9. Assignment of flaviviruses to antigenic complexes*
Antigenic complex
Tick-borne encephalitis
Rio Bravo
Japanese encephalitis
Tyuleniy
Ntaya
Uganda S
Dengue
Modoc
Viruses
(Russian spring-summer encephalitis, Central European encephalitis), Omsk
haemorrhagic fever, louping ill, Kyasanur Forest disease, (Langat, Phnom Penh
bat, Carey Island), Negishi, Powassan, Karshi, Royal Farm
Rio Bravo, Entebbe bat, Dakar bat, Bukalasa bat, Saboya, Apoi
Japanese encephalitis, Murray Valley encephalitis, Kokobera, Alfuy, Stratford, St
Louis encephalitis, Usutu, West Nile, Kunjin, Koutango
Tyuleniy, Saumarez Reef, Meaban
Ntaya, (Tembusu, Yokose), (Israel turkey meningoencephalitis, Bagaza)
Uganda S, Banzi, Bouboui, Edge Hill
Dengue 1, dengue 2, dengue 3, dengue 4
Modoc, Cowbone Ridge, Jutiapa, Sal Vieja, San Perlita
* Viruses more closely related to each other than to others of the antigenic complex are in parentheses.
32-fold differences in both directions, were not included in these tables; such reactions were
considered only to signify membership of the serogroup. Final determinations of antigenic
relationships were based on visual inspection of tabulated results, and assignment to a specific
antigenic complex was by subjective determination of relatedness in these and other tests (de
Madrid & Porterfield, 1974; Varelas-Wesley & Calisher, 1982). Results of these tests allowed the
separation of 49 flaviviruses into eight antigenic complexes (Table 9), supporting and extending
the findings of de Madrid & Porterfield (1974) and Varelas-Wesley & Calisher (1982).
Aroa (Aroa and Bussuquara viruses most closely related to each other by complement fixation
tests; J. Casals, cited in American Committee on Arthropod-Borne Viruses, 1985) (homologous
antibody titre, 1280), Bussuquara (640), Cacipacore (>/1280), Gadgets Gully (160), Ilheus (640),
Jugra (40), Kadam (antibody to Kadam virus neutralized Apoi and Entebbe bat viruses,
suggesting membership of Kadam virus in the Rio Bravo complex) (>~640), Montana Myotis
leukoencephalitis (Montana Myotis leukoencephalitis and Tamana bat viruses most closely
related to each other by haemagglutination inhibition tests; R. E. Shope, unpublished data)
(160), Naranjal (320), Rocio (640), Sepik (Sepik and Wesselsbron viruses most closely related to
each other by complement fixation and mouse neutralization tests; G. M. Woodroofe, cited in
de Madrid & Porterfield, 1974) (640), Sokuluk (5120), Spondweni (640), Tamana bat (160),
Wesselsbron (5120), yellow fever (1280) and Zika (2560) viruses could not be assigned to an
antigenic complex because they did not cross-react significantly with other flaviviruses.
DISCUSSION
These tests were performed with only one immune reagent for each virus. Homologous
antibody preparations with low titres do not allow detection of weak heterologous reactivity, and
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 22:23:38
42
c . H . CALISHER AND OTHERS
variation between individual reagents, immunization schedules, host animals, cell culture
systems and virus passage levels may influence the sensitivity and specificity of polyclonal
antibody preparations.
In spite of these drawbacks, a semblance of order between these viruses can be observed.
Antigenic complexes approximate the ecological categories of these viruses, as determined by
geographical distribution and vertebrate host or arthropod vector association. For example, the
flaviviruses may be generally associated with ticks (Tables 1 and 4), bats (Table 2), mosquitoes
(Tables 3, 5, 6 and 7) or rodents (Table 8), and even more generally associated with Europe and
Asia (Table 1), Africa (Tables 2 and 6), Australia and nearby Asia (Table 3), sea coasts (Table 4)
and North America (Table 8). Viruses assigned to the dengue antigenic complex cause clinically
indistinguishable illnesses in humans. Although such subdivisions provide data sufficient to
devise intriguing evolutionary hypotheses, they may also be considered the result of viruscollecting bias. Tick-borne flaviviruses (Tables 1 and 4) may belong to a single antigenic
complex. Complement fixation tests (Chastel et al., 1985) suggest a closer relationship between
Tyuleniy, Saumarez Reef and Meaban viruses than between these and other tick-borne
flaviviruses, but N tests did not support this observation; we did not compare Meaban virus
with any but Tyuleniy and Saumarez Reef viruses.
Antigenic relationships between viruses of the dengue antigenic complex are representative
of difficulties inherent in taxonomic segregation of flaviviruses. Whereas studies with
monoclonal antibodies confirm by immunofluorescence reaction that the dengue viruses form
an antigenic complex (Henchal et al., 1983), the data (Table 7) demonstrate a significant lack of
cross-neutralization. Alternatively, Edge Hill virus, which our data suggest belongs with the
Uganda S complex viruses (Table 6), has been shown not only to share epitopes with the dengue
complex viruses (Henchal et al., 1983) but also to have a high degree of RNA homology with
dengue 2 virus (Blok et al., 1984).
Conflicting data (results not shown) regarding the relationships between Kokobera virus and
both the Japanese encephalitis antigenic complex viruses (Table 3) and Israel turkey
meningoencephalitis and Tembusu of the Ntaya antigenic complex (Table 5) allude to the
presence of epitopes on Kokobera virus common to members of both antigenic complexes; such
ambiguity may be due to the possibility that viruses in both sets are simply more distant
members of a single, larger complex.
Westaway (1966) determined by N tests that Stratford virus is not related to Japanese
encephalitis, Murray Valley encephalitis or St Louis encephalitis viruses, and de Madrid &
Porterfield (1974) demonstrated a cross-reaction of Stratford virus only with a broadly crossreactive antibody to Murray Valley encephalitis virus; these authors also reported crossreactions between Kokobera and Stratford viruses. In fact, the results of de Madrid &
Porterfield (1974) indicated that Kokobera virus is a subtype of Stratford virus. We detected a
one-way cross between Kokobera virus (homologous antibody titre, 1280) and Stratford virus
(titre of antibody to Kokobera virus with Stratford virus, 160). So too, placement of Carey Island
and Phnom Penh bat viruses in the tick-borne encephalitis antigenic complex is tenuous, being
based on the neutralization of Langat and Phnom Penh bat viruses by antibody to Carey Island
virus. There obviously are no satisfactory solutions in such situations. These apparent
inconsistencies can be resolved by tests with additional polyclonal serum samples, with IgM
antibodies (Westaway, 1968), by epitopic analyses using monoclonal antibodies and by peptide
mapping. Results of tests using such reagents as we used lead to conclusions that must be
considered the best available, but not the only ones possible. The serological data presented here
will be subject to further experimentation and scrutiny in the future, but flavivirus nucleotide
sequence data are needed to provide final determination of genetic relatedness. The flaviviruses
have been justly characterized as an 'antigenic hall of mirrors' (K. M. Johnson, personal
communication).
REFERENCES
AMERICAN COMMITTEE ON ARTHROPOD-BORNEVIRUSES(1985). International Catalogue of Arboviruses Including
Certain Other Viruses of Vertebrates, 3rd edn. Edited by N. Karabatsos. American Society of Tropical
Medicine and Hygiene.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 22:23:38
Antigenic classification o f flaviviruses
43
BLOK, J., HENCHAL, E. A. & GORMAN, B. M. (1984). Comparison of dengue viruses and some other flaviviruses by
c D N A - R N A hybridization analysis and detection of a close relationship between dengue virus serotype 2
and Edge Hill virus. Journal of Generul Virology 65, 2173-2181.
CALISHER, C. H., BRANDT, W., CASALS,J., SHOPE, R. E., TESH, R. B. & WIEBE, M. E. (1980). R e c o m m e n d e d antigenic
classification of registered arboviruses. I. Togaviridae, Alphaviruses. lntervirology 14, 229-232.
CHASTEL, C., MAIN, A. J., GUIGUEN, C., LE LAY, G., QUILLIEN, M. C., MONNAT,J. Y. & BEAUCOURNU,J. C. (1985). The
isolation of M e a b a n virus, a new Flavivirus from the seabird tick Ornithodoros (Alectorobius) maritimus in
France. Archives of Virology 83, 129-140.
CLARKE, D. H. (1962). Antigenic relationships a m o n g viruses of the tick-borne encephalitis complex as studied by
antibody absorption and agar gel precipitin techniques. In Biology of Viruses of the Tick-borne Encephalitis
Complex. Proceedings of a Symposium, Smolenice, 1960, pp. 67-75. Edited by H. Libikova. Prague:
Czechoslovak A c a d e m y of Sciences.
CLARKE,D. H. (1964). Further studies on antigenic relationships a m o n g viruses of the group B tick-borne complex.
Bulletin of the World Health Organization 31, 45-56.
DE MADRID, A. T. & PORTERFIELD, J. S. (1974). The flaviviruses (group B arboviruses): a cross-neutralization study.
Journal of General Virology 23, 91-96.
HEINZ, F. X. & KUNZ, C. (1981). Homogeneity of the structural glycoprotein from European isolates of tick-borne
encephalitis virus: comparison with other flaviviruses. Journal of General Virology 57, 263-274.
HEINZ, F. X., BERGER, R., TUMA, W. & KUNZ, C. (1983). A topological and functional model of epitopes on the
structural glycoprotein of tick-borne encephalitis virus defined by monoclonal antibodies. Virology 126,
525-537.
HENCHAL, E. A., McCOWN,J. M., SEGUIN, M. C., GENTRY, M. K. & BRANDT,W. E. (1983). Rapid identification of dengue
virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay. American Journal of
Tropical Medicine and Hygiene 32, 164-169.
LINDSEY, H. S., CALISHER,C. H. & MATHEWS,J. H. (1976). Serum dilution neutralization test for California group virus
identification and serology. Journal of Clinical Microbiology 4, 503-510.
MATTHEWS, R. E. F. (1982). Classification and nomenclature of viruses. Intervirology 15, 1-199.
PORTERFIELD, J. S., CASALS,J., CHUMAKOV,M. P., GAIDAMOVICH,S. YA., HANNOUN,C., HOLMES,I. H., HORZINEK, M. C.,
MUSSGAY, M., OKER-BLOM, N., RUSSELL, P. K. & TRENT, D. W. (1978). Togaviridae. Intervirology 9, 129-148.
STEPHENSON, J. R., LEE, J. M. & WILTON-SMITH,P. D. (1984). Antigenic variation a m o n g m e m b e r s of the tick-borne
encephalitis complex. Journal of General Virology 65, 81 89.
THEILER, M. & DOWNS, W. G. (1973). The Arthropod-borne Viruses of Vertebrates. New H a v e n & London: Yale
University Press.
TIKASINGH,E. S., SPENCE, L. & DOWNS, W. G. (1966). The use of adjuvant and Sarcoma 180 cells in the production of
mouse h y p e r i m m u n e ascitic fluids to arboviruses. American Journal of Tropical Medicine and Hygiene 15,
219-226.
TRENT, D. W. (1977). Antigenic characterization of flavivirus structural proteins separated by isoelectric focusing.
Journal of Virology 22, 6 0 8 ~ 1 8 .
VARELAS-WESLEY,I. & CALISHER,C. H. (1982). Antigenic relationships of flaviviruses with undetermined arthropodborne status. American Journal of Tropical Medicine and Hygiene 31, 1273-1284.
WESTAWAY, E. G. (1966). Assessment and application of a cell line from pig kidney for plaque assay and
neutralization tests with twelve group B arboviruses. American Journal of Epidemiology 84, 439-456.
WESTAWAY,E. G. (1968). Greater specificity of 19S than 7S antibodies on haemagglutination inhibition tests with
closely related group B arboviruses. Nature, London 219, 78-79.
WESTAWAY,E. G., BRINTON, M. A., GAIDAMOVICH,S. YA., HORZINEK, M. C., IGARASHI,A., K,~[RI.~INEN, L., LVOV, D. K.,
PORTERFIELD, J. S., RUSSELL, P. K. & TRENT, D. W. (1985). Flaviviridae. Intervirology 24, 183-192.
(Received 28 June 1988)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 22:23:38