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,--.

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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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