111111 i 11111 il]11111 il[ill 1111 - dticform approved report documentation page om no 004-01881....
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AD-A239 227111111 I 11111 il]11111 Il[Ill 1111AD_________
CONTRACT NO: DAMD17-90-C-0050
TITLE: MOLECULAR STUDIES OF ALPHAVIRUS IMMUNOGENICITY
PRINCIPAL INVESTIGATOR: James H. Strauss, Ph.D.
CONTRACTING ORGANIZATION: California Institute of Technology1201 E. California BoulevardPasadena, California 91125
REPORT DATE: May 1, 1991
* A6G06 1991;TYPE OF REPORT: Annual Report L
PREPARED FOR: U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDFort Detrick, Frederick, Maryland 21702-5012
DISTRIBUTION STATEMENT: Approved for public release;distribution unlimited
The findings in this report are not to be construed as anofficial Department of the Army position unless so designated byother authorized documents.
91-06858,, , ,,,9 1 0 05 0 2,--.
Form Approved
REPORT DOCUMENTATION PAGE OM No 004-0188
1. AGENCY USE ONLY (Leave blank) 2. REPORT DATE 3. REPORT TYPE AND DATES COVERED
1 May 1991 Annual Report (3/30/90 - 3/29/914. TITLE AND SUBTITLE S. FUNDING NUMBERS
MOLECULAR STUDIES OF ALPHAVIRUS IMMUNOGENICITY Contract No.DAMD1 7-90-C-0050
6. AUTHOR(S) 61102A
James H. Strauss, Ph.D. 3M161102BS12.AB.117WUDA346101
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
California Institute of Technology REPORT NUMBER
1201 E. California BoulevardPasadena, California 91125
9. SPONSORING, MONITORING AGENCY NAME(S) AND AGORESS(ES) 10. SPONSORING MONITORING
U.S. Army Medical Research and Development Command AGENCY REPORT NUMBER
Fort DetrickFrederick, Maryland 21702-5012
11. SUPPLEMENTARY NOTES
1Za. OISTRiMUTION AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE
Approved for public release; distribution unlimited
13. ABSTRACT (Maximum 200 words)
Ockelbo virus, first isolated in 1982 in Sweden, causes arthriti fever andrash in man. We have obtained the complete nucleotide sequence ofOckelbo virus and compared this sequence to that of other strains of Sindbisvirus. Partial sequence analysis of five other strains of Sindbis virus wasalso performed. Three principal conclusions arise from our data. (1)Ockelbo is virtually identical to the causitive agents of Karelian Fever ofRussia and of Pogosta disease of Finland. (2) These agents are closelyrelated to South African strains of Sindbis virus, and Ockelbo was probablyintroduced into northern Sweden from Africa in the 1960's, followed byspread to Russia and Finland. (3) There exist an European-African groupof closely related Sindbis viruses and an Asian-Australian group of Sindbisviruses.
14. SUBJECT TERMS 15. NU.BER OF PAGES
Alphavirus; Sindbis Virus; Ockelbo Disease; BD; RAI; AntigenicEpitope; immongencity 16. PRICE CODE
17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACT
OF REPORT OF THIS PAGE OF ABSTRACT
Unclassified Unclassified Unclassified Unlimited
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A - -2
FOREWORD
Opinions, interpretations, conclusions and recommendations arethose of the author and are not necessarily endorsed by the USArmy.
Where copyrighted material is quoted, permission has beenobtained to use such material.
Where material from documents designated for limiteddistribution is quoted, permission has been obtained to use thematerial.
Citations of commercial organizations and trade names inthis report do not constitute an official Department of Armyendorsement or approval of the products or services of theseorganizations.
_ In conducting research using animals, the investigator(s)adhered to the "Guide for the Care and Use of Laboratory Animals,"prepared by the Committee on Care and Use of Laboratory Animals ofthe Institute of Laboratory Resources, National Research Council(NIH Publication No. 86-23, Revised 1985).
-For the protection of human subjects, the investigator(s)adhered to policies of applicable Federal Law 45 CFR 46.
In conducting research utilizing recombinant DNA technology,the investigator(s) adhered to current guidelines promulcated bythe National Institutes of Health.
,P'I - ignature DATE
Aooession For
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Table of Contents
Page
Front Cover 1
Report Documentation Page 2
Foreword 3
Table of Contents 4
Report
Introduction 5
Methods Used 7
Complete Nucleotide Sequence of Ockelbo Virus 7
3' Terminal Nudeotide Sequence of Other Strains of Sindbis Virus 15
Conclusions 18
References 19
P3,, e 4
Introduction
The alphaviruses are a widespread group of human pathogens that are endemic and
epidemic in many parts of the world (Chamberlain, 1980; Griffin, 1986; Peters and Dalrymple,
1990). They are mosquito-bome and are particularly prevalent in tropical and subtropical areas of
the world, but alphaviruses pathogenic for man are also present in temperate and even Arctic areas.
Many alphaviruses are capable of causing fever, rash and arthralgia in man that in some cases can
be disabling for extended periods of time. Many of the New World alphaviruses can cause
encephalitis in man. We wish to determine the relationships of alphaviruses and strains of
alphaviruses to one another and to search for emerging viruses.
The prototype alphavirus, Sindbis virus, is widespread throughout the Old World,
occurring in Australia, Southeast Asia, India, Europe and Africa (Niklasson, 1988). Strains of
Sindbis virus in Northern Europe, referred to as Ockelbo virus and Karelian fever virus, cause an
illness characterized by polyarthritis whose symptoms can persist for months or years. This illness
first appeared in Northern Europe in the latter half of the 1960's and a virus was first isolated from
female Culiseta mosquitos collected in the endemic area in the summer of 1982 (Niklasson et al.,
1984). Serological studies with convalescent sera from patients with Ockelbo disease showed that
this virus was the causative agent of Ockelbo disease and that it was closely related t, Sindbis
virus. Similar diseases were also found in Finland and the Karelian region of the So% let Union in
the early 1980's and were called Pogosta disease and Karelian fever, respectively. Kareiian fever
virus was also isolated in 1982 and shown to be nearly identical to Ocke'bo virus (Lvov et al.,
1984,1988). The causative agent of Pogosta disease was also found to be closely related to
Ockelbo virus and Sindbis virus (Calisher et al., 1985). Thus, we have the phenomenon of a new
virus appearing in Northern Europe and apparently spreading, infecting increasing numbers of
Page 5
people. It is also of interest that this Northern European strain of Sindbis virus causes an illness
whose clinical features are most closely related to those caused by Ross River virus, a virus
restricted to Australia and the Southern Pacific region that is not closely related to Sindbis virus.
Strains of Sindbis virus in Southern Africa are also known to be virulent for man and have
caused epidemics of human illness. The pattern of clinical features in these illnesses is similar to
that caused by Old World alphaviruses such as chickengunya and O'Nyong-Nyong, but the
severity of the disease is, in general, less.
Sindbis virus is antigenically related to the New World Western equine encephalitis virus
which, as the name implies, is capable of causing encephalitis in man. We recently found that
Western equine encephalitis virus is recombinant between a Sindbis-like virus, presumably found
somewhere in the New World, and the New World Eastern equine encephalitis virus. Its
encephalogenic properties probably originated from the Eastern equine encephalitis virus parent
(Hahn et al., 1988).
We wish to explore the relationships of these various strains of Sindbis virus to one
another and to determine, if possible, why Sindbis virus disease seems to be emerging as a more
widespread human threat. For this purpose, we obtained the complete nucleotide sequence of the
prototypic Ockelbo virus (Edsbyn 82-5) and obtained partial sequences of several geographical
isolates of Sindbis virus. A full description of these results has appeared inVirology 182:753-764
(1991). A preprint of this paper, entitled "Structure of the Ockelbo virus genome and its
relationship to other Sindbis viruses", by Y. Shirako, B. Niklasson, J. M. Dalrymple, E. G.
Strauss, and J. H. Strauss, was submitted to the U.S. Army Medical Research and Development
Command at the time of submission to the journal.
Pacq*: 6
Methods Used
The seven strains of Sindbis virus used in this study and their original sources are
described in Table 1. In addition to the Edsbyn 82-5 strain of Ockelbo isolated in 1982 in Edsbyn
Village, Sweden (Niklasson, et al., 1984), strains of Sindbis isolated in 1952 in Egypt (Taylor et
al., 1955), 1953 in India (Shah et al., 1960), 1963 in South Africa (Malherbe et al., 1963), 1975
in Australia, and strains of Ockelbo/Karelian fever virus isolated in 1983 in Sweden (Niklasson et
al., 1984) and the USSR (Lvov et al., 1984), were included. Viruses were grown either in BHK-
21 cells or in secondary chicken embryo fibroblasts and RNA was isolated from sucrose density
gradient purified virus preparations with SDS-phenol as previously described (Ou et al., 1981).
cDNA clones from the viruses were produced using standard methods (Sambrook et al.,
1989). First-strand cDNA was made using oligo(dT) as a primer and second-strand synthesis was
by the method of Gubler and Hoffman (Gubler and Hoffman, 1983). In some cases HindII
fragments of the cDNA were cloned into vector pGEM3Z. In other cases, EcoRI vectors were
added to the double-stranded cDNA and the cDNA cloned into the EcoRI site of pGEM3Z.
DNA sequencing and RNA sequencing used standard technology that is in common use in
our laboratory (Hahn et al., 1989; Rice et al., 1985; Shirako and Strauss, 1990; Strauss et al.,
1984).
Complete Nucleotide Sequence of Ockelbo Virus
cDNA representing the complete genome of Ockelbo virus was obtained and sequenced. In
addition, the sequence of the 5' end, the 3' end and some internal sequences were obtained by
directly sequencing virus genomic RNA using a dideoxy method. The complete nucleotide
sequence obtained and the deduced amino acid sequence of the proteins encoded in the viral
Page 7
Table 1 Sindbis-like Viruses Pertinent to this Study
Name Strain Source Year Location Reference
Ockelbo Edsbyn 82-5 Pooled mosquitos 1982 Edsbyn village, Niklasson et al.,(Culiseta spp.) Sweden (1984)
Ockelbo Edsbyn 83M107 Mosquito 1983 Edsbyn village,(Culiseta morsitans) Sweden
Karelian LEIV 9298 Mosquito 1983 Central Karelia, Lvov et al.,Fever (Aedes communis) USSR (1988)
Sindbis Girdwood Human 1963 South Africa Malherbe et al.,(1963)
Sindbis A-1036 Mite 1953 India Shah et al.,(Bdellonyssus bursa) (1960)
Sindbis MRM18520 Mosquito 1975 Queensland,(unidentified) Australia
Sindbis AR339 Mosquito 1952 Egypt Taylor et al.,(Culex univittatus) (1955)
Page 8
genome of Ockelbo virus (Edsbyn 82-5) are shown in Figure 1. The viral genome is 11,708
nucleotides in length excluding the 5' terminal cap and the 3' terminal poly(A) tract. The genome
organization is virtually identical to that of the Sindbis virus AR339 strain (Strauss et al., 1984)
isolated in Sindbis, Egypt in 1952 (Taylor et al., 1955). Compared to AR339, Ockelbo nsP3
contains a deletion of 9 nucleotides and 2 separate insertions of 6 and 9 nucleotides in the C-
terminal half, and there are 3 single nucleotide insertions and deletions of the 3' nontranslated
region. Otherwise the numbers of nucleotides translated into amino acids in each region were
exactly the same for these two strains of Sindbis virus. Overall, as illustrated schematically in
Figure 2, there are 672 nucleotide differences between the two viruses (5.7% divergence) that
result in 97 amino acid changes (2.6% divergence). Thus, more than 85% of the nucleotide
changes are silent. Only proteins E2 and 6K and the C-terminal domain of nsP3 show an amino
acid sequence divergence that is significantly higher th:n the average divergence between the two
viruses (2.6% divergence averaged over the entire genome or 1.7% excluding these three
domains). The C-terminal half of nsP3 is not conserved in alphaviruses (Strauss et al., 1988) and
the 6K protein exhibits relatively low conservation so that the divergence in these regions is
probably not significant. However, E2 is a major antigenic determinant, and changes in E2 have
been associated with changes in virulence (Lustig et al., 1988; Olmsted et al., 1986; Strauss et al.,
1991; Tucker and Griffin, 1991).
These changes in glycoprotein E2 are examined in more detail in Table 2. Differences in
E2 among six different strains of Sindbis virus (Ockelbo virus, four strains of Sindbis virus
derived from isolate AR339, isolated in Sindbis, Egypt in 1952, and Sindbis isolate SAAR86,
isolated in South Africa in 1963) are shown. The residues at positions 172, 209, 212, and 216 are
known to be important determinants of the antigenicity of the virus (Strauss et al., 1991), and the
differences at these positions have important implications for the reactivity of the different viruses
with neutralizing antibodies. The residues at 55 and 172 are known to be important determinants
of the neurovirulence of the virus in a mouse model (Lustig et al., 1988), and it is possible that the
Page 9
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Pag ci
402 L A P N A V I P T S L A L CL C C V F1 S A N A TIFUTC T M S Y S W SC NC S 99F P98 40 9959CA iGC UA CA UG GGA UUCGIUAGUGCI~GUAAG AACAGGULU ACG
19 W V 0 L C I P L A A El I V L M A C C S C C L P P L V V A G ACCY L A K '4 0 A F E 39960 UGGCA IGUCAAC UGCC2CUGJUAGGUIUCCJCGCGCUUUGGUGCG. ACGCAGUGCCUCACU10079
4 A T T V P N V P a I P Y K A L V E R A G Y A P I. N L E I T V M S S E L P 5 T N 43
A4 a E Y IT C K FITT V V P S P K V K C COGS L FC0 OP A A H A [V I TC K V F G 83
84 V Y P F " W G S a0 C F C 0 A V V 8 5 A 0 CA) 01030G5ACC~AGGGAGS.AUUUUCAAUAACGCGUAGGGCUCUGAUUACGUGCGEUACCCCGCAU 10439
124 V H T A A M K V G L R I V Y G N I T S P L 0 V Y V N G V I P G T S K 0 L K V I A 153
194 G P I S A S P 1U P FUC0 H K V V I H R CG CL V Y N Y 0D PF P E Y 0G A M K P G [ ] FUUG 0 1 12;3
204 2 A I S 0 L I A S T 0 1 9 L L K P S A K< N V H V P YV 1 0 A g' S G F K M W 243
244 K N N S G " P L C0 F I A P F 0 C K I A V N P L A A V 0) C S Y G N I P I S 1 0 1 P 293
284 N A A F I P T S 0 A P L V S I V K C V S KE A 0 F G 0 U A T L G D 32310 920 AA CGCUGCC UUU AUCAGGA0AJCAGAUGCACCACUGUCUjCAACAG UC AAAUU GVUCACU GA GOGCACUUAUUOCA CAGA C UUCGG C GG0AUGGCC ACC CUGCAGUAUGUAUC CGAC 11 039
324 a C P V M S S S I A I L 0 E S T V H V L E K G A V I V V F S I A S P 0 A 3631040 CGCGAA0GACA AUGC CCUGUA CAUUCGC AUUC A CACAGCA ACCCCA AGAGUOG CG AG UWCAUGUC C U0 GAGA AAGGAG C GGUGACAUCCFG CA0CGGGCAC G1 5
384 N F I V S L C G K K T I C N A E C K P P A 0 H I V S T P H K N 0 0 E F 0 A A I S 4032118s0 AA C UUC AUGUAUCGCUGUGI:GGUAAGAAGA CA ACAU GC AA UGCAGAAU U AAACCACCA ,UGACCAUAUCGUGAGCACCCCGCACAAAAAUGCAGAUCACGCUUA1 7
404 K I S W S A F A L 0 0 A S 5 L L I I 0 L E) I F A C S 4 M I S T R R 09
A
11940 CC CGGCGAA2UL UUUUUUAUACAAAUUGJIACAI' 11708
1. Complete sequence of the Ockelbo virus. The sequence is shown from 5' to 3' and translatedig the single letter amino acid code. Nucleotides different from those in HRSP are underlined,changed amino acids are boxed. Deletions relative to HR are indicated by solid triangles
iting upward and the number of residues deleted. Insertions have both amino acids andleotides boxed together, and an open triangle pointing downward. Termination codons areffled Am (Amber, UAG) or Op (Opal, UGA) as appropriate. Nucleotides are numbered 5' to 3';no acid numbering begins again at the beginning of each final protein product.
Page 12
CO)
C-1 C
U) r
U).
Page 13
Table 2. Amino Acid Substitutions in Glycoprotein E2 in Sindbis Strains
RESIDUE AR339 SA. AR86 e OCKELBO f
HRSpa DGb ASC Rjd
1 S S S R S S3 I T T T T T
23 V E E E E E29 V V V V I I55 Q Q Q Q Q K61 A A A A S T69 L L L L L F70 K K E E E E
116 V V V V A A126 L L L L M M172 R G G G G G209 G R G G G G212 S S S S T T216 E E K E E E243 L L L L S L247 D D D D A A277 I I I I V I312 V V V V I I375 T T T T A A386 V V V V A A
a Sequence of HRSP is from Strauss et al. (1984).
b Sequence is from Lustig et al. (1988) of the SV1A strain from the laboratory of Diane
Griffin.C Sequence from Strauss et al. (1991 in press) of the strain used by A. Schmaljohn for
the isolation of antigenic variants (Stec et al. 1986).
d Sequence from Davis et al. (1986) of the laboratory strain of Robert Johnston.
e Sequence of the SA AR86 strain from Russell et al. (1989).
f Sequence from Shirako et al. (1991).
Page 14
amino acid difference at position 55 between Ockelbo and the other Sindbis strains might be
responsible in part for the increased virulence of Ockelbo virus compared to other strains of
Sindbis virus.
3' Terminal Nucleotide Sequence of Other Strains of Sindbis Virus
To ascertain the relationships among Sindbis virus strains present in nature, RNA sequence
was obtained for a number of isolates of Sindbis virus that differ in their geographic source and in
their disease symptomology in man (the strains and their source are shown in Table 1). The 420
nucleotides at the 3'-terminus were sequenced for each isolate. These sequences are shown in
Figure 3. The sequence identity throughout this region is greater than 80% for all viruses
examined, and the sequence organization is identical in all cases except for a few scattered
nucleotide insertions and deletions. In the 3' nontranslated region there are three highly conserved
repeats that are 40 nucleotide long (boxed in the figure). The sequences within these repeated
elements are more highly conserved than the sequences outside these elements. As an example of
this, comparing the Australian and AR339 strains, there are 49 differences in the 3' nontranslated
region outside the repeated elements (24.1% divergence), but only 7 changes within the elements
(5.8% divergence), for an overall divergence of 18.1%. Similarly, comparing the Ockelbo '82
isolate with AR339, there are 13 changes outside the repeated elements (6.4% divergence) but only
3 within the elements (2.5% divergence), for an overall divergence of 4%.
The relationships among the virus sequences are shown diagrammatically in Figure 4.
From this figure the number of nucleotide differences in the 3' terminal 420 nucleotides between
any two strains of Sindbis virus can be computed. Three points are immediately obvious from a
study of this diagram. One is that the Sindbis strains analyzed can be divided into a European-
African group and an Asian-Australian group. The Asian-Australian group differs from the
European-African group in 17% of the nucleotides sequenced, whereas within the European-
Page 15
11290 11300 11310 11320 11330 13340
W S W L F A AL F S I L L I CAR339 (HR) UGCAGUUGGCUCUUUGCCCUUUUCGGCGGCGCCUCCUCGCUAUUAAUUAUAGGACUUAUG
Ockelbo 82 -------------------- ---------------------------- C
Ockelbo 83 ------------------------------------------------------ -- C-Karelia 83 --------------------------------------------- ------- --- -C-G ird w ood 63 -. -- - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . .
India53 -. .... .. . AU-A- U- G A --- A--C--U---G G AAustralla75 ---- C- C ----------- A--A--U--G--A--U--A- C- G- -G - G A- -
11350 11360 11370 11380 11390 11400I I1 I I I II F A C S I F L T S T R R Op
AR339 (HR) AUUUUUGCUUGCAGCAUGAUGCUGACUAGCACACGAAGAUGACCGCUACGCCCCAAUGAU
Ockelbo 82 ------------------------------------------------- COckelbo 83 C-----------------------------------------------------------CKarelia 83 .----------------------------------------------------------- CGirdwood 63 -------------------------- - ---------------- -- ? ? ? ------ .. CIndia 53 - ------ GCUC -- A -- C -- C---- -??? - --- CAustralia 75 - ------ GCUU -------- A - ------ -------- ??- ? ------------- C
11410 11420 11430 11440 11450 11460I I I I I I
AR339 (HR) CCGACCAGC AAACUCGAUGUACUUCCGAGGAACUGAUGUCCAUAAUCC AUCAGGCUGGU
O c k e lb o 8 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ockelbo 83
Karelia 83Girdwood 63 -.--------------India 53 -- - C- --- A -------- C - ------- G -Australia 75 -U -- C- A ------------- -- C - -G
11170 11180 11 190 '110 11oo - 10T 11 20
AR339 (HR) ACAUUAGAUCCCCGCUUACCGCGGG CAAUAUAGCAACACUA AAAACUCGAUGUACUUCC
Ockelbo 82 -U - U- ---------- - ----- C--------C----------- -- - --
Ockelbo 83 -U------------------ U ----- -------------- C --------- C---------Karelia 83 U C--- C - ----- -------------------- C C - - -
Girdwood 63 -U ? ? ? ? ------------- CIndia 53 -- ???-ACCAGA - C-UU-C --- G--G-C --------- CAustralia 75 A -A-CAGA--- - UCC ---- G--G -- -------------- C--
11530 11540 11550 11560 11570 11580I I I I I IAR339 (HR) CACGAAUCGCA(,UGCAUAAUGCCGCCAGUUUUCCACAUAACCACUAUAUUAACCAUUU
Ockelbo 82 -- - U --Ockelbo 83 ----- ---. . .... ......... U -Karelia 83 -U - - -Girdwood 63 -.---- A - - -U - ---- ------ - _ -
India 63 U C -A .-- C-??-UU-U --- UU-- - -U AAustralia 75 -- U . A -- C--C-U--U U -U- - UU A
115.90 11600 11610 11620 11630 11640I I I I I I
AR339 (HR) AUCUAGCGGACGCCA AAAACUCAAUGUAUUUCUGAGGAAGCG[GGUGCAUAAUCJC ACC
Ockelbo 82 U - --- G - - -- A ---------------U-Ockelbo 83 U - G- A . .-------- C -UKarelia 83 U C - A C -U
Girdwood 83 U A --- U-India 3 AG UA AAustralia 75 -CAG UA A -
11650 11660 11670 11680 11690 11700I I I I I 1
AR339 (HR) ACCCUCULCAUAAC UUUUAUUA UUUCUUUUA UUAAUCAACAAAAUUUUGUUUUUAACAUUC -poly(A)
Ockelbo 82 C UU .... .
Ockelho .3 U .Karella 83 U UCirdwood 63 UIndia M3 U A CAAC U UG UU U NNNNAustralia75 U AA -UCAA- - - U ....--- U -UU U .NNNN
Figure 3. Sequence of the 3' end of several Sindbis viruses. The sequences of Ockelbo 83M107, Karelian fever, and the SouthAfrican Girdwood were determined from cloned cDNA. Those of Indian A1036 and Australian MRM18520 were determineddirectly from RNA by dideoxy sequencing using T12 GA primer. The sequence of Ockelbo 82 is from Fig. 1 and that for AR339(HRSP) is from Strauss et al. (1984). Gaps have been introduced as necessary to maintain the alignment. Three repeatedsequence elements of 40 nucleotides are boxed. The translated amino acid sequence and nucleotide numbers are for AR339 (11hR);and amino acid that differs in any of the other viruses is boxed.
Page 16
KF Aust.
OCK OCK 1983 1975
AR339 S.Af. 1983 nd1952 1963 21 1 Ini
2 3 1953
7 1510
29
25
Figure 4. Relationships among strains of Sindbis virus. Thevertical distances indicate the number of nucleotide differencesbetween any two strains in the 3'terminal 420 nucleotides. Thehorizontal distances are arbitrary. Nucleotide differences betweenany two strains can be determined quite accurately by summingthe numbers on the vertical branches between them.
Page 17
African group the maximum divergence is about 5%. The second obvious point is the relationship
between thc Karelian fever and Ockelbo viruses. These are different isolates of what is effectively
the same virus. The third point isthat Ockelbo virus is more closely related to the South African
strain isolated in 1963 than it is to the Egyptian strain isolated in 1952, which has important
implications for the origin and spread of Ockelbo disease.
Conclusions
Ockelbo virus and Karelian fever virus are the same virus, and we assume that this same
strain of Sindbis virus also causes Pogosta disease. The Ockelbo strain of Sindbis virus is very
closely related to the South African strains of Sindbis virus, more so than to other European-
African subgroup Sindbis viruses such as Egyptian AR339. This close relationship is evident
either from comparisons of glycoprotein E2 among various Sindbis viruses, or from comparisons
of 3'-terminal sequences. The closeness of this relationship is illustrated by the fact that in the 3'
nontranslated region, Ockelbo and the South African strain examined demonstrate a sequence
divergence of only 4%. Since these viruses were isolated twenty years apart, the maximum rate of
sequence divergence in this region could be no more than 0.2% per year. Since it seems unlikely
that the Girdwood strain of South African Sindbis is the direct ancestor of Ockelbo, the actual rate
of divergence is probably less. Such a divergence rate is low in comparison to rates established for
a number of other RNA viruses (Steinhauer and Holland, 1987; Strauss and Strauss, 1988).
Thus, it seems clear that South African and Northern European strains of Sindbis virus have not
been separated for long. It is also of note that the South African strains of Sindbis virus have been
implicated in human disease, as has Ockelbo virus. It seems likely that Ockelbo originated in
South Africa and was introduced into Sweden by migratory birds or by the activities of man,
perhaps in the 1960's, and then spread to Finland and the Karelian region of the Soviet Union in
the 1980's. Thus, it appears that virulent strains of Sindbis virus have the potential to spread to
Page 18
other regions of the world and cause epidemics of febrile illness that can be of moderate severity
because of associated arthralgia.
It is also important that repeats of a sequence element found in the 3' nontranslated region
of Sindbis viruses are much more highly conserved than sequences outside these elements. The
function of the 3' nontranslated region, and in particular the repeated sequence elements, is
unknown, but the conservation of these elements among Sindbis viruses makes clear that they play
an important role in viral replication, and presumably are important for viral RNA replication. We
have previously shown that site-specific mutants in the 3' nontranslated region have different
effects in mosquito cells and chicken cells, implying that host cell proteins bind to this region to
promote replication (Kuhn et al., 1990). The fact that the repeated elements are present in three
copies and exhibit a high degree of concentration suggest that they might serve as binding sites for
interactions with host cell proteins. These repeated sequence elements differ among different
alphaviruses, but equivalent sequences are found in all alphaviruses. Conserved sequence
elements could be useful for diagnostic purposes, although other conserved domains of the virus
are probably more useful.\
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