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Genetic Relatedness of Sindbis Virus Strains from Europe, Middle East, and Africa
VIROLOGY
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SHORT COMMUNICATION Genetic Relatedness of Sindbis Virus Strains from Europe, Middle East, and Africa ¨ M,*,1 OTO KOZUCH,† and LARS O. MAGNIUS* HELE´NE NORDER,* JAN O. LUNDSTRO *Department of Virology, Swedish Institute for Infectious Disease Control, S-105 21 Stockholm, Sweden; and †Institute of Virology, Slovak Academy of Sciences, 842 46 Bratislava, Slovak Republic Received January 16, 1996; accepted June 12, 1996 The relatedness of 40 strains of Sindbis virus (SIN) from Europe, the Middle East, and Africa was investigated by limited sequencing within the gene encoding the E2 glycoprotein corresponding to amino acid residues 117 to 229 and encompassing one of the major neutralization epitopes. Phylogenetic analyses using distance matrix and parsimonious methods identified two major genetic clusters of western SIN strains, although the variability was less than that of the corresponding region for Venezuelan equine encephalomyelitis virus with a maximum divergence of 12.4% versus 28.5%, respectively. One cluster comprising 19 strains included the HR derivate of the Egypt SIN prototype, AR339, and strains from Israel, SaudiArabia, Italy, Slovak Republic, Azerbaijan, as well as three Swedish strains. Another cluster of 17 strains included the Ockelbo virus (OCK) prototype, Edsbyn 5/82, and the majority of SIN strains from northern Europe including strains from Sweden, Norway, and Karelia, as well as two strains from South Africa. A third cluster, supported by the Neighbor Joining method, was made up of four strains from South Africa, Uganda, and Cameroon. Residue 212, either Ser or Thr, previously appointed important for the differences in neutralization assays between SIN and Edsbyn 5/82, respectively, correlated with the two major genetic clusters, but was a Thr for two of the three Swedish strains in the SIN prototype cluster, and a Ser in one Swedish and one Karelian strain in the OCK cluster. The finding of strains similar to prototype SIN in Middle Sweden and of strains in South Africa relating to the northern cluster of SIN strains supports the notion of the dispersal of SIN by migrating birds as previously suggested for New World alphaviruses. q 1996 Academic Press, Inc.
Sindbis virus (SIN) is the prototype virus of the genus Alphavirus in the family Togaviridae. It has an approximately 11.7-kb plus strand RNA genome. The structural proteins, i.e., the nucleocapsid protein, and two membrane associated glycoproteins, E1 and E2, correspond to a polyprotein encoded by a subgenomic RNA transcript homologous to the 3* end of the complete genome (48). Phylogenetic analyses have shown that the New World alphaviruses form one branch and that the Old World alphaviruses constitute another branch of this genus (1, 25, 37), and also that the western equine encephalomyelitis virus (WEE) is a recombinant between SIN and eastern equine encephalomyelitis virus (EEE) (14, 53). Recently, the systematics of the alphaviruses revised on sequence data suggests four major complexes: the SIN complex, the Semliki Forest virus (SF) complex, the EEE/ Venezuelan equine encephalomyelitis virus (VEE) complex, and the recombinant alphavirus complex (48). A considerable amount of information is available on the genetic variability of EEE, VEE, and WEE in the New World (21–23, 46, 50–55). Less information has been gathered with regard to SIN and Ross River virus (RR) in
the Old World (36, 40, 45, 44), although SIN has been shown to be widely dispersed being isolated from Europe, Africa, as well as East Asia (36). Recently, Aura virus, a South-American alphavirus, was shown by sequencing of its complete genome to belong to the SIN complex (42), previously indicated by serological cross reactivity (2, 18). To increase the knowledge on the genetic variability of SIN, and to provide further genetic support for the previously suggested relation between North-European and African SIN strains (27, 45), we undertook the limited sequencing of the gene corresponding to the E2 glycoprotein of SIN strains from Europe, Africa, and the Near East. This gene was chosen, since encoding one of the envelope proteins, it is the most variable alphavirus gene (48). The sequenced region of the E2 glycoprotein also contains one of the major epitopes for virus neutralization. Isolates of SIN, originally isolated in 12 different countries, were kindly provided by Dr. Bruce Francy (CDC, Fort Collins, CO), Dr. Alan Schmaljohn (USAMRIID, Fort Detrick, Frederick, MD), Dr. Gunnar Hoddevik (Statens Institut fo¨r Folkehelkse, Oslo, Norway), or isolated in Sweden during previous studies (9, 26, 33). The 40 SIN strains, included in the study, with their location and source are listed in Table 1. These strains were isolated from 1952 to 1992 at various sites in northern, central,
1 To whom correspondence and reprint requests should be addressed. Present address: at Department of Zoology, Uppsala University, Villava¨gen 9, S-752 36 Uppsala, Sweden.
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Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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TABLE 1 Strains of Sindbis Virus Analyzed in This Study Strain Edsbyn 5/82 83M107 83M108 84M137 84M140 85M68 85M78 85M94 85M134 86-486 86-520 86-737 86-743 86-752 86-828 90B146 LEIV-9298 Vemork1/92 R33 Acrocephalus AS-2A1 R2 SK 2 Cricetus Gresikova AZ-16 1038 M-1855 Ar 339 SA80-62 SA80-66 SA80-231 SA80-287 MP 684 Y-251 AR 86 Girdwood AR 6071 AR 18132 AR 18141
Passage No.a Unknown sm1, v1 v2 v2 v1 v1 v1 v1 v1 v2 v2 v2 v2 v2 v2 v1 sm2, v1 v4 sm4, v1 Unknown Unknown sm7, v1 sm7, v1 Unknown Unknown p1, v1 Unknown Unknown Unknown d1, v1 d1, v1 d1, v1 v2 Unknown Unknown Unknown p3, sm1, v1 Unknown sm1, v1 Unknown
Source
Year
Location
Reference
Culiseta sp. Cs. morsitans Culex sp. Cs. morsitans Cs. morsitans Cs. morsitans Aedes cinereus Cx. sp. Cx. sp. Cx. sp. Cx. sp. Ae. cinereus Ae. cinereus Cx. sp. Cs. morsitans Anas plathyrhynchus Ae. sp. Ae. sp. Acrocephalus scirpaceus — — Rana ridibunda Cricetus cricetus — Hyalomma marginatum Nycticorax nycticorax Streptopelia turtur Cx. molestus Cx. sp. Cx. tritaeniorhynchus Cx. univittatus Cx. univittatus Cx. univittatus Mansonia fuscopennata Ms. africana Cx. sp. Homo sapiens Cx. univittatus Cx. univittatus Cx. univittatus
1982 1983 1983 1984 1984 1985 1985 1985 1985 1985 1985 1985 1985 1985 1985 1990 1983 1992 1971 — — 1975 1972 — 1975 1977 1964 1967 1952 1980 1980 1980 1980 1958 1969 1954 1963 1964 1974 1976
Sweden, Edsbyn Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Bergsa˚ker Sweden, Bergsa˚ker Sweden, Bergsa˚ker Sweden, Bergsa˚ker Sweden, Bergsa˚ker Sweden, Bergsa˚ker Sweden, Boda bruk Russia, Karelia Norway, Rjukan Slovac Republic — — Slovac Republic Slovac Republic — Italy, Sicily Russia, Azerbaijan Israel, Rubin Israel, Hadera Egypt, Sindbis Saudi Arabia Saudi Arabia Saudi Arabia Saudi Arabia Uganda, Entebbe Cameroon South Africa, Spring S. Africa, Johannesburg S. Africa, Johannesburg South Africa, Upington South Africa
33 9 9 9 9 9 9 9 9 9 9 9 9 9 9 26 28 Hoddevik et al., unpublished 6 —b —b 17 12 —b 13 10 34 35 49 58 58 58 58 60 —c 56 29 —c 30 31
a
Suckling mice (sm), vero cells (v), duck embryo cells (d), passage in undefined cell or animal (p). Numbers denote number of passages. Passages provided by Dr A. Schmaljohn of the strain above brought to U.S.A. by Dr Gresikova in 1970. c Strains provided by Dr A. Schmaljohn. b
and southern Europe, the Middle East, and sub-Saharan Africa. The virus specimens were cultured in Vero cells. The growth medium for the cells was Eagle’s minimal essential medium supplemented with 2% fetal bovine serum, 200 units/ml of penicillin, and 200 mg/ml of streptomycin. Confluent Vero cell cultures were inoculated with approximately 103 plaque-forming units (PFU) of each stock virus. After showing 80% cytopathogenic effect, the culture supernatant was harvested and stored at 0707 until used. Briefly the utilized laboratory procedures were as follows. For extraction of RNA 100 ml of culture supernatant
were treated with 2 mg proteinase K in a buffer containing 0.5% sodium dodecyl sulfate, 10 mM Tris, pH 8.4, and 100 ng tRNA for 2 hr at 377. After phenol and phenol/ chloroform extraction the nucleic acids were ethanol precipitated. The RNA was dissolved in 20 ml diethylpyrocarbonate-treated water. Five microliters of dissolved RNA was used for cDNA synthesis using MMLV reverse transcriptase and primer A2 (5*-TGGGCAACAGGGACCATGCA-3*). The cDNA was amplified by PCR using 10 pmol of the primers A1 (5*-AAAGGATACTTTCTCCTCGC3*) and A2, 200 mM dNTPs, 1 U Taq polymerase, 10 mM Tris–HCl, pH 8.3, 50 mM KCl, and 2.5 mM MgCl2 . The PCR was performed for 40 cycles with denaturation at
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FIG. 1. (a) Phylogenetic tree relating 58 alphavirus strains including 41 strains of Sindbis from Europe, Middle East, and Africa and strains of the alphaviruses Venezuelan equine encephalomyelitis, eastern equine encephalomyelitis, Ross River, O’Nyong-nyong, western equine encephalomyelitis, and Aura. The dendrogram was based on the alignment of 341 nucleotides of the E2 gene using the Neighbor-Joining method corrected for multiple hits (Kimura 2-parameter). (b) The same dendrogram as in a. where only the branch with the 41 Sindbis strains is shown.
947, annealing at 557, and extension at 727, each step for 1 min. The primers A1 and A2 correspond to positions 8919–8938 and 9426–9445, respectively, in the SIN sequence (47). The oligonucleotides and the excess of dNTPs were removed from the amplified product with Magic PCR Preps (Promega Corp., Madison, WI). The purified products (0.5 pmol) were sequenced in both directions according to Casanova et al. (3) using 10 pmol of A1 and A2 as primers. Multiple nucleotide and amino acid sequence alignments were performed using the CLUSTAL V program (15). The nucleotide and deduced amino acid differences between the sequences were calculated in the DNA Star program. The phylogenetic trees for the nucleic acid sequences were created using the programs DNAdist together with Neighbor using the UPGMA, Neighbor-Joining, and Fitch Margoliash methods for distance matrix algorithms and DNA-PARS for maximum parsimony in the PHYLIP package version 3.5 (8). The nucleotide sequences of the 39 strains, two SIN sequences retrieved from GenBank (41, 47), and the published sequences of seven VEE strains (16, 19–21), three EEE strains (4, 53, 59), WEE (14), Aura virus (42), RR (7), SF (11), and ONN (25) were aligned and subjected to phylogenetic comparisons. A phylogenetic analysis constructed from the nucleo-
tide sequence data comparing the sequenced region for all the analyzed alphavirus genomes by Neighbor Joining is shown in Fig. 1a. The results were in agreement with those based on partial genomes with Aura and WEE being located on the SIN branch, while ONN, SF, and RR formed a separate cluster, with both of the Old World alphavirus clusters being distinct from the New World alphavirus cluster. The genetic variability of the sequenced region was considerably less for the SIN strains compared to the corresponding fragment for the VEE strains with maximum divergences of 12.4 and 28.5%, respectively. Phylogenetic tree constructions using four different methods (Neighbor Joining, Fitch Margoliash methods, maximum parsimony, and UPGMA) defined two major clusters of SIN strains. A third cluster, supported by Neighbor Joining, was less evident by other methods (Fig. 1b). The nucleotide divergence was 4.4–12.4% between strains belonging to different clusters and 0–7.4% between strains within the same cluster. One cluster was made up of 19 sequences comprising two sequences of the SIN prototype including all strains from Middle and South Europe (six from Slovak Republic representing three original isolates and one from Italy), all the isolates from the Near East (four from Saudi-Ara-
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FIG. 2. Alignment of the deduced amino acid sequence, residues 117 to 229, according to Shirako et al. (45), of the E2 gene in 41 Sindbis virus strains from Europe, Middle East, and Africa, and WEE and Aura. Deviations from the amino acid sequence of the Sindbis HR strain are indicated.
bia and two from Israel), one from Azerbaijan, and three from Sweden. The other cluster was made up of 15 North European strains, including the Ockelbo (OCK) prototype, Edsbyn 5/82, 12 other Swedish strains, one strain from Karelia, and one from Norway, as well as two isolates from South Africa, AR86 and AR6071. The third cluster contained four African isolates, AR18132 and AR18141 from South Africa, MP 684 from Uganda, and Y-251 from Cameroon. Another strain from South-Africa, Girdwood, did not give any consistent association to the previous clusters in the phylogenetic analysis. Most of the variable nucleotides, 78.7%, in the sequences obtained for the SIN strains were synonymous when compared to the published HR strain of the SIN prototype AR339, corresponding to a ratio between synonymous and nonsynonymous substitutions of 3.7. The deduced amino acid sequence similarity was high among all the examined strains, with sequence identities between 88.8 and 100%. The maximum divergence of the sequenced fragment of SIN compared to ONN, RR, VEE, EEE, Aura, and WEE was 72.4, 64.7, 66.4, 60.6, 43.4, and 28.3%, respectively. The deduced amino acid residues within the region of the E2 glycoprotein sequenced by us is shown in Fig. 2. The glycosylation site at amino acid residue 196 was conserved for all strains. Five resi-
dues, a Val187, a Gly194, and three cysteinic residues at positions 201, 203, and 220, invariant for all alphavirus E2 glycoproteins, were all present in the herein sequenced SIN strains. As mentioned the region of the E2 glycoprotein sequenced by us encompasses one of the major neutralization epitopes. Residue 212, either Ser or Thr, has previously been appointed important for the differences in neutralization assays between SIN prototype and OCK, respectively (45). The substitutions at residue 212 parallelled but did not completely agree with the two major genetic clusters and was a Thr for two of the three Swedish strains in the SIN prototype cluster and a Ser in one Swedish and the Karelian strain in the OCK cluster. Thus, we found Thr212 in 15 of 18 north European strains and in four of 21 strains from South and Central Europe, Middle East, and Africa. In this context it is worth mentioning that the Slovak strains R33, SK2, and R2 with low passage numbers were more similar to each other in the compared part of the E2 region than to their derivates Acrocephalus, AS-2A1, and Cricetus that have drifted as compared to their respective parenteral strain (Fig. 1b). Interestingly, a Lys216 present in both R33 and SK2 in early passages, was lost in two of the three of their derivates (Fig. 2). As pointed
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out SIN virus tend to accumulate mutations on passage as an adaption to cell culture conditions (32). To a high extent the phylogenetic tree obtained with the analyzed fragment within the E2 region reflected a previous phylogenetic tree based on partial alphavirus genomes (48). The Aura and WEE sequences diverged considerably from the SIN sequences, although less so than the other Old World alphaviruses, RR, ONN, and SF. Our phylogenetic analyses has identified two major genetic clusters of western SIN strains, although the variability was less than that of the corresponding region for VEE. However, when sequence information on eastern SIN strains will become available, the variability of SIN is assumed to increase significantly. The two major clusters of western SIN strains to a high extent parallelled their geographical origin. Thus, one cluster including the SIN prototype from Egypt comprised all the strains from the Mediterranean, Middle and South East Europe, and Saudi-Arabia. The other cluster including the OCK prototype, Edsbyn 5/82, comprised all but three Swedish strains, originating from northern Europe as well as two strains from South Africa. The three Swedish strains in the first cluster all derived from the same village in Middle Sweden. Future research has to settle, whether the presence of SIN prototype-like strains in Northern Europe and North European strains in South Africa represent temporary imports, possibly, by migrating birds or stable introductions. Also the existence has to be confirmed of a third cluster of western SIN strains with its origin in sub-Saharan Africa. The distinct clustering of north European SIN strains indicate that influx of SIN virus strains from sub-Saharan Africa is not the main mechanism for initiating the virus enzootic transmission cycle in birds and mosquitoes in northern latitudes. However, intercontinental exchange of SIN strains are indicated by two South African strains being most closely related to the north European strains and that three Swedish strains are more related to the strains in the SIN prototype cluster. Long-distance transport of SIN strains might be accomplished by infected mosquitoes being transported by human activities. In fact intercontinental transport of mosquitoes in aircraft and in ships has been convincingly documented (38, 57), but the magnitude of direct transport of mosquitoes between Scandinavia and sub-Saharan Africa is difficult to estimate. Another transport mechanism could be by migrating birds, since alpha viruses may cause latent and periodically reactivated viremia in birds. This is supported by an early finding that WEE, although not present in the blood, could be isolated from tissues of passerine birds infected 8 to 10 months earlier (39). The finding that SIN RNA may persist for 17 months in the central nervous system of experimentally infected mice provides further support for latent and periodically reactivated alphavirus viremia in vertebrates (24). Extended studies are needed to clarify whether migrating birds are actually carrying
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latent alphavirus within their bodies, and ultimately positive finding should be evaluated experimentally. ACKNOWLEDGMENTS We are grateful to Dr. Hoddevik for providing us with a Norwegian Sindbis virus isolate and to Dr. Ru¨menapf for providing us with the Aura sequence as a datafile. This work was supported by the Swedish Medical Research Council Grant No. K95-11019-02B (H. Norder), and by grants from the Swedish Medical Association (J. O. Lundstro¨m). The nucleotide sequences reported in this paper will appear in EMBL, GenBank, and DDBJ Nucleotide Data Bases under the accession numbers U62443–U62480.
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SHORT COMMUNICATION Genetic Relatedness of Sindbis Virus Strains from Europe, Middle East, and Africa ¨ M,*,1 OTO KOZUCH,† and LARS O. MAGNIUS* HELE´NE NORDER,* JAN O. LUNDSTRO *Department of Virology, Swedish Institute for Infectious Disease Control, S-105 21 Stockholm, Sweden; and †Institute of Virology, Slovak Academy of Sciences, 842 46 Bratislava, Slovak Republic Received January 16, 1996; accepted June 12, 1996 The relatedness of 40 strains of Sindbis virus (SIN) from Europe, the Middle East, and Africa was investigated by limited sequencing within the gene encoding the E2 glycoprotein corresponding to amino acid residues 117 to 229 and encompassing one of the major neutralization epitopes. Phylogenetic analyses using distance matrix and parsimonious methods identified two major genetic clusters of western SIN strains, although the variability was less than that of the corresponding region for Venezuelan equine encephalomyelitis virus with a maximum divergence of 12.4% versus 28.5%, respectively. One cluster comprising 19 strains included the HR derivate of the Egypt SIN prototype, AR339, and strains from Israel, SaudiArabia, Italy, Slovak Republic, Azerbaijan, as well as three Swedish strains. Another cluster of 17 strains included the Ockelbo virus (OCK) prototype, Edsbyn 5/82, and the majority of SIN strains from northern Europe including strains from Sweden, Norway, and Karelia, as well as two strains from South Africa. A third cluster, supported by the Neighbor Joining method, was made up of four strains from South Africa, Uganda, and Cameroon. Residue 212, either Ser or Thr, previously appointed important for the differences in neutralization assays between SIN and Edsbyn 5/82, respectively, correlated with the two major genetic clusters, but was a Thr for two of the three Swedish strains in the SIN prototype cluster, and a Ser in one Swedish and one Karelian strain in the OCK cluster. The finding of strains similar to prototype SIN in Middle Sweden and of strains in South Africa relating to the northern cluster of SIN strains supports the notion of the dispersal of SIN by migrating birds as previously suggested for New World alphaviruses. q 1996 Academic Press, Inc.
Sindbis virus (SIN) is the prototype virus of the genus Alphavirus in the family Togaviridae. It has an approximately 11.7-kb plus strand RNA genome. The structural proteins, i.e., the nucleocapsid protein, and two membrane associated glycoproteins, E1 and E2, correspond to a polyprotein encoded by a subgenomic RNA transcript homologous to the 3* end of the complete genome (48). Phylogenetic analyses have shown that the New World alphaviruses form one branch and that the Old World alphaviruses constitute another branch of this genus (1, 25, 37), and also that the western equine encephalomyelitis virus (WEE) is a recombinant between SIN and eastern equine encephalomyelitis virus (EEE) (14, 53). Recently, the systematics of the alphaviruses revised on sequence data suggests four major complexes: the SIN complex, the Semliki Forest virus (SF) complex, the EEE/ Venezuelan equine encephalomyelitis virus (VEE) complex, and the recombinant alphavirus complex (48). A considerable amount of information is available on the genetic variability of EEE, VEE, and WEE in the New World (21–23, 46, 50–55). Less information has been gathered with regard to SIN and Ross River virus (RR) in
the Old World (36, 40, 45, 44), although SIN has been shown to be widely dispersed being isolated from Europe, Africa, as well as East Asia (36). Recently, Aura virus, a South-American alphavirus, was shown by sequencing of its complete genome to belong to the SIN complex (42), previously indicated by serological cross reactivity (2, 18). To increase the knowledge on the genetic variability of SIN, and to provide further genetic support for the previously suggested relation between North-European and African SIN strains (27, 45), we undertook the limited sequencing of the gene corresponding to the E2 glycoprotein of SIN strains from Europe, Africa, and the Near East. This gene was chosen, since encoding one of the envelope proteins, it is the most variable alphavirus gene (48). The sequenced region of the E2 glycoprotein also contains one of the major epitopes for virus neutralization. Isolates of SIN, originally isolated in 12 different countries, were kindly provided by Dr. Bruce Francy (CDC, Fort Collins, CO), Dr. Alan Schmaljohn (USAMRIID, Fort Detrick, Frederick, MD), Dr. Gunnar Hoddevik (Statens Institut fo¨r Folkehelkse, Oslo, Norway), or isolated in Sweden during previous studies (9, 26, 33). The 40 SIN strains, included in the study, with their location and source are listed in Table 1. These strains were isolated from 1952 to 1992 at various sites in northern, central,
1 To whom correspondence and reprint requests should be addressed. Present address: at Department of Zoology, Uppsala University, Villava¨gen 9, S-752 36 Uppsala, Sweden.
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Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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TABLE 1 Strains of Sindbis Virus Analyzed in This Study Strain Edsbyn 5/82 83M107 83M108 84M137 84M140 85M68 85M78 85M94 85M134 86-486 86-520 86-737 86-743 86-752 86-828 90B146 LEIV-9298 Vemork1/92 R33 Acrocephalus AS-2A1 R2 SK 2 Cricetus Gresikova AZ-16 1038 M-1855 Ar 339 SA80-62 SA80-66 SA80-231 SA80-287 MP 684 Y-251 AR 86 Girdwood AR 6071 AR 18132 AR 18141
Passage No.a Unknown sm1, v1 v2 v2 v1 v1 v1 v1 v1 v2 v2 v2 v2 v2 v2 v1 sm2, v1 v4 sm4, v1 Unknown Unknown sm7, v1 sm7, v1 Unknown Unknown p1, v1 Unknown Unknown Unknown d1, v1 d1, v1 d1, v1 v2 Unknown Unknown Unknown p3, sm1, v1 Unknown sm1, v1 Unknown
Source
Year
Location
Reference
Culiseta sp. Cs. morsitans Culex sp. Cs. morsitans Cs. morsitans Cs. morsitans Aedes cinereus Cx. sp. Cx. sp. Cx. sp. Cx. sp. Ae. cinereus Ae. cinereus Cx. sp. Cs. morsitans Anas plathyrhynchus Ae. sp. Ae. sp. Acrocephalus scirpaceus — — Rana ridibunda Cricetus cricetus — Hyalomma marginatum Nycticorax nycticorax Streptopelia turtur Cx. molestus Cx. sp. Cx. tritaeniorhynchus Cx. univittatus Cx. univittatus Cx. univittatus Mansonia fuscopennata Ms. africana Cx. sp. Homo sapiens Cx. univittatus Cx. univittatus Cx. univittatus
1982 1983 1983 1984 1984 1985 1985 1985 1985 1985 1985 1985 1985 1985 1985 1990 1983 1992 1971 — — 1975 1972 — 1975 1977 1964 1967 1952 1980 1980 1980 1980 1958 1969 1954 1963 1964 1974 1976
Sweden, Edsbyn Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Sa¨ssman Sweden, Bergsa˚ker Sweden, Bergsa˚ker Sweden, Bergsa˚ker Sweden, Bergsa˚ker Sweden, Bergsa˚ker Sweden, Bergsa˚ker Sweden, Boda bruk Russia, Karelia Norway, Rjukan Slovac Republic — — Slovac Republic Slovac Republic — Italy, Sicily Russia, Azerbaijan Israel, Rubin Israel, Hadera Egypt, Sindbis Saudi Arabia Saudi Arabia Saudi Arabia Saudi Arabia Uganda, Entebbe Cameroon South Africa, Spring S. Africa, Johannesburg S. Africa, Johannesburg South Africa, Upington South Africa
33 9 9 9 9 9 9 9 9 9 9 9 9 9 9 26 28 Hoddevik et al., unpublished 6 —b —b 17 12 —b 13 10 34 35 49 58 58 58 58 60 —c 56 29 —c 30 31
a
Suckling mice (sm), vero cells (v), duck embryo cells (d), passage in undefined cell or animal (p). Numbers denote number of passages. Passages provided by Dr A. Schmaljohn of the strain above brought to U.S.A. by Dr Gresikova in 1970. c Strains provided by Dr A. Schmaljohn. b
and southern Europe, the Middle East, and sub-Saharan Africa. The virus specimens were cultured in Vero cells. The growth medium for the cells was Eagle’s minimal essential medium supplemented with 2% fetal bovine serum, 200 units/ml of penicillin, and 200 mg/ml of streptomycin. Confluent Vero cell cultures were inoculated with approximately 103 plaque-forming units (PFU) of each stock virus. After showing 80% cytopathogenic effect, the culture supernatant was harvested and stored at 0707 until used. Briefly the utilized laboratory procedures were as follows. For extraction of RNA 100 ml of culture supernatant
were treated with 2 mg proteinase K in a buffer containing 0.5% sodium dodecyl sulfate, 10 mM Tris, pH 8.4, and 100 ng tRNA for 2 hr at 377. After phenol and phenol/ chloroform extraction the nucleic acids were ethanol precipitated. The RNA was dissolved in 20 ml diethylpyrocarbonate-treated water. Five microliters of dissolved RNA was used for cDNA synthesis using MMLV reverse transcriptase and primer A2 (5*-TGGGCAACAGGGACCATGCA-3*). The cDNA was amplified by PCR using 10 pmol of the primers A1 (5*-AAAGGATACTTTCTCCTCGC3*) and A2, 200 mM dNTPs, 1 U Taq polymerase, 10 mM Tris–HCl, pH 8.3, 50 mM KCl, and 2.5 mM MgCl2 . The PCR was performed for 40 cycles with denaturation at
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FIG. 1. (a) Phylogenetic tree relating 58 alphavirus strains including 41 strains of Sindbis from Europe, Middle East, and Africa and strains of the alphaviruses Venezuelan equine encephalomyelitis, eastern equine encephalomyelitis, Ross River, O’Nyong-nyong, western equine encephalomyelitis, and Aura. The dendrogram was based on the alignment of 341 nucleotides of the E2 gene using the Neighbor-Joining method corrected for multiple hits (Kimura 2-parameter). (b) The same dendrogram as in a. where only the branch with the 41 Sindbis strains is shown.
947, annealing at 557, and extension at 727, each step for 1 min. The primers A1 and A2 correspond to positions 8919–8938 and 9426–9445, respectively, in the SIN sequence (47). The oligonucleotides and the excess of dNTPs were removed from the amplified product with Magic PCR Preps (Promega Corp., Madison, WI). The purified products (0.5 pmol) were sequenced in both directions according to Casanova et al. (3) using 10 pmol of A1 and A2 as primers. Multiple nucleotide and amino acid sequence alignments were performed using the CLUSTAL V program (15). The nucleotide and deduced amino acid differences between the sequences were calculated in the DNA Star program. The phylogenetic trees for the nucleic acid sequences were created using the programs DNAdist together with Neighbor using the UPGMA, Neighbor-Joining, and Fitch Margoliash methods for distance matrix algorithms and DNA-PARS for maximum parsimony in the PHYLIP package version 3.5 (8). The nucleotide sequences of the 39 strains, two SIN sequences retrieved from GenBank (41, 47), and the published sequences of seven VEE strains (16, 19–21), three EEE strains (4, 53, 59), WEE (14), Aura virus (42), RR (7), SF (11), and ONN (25) were aligned and subjected to phylogenetic comparisons. A phylogenetic analysis constructed from the nucleo-
tide sequence data comparing the sequenced region for all the analyzed alphavirus genomes by Neighbor Joining is shown in Fig. 1a. The results were in agreement with those based on partial genomes with Aura and WEE being located on the SIN branch, while ONN, SF, and RR formed a separate cluster, with both of the Old World alphavirus clusters being distinct from the New World alphavirus cluster. The genetic variability of the sequenced region was considerably less for the SIN strains compared to the corresponding fragment for the VEE strains with maximum divergences of 12.4 and 28.5%, respectively. Phylogenetic tree constructions using four different methods (Neighbor Joining, Fitch Margoliash methods, maximum parsimony, and UPGMA) defined two major clusters of SIN strains. A third cluster, supported by Neighbor Joining, was less evident by other methods (Fig. 1b). The nucleotide divergence was 4.4–12.4% between strains belonging to different clusters and 0–7.4% between strains within the same cluster. One cluster was made up of 19 sequences comprising two sequences of the SIN prototype including all strains from Middle and South Europe (six from Slovak Republic representing three original isolates and one from Italy), all the isolates from the Near East (four from Saudi-Ara-
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FIG. 2. Alignment of the deduced amino acid sequence, residues 117 to 229, according to Shirako et al. (45), of the E2 gene in 41 Sindbis virus strains from Europe, Middle East, and Africa, and WEE and Aura. Deviations from the amino acid sequence of the Sindbis HR strain are indicated.
bia and two from Israel), one from Azerbaijan, and three from Sweden. The other cluster was made up of 15 North European strains, including the Ockelbo (OCK) prototype, Edsbyn 5/82, 12 other Swedish strains, one strain from Karelia, and one from Norway, as well as two isolates from South Africa, AR86 and AR6071. The third cluster contained four African isolates, AR18132 and AR18141 from South Africa, MP 684 from Uganda, and Y-251 from Cameroon. Another strain from South-Africa, Girdwood, did not give any consistent association to the previous clusters in the phylogenetic analysis. Most of the variable nucleotides, 78.7%, in the sequences obtained for the SIN strains were synonymous when compared to the published HR strain of the SIN prototype AR339, corresponding to a ratio between synonymous and nonsynonymous substitutions of 3.7. The deduced amino acid sequence similarity was high among all the examined strains, with sequence identities between 88.8 and 100%. The maximum divergence of the sequenced fragment of SIN compared to ONN, RR, VEE, EEE, Aura, and WEE was 72.4, 64.7, 66.4, 60.6, 43.4, and 28.3%, respectively. The deduced amino acid residues within the region of the E2 glycoprotein sequenced by us is shown in Fig. 2. The glycosylation site at amino acid residue 196 was conserved for all strains. Five resi-
dues, a Val187, a Gly194, and three cysteinic residues at positions 201, 203, and 220, invariant for all alphavirus E2 glycoproteins, were all present in the herein sequenced SIN strains. As mentioned the region of the E2 glycoprotein sequenced by us encompasses one of the major neutralization epitopes. Residue 212, either Ser or Thr, has previously been appointed important for the differences in neutralization assays between SIN prototype and OCK, respectively (45). The substitutions at residue 212 parallelled but did not completely agree with the two major genetic clusters and was a Thr for two of the three Swedish strains in the SIN prototype cluster and a Ser in one Swedish and the Karelian strain in the OCK cluster. Thus, we found Thr212 in 15 of 18 north European strains and in four of 21 strains from South and Central Europe, Middle East, and Africa. In this context it is worth mentioning that the Slovak strains R33, SK2, and R2 with low passage numbers were more similar to each other in the compared part of the E2 region than to their derivates Acrocephalus, AS-2A1, and Cricetus that have drifted as compared to their respective parenteral strain (Fig. 1b). Interestingly, a Lys216 present in both R33 and SK2 in early passages, was lost in two of the three of their derivates (Fig. 2). As pointed
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out SIN virus tend to accumulate mutations on passage as an adaption to cell culture conditions (32). To a high extent the phylogenetic tree obtained with the analyzed fragment within the E2 region reflected a previous phylogenetic tree based on partial alphavirus genomes (48). The Aura and WEE sequences diverged considerably from the SIN sequences, although less so than the other Old World alphaviruses, RR, ONN, and SF. Our phylogenetic analyses has identified two major genetic clusters of western SIN strains, although the variability was less than that of the corresponding region for VEE. However, when sequence information on eastern SIN strains will become available, the variability of SIN is assumed to increase significantly. The two major clusters of western SIN strains to a high extent parallelled their geographical origin. Thus, one cluster including the SIN prototype from Egypt comprised all the strains from the Mediterranean, Middle and South East Europe, and Saudi-Arabia. The other cluster including the OCK prototype, Edsbyn 5/82, comprised all but three Swedish strains, originating from northern Europe as well as two strains from South Africa. The three Swedish strains in the first cluster all derived from the same village in Middle Sweden. Future research has to settle, whether the presence of SIN prototype-like strains in Northern Europe and North European strains in South Africa represent temporary imports, possibly, by migrating birds or stable introductions. Also the existence has to be confirmed of a third cluster of western SIN strains with its origin in sub-Saharan Africa. The distinct clustering of north European SIN strains indicate that influx of SIN virus strains from sub-Saharan Africa is not the main mechanism for initiating the virus enzootic transmission cycle in birds and mosquitoes in northern latitudes. However, intercontinental exchange of SIN strains are indicated by two South African strains being most closely related to the north European strains and that three Swedish strains are more related to the strains in the SIN prototype cluster. Long-distance transport of SIN strains might be accomplished by infected mosquitoes being transported by human activities. In fact intercontinental transport of mosquitoes in aircraft and in ships has been convincingly documented (38, 57), but the magnitude of direct transport of mosquitoes between Scandinavia and sub-Saharan Africa is difficult to estimate. Another transport mechanism could be by migrating birds, since alpha viruses may cause latent and periodically reactivated viremia in birds. This is supported by an early finding that WEE, although not present in the blood, could be isolated from tissues of passerine birds infected 8 to 10 months earlier (39). The finding that SIN RNA may persist for 17 months in the central nervous system of experimentally infected mice provides further support for latent and periodically reactivated alphavirus viremia in vertebrates (24). Extended studies are needed to clarify whether migrating birds are actually carrying
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latent alphavirus within their bodies, and ultimately positive finding should be evaluated experimentally. ACKNOWLEDGMENTS We are grateful to Dr. Hoddevik for providing us with a Norwegian Sindbis virus isolate and to Dr. Ru¨menapf for providing us with the Aura sequence as a datafile. This work was supported by the Swedish Medical Research Council Grant No. K95-11019-02B (H. Norder), and by grants from the Swedish Medical Association (J. O. Lundstro¨m). The nucleotide sequences reported in this paper will appear in EMBL, GenBank, and DDBJ Nucleotide Data Bases under the accession numbers U62443–U62480.
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