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National Institute of Virology, 130/1 Sus Road, Pashan, Pune 411021, India
Correspondence
Vidya A. Arankalle
v_arankalle{at}yahoo.co.in
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the full genome sequences of eight CHIK virus isolates determined in this study are EF027134–EF027141.
Supplementary tables are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), in Indonesia during 2001–2003 (Laras et al., 2005) and in India during 2005–2006 (Yergolkar et al., 2006), after gaps of 39, 20 and 32 years, respectively. CHIK is emerging as an important infection in South-East Asia and the Pacific region (Thaikruea et al., 1997; Mackenzie et al., 2001; Kit, 2002). Recently, massive outbreaks of CHIK have been reported from many islands of the Indian Ocean (WHO, 2006).
CHIK virus infection is characterized by fever, headache, rash, nausea, vomiting, myalgia and arthralgia. The virus was first isolated during an outbreak in Tanzania in 1952 (Ross, 1956). CHIK outbreaks in east, south, west and central Africa have been documented. The virus appears to have spread from Africa to other parts of the world and caused epidemics in the Asian tropics (Powers et al., 2000). In Africa, the virus is maintained in a sylvatic cycle involving wild primates and many species of Aedes mosquito. Aedes albopictus is considered to be the vector in Reunion and other islands of the Indian Ocean. Although both Aedes aegypti and A. albopictus mosquitoes are prevalent in India, the former is the main vector (Yergolkar et al., 2006).
CHIK virus belongs to the genus Alphavirus of the family Togaviridae. Phylogenetic analyses based on E1 gene sequences grouped CHIK viruses isolated worldwide into three genotypes: Asian, east/central/south African (ECSA) and west African (Powers et al., 2000; Schuffenecker et al., 2006). The complete nucleotide sequence for the African prototype strain, S27, was determined and the presence of an internal polyadenylation [I-poly(A)] site and repeated sequence elements within the 3' non-translated region (3'-NTR) was observed (Khan et al., 2002). Full-genome sequences for the Ross and Senegal strains have been determined. However, similar data for Asian strains are not available. Recently, several isolates from Reunion and other islands have been sequenced (Schuffenecker et al., 2006).
In India, the first CHIK outbreak was recorded in 1963 in Kolkata (Calcutta) (Shah et al., 1964), followed by epidemics in eastern coastal areas, namely Chennai (Madras), Pondicherry and Vellore in 1964, Visakhapatnam, Rajmundry and Kakinada in 1965 (Rao, 1966), Nagpur in 1965 (Rodrigues et al., 1972) and Barsi in 1973 (Padbidri & Gnaneswar, 1979). In Chennai alone, nearly 400 000 cases were recorded in 1964. In Nagpur, the incidence in certain wards was as high as 40–70 %. In view of the long absence of CHIK epidemics, it was postulated that CHIK virus had disappeared from India and South-East Asia (Burke et al., 1985; Pavri, 1986). Serological surveys supported this view (Neogi et al., 1995).
Several states in India experienced massive outbreaks of CHIK during 2005–2006. Initially, two southern states [Andhra Pradesh (AP) and Karnataka] and one western state (Maharashtra) were affected (Yergolkar et al., 2006). The outbreak continued with reports of a large number of cases from several other states (Rajasthan, Gujarat, Tamilnadu, Orissa and Madhya Pradesh).
We determined the full-genome sequences of eight CHIK virus isolates: one from each of five states affected during the 2005–2006 episode, one isolate from mosquitoes in 2000 (Yawat, Maharashtra state) and two strains isolated during the epidemics in 1963 and 1973. An attempt has been made to determine the association, if any, of mutations in the genome with the increased transmissibility of the virus, leading to a large epidemic affecting 13 states of the country within a year.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. These include two strains of the Asian genotype isolated in 1963 from Kolkata, eastern India (IND-63-WB1), and in 1973 from Barsi, western India (IND-73-MH5), along with one 2005–2006 epidemic isolate from each of five states in India, i.e. three from the states affected earlier, IND-06-KA15, IND-06-AP3 and IND-06-MH2, and two from the states affected 3 months later, IND-06-TN1 and IND-06-RJ1. One isolate recovered from a mosquito in 2000 from western India (ECSA genotype, IND-00-MH4) was also sequenced. The older isolates were obtained from the virus repository of the National Institute of Virology, Pashan, India, reconstituted and used. CHIK viruses were isolated according to protocols described previously (Yergolkar et al., 2006). Supplementary Table S1, available in JGV Online, gives details of the additional CHIK virus strains included in the study.
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). Briefly, viral RNA was isolated by using a QIAamp Viral RNA Mini kit (Qiagen) according to the manufacturer's instructions, followed by the SuperScript II protocol (Invitrogen). Amplified fragments were visualized by ethidium bromide agarose gel staining, extracted from the gels and both strands were sequenced by using a BigDye Terminator Cycle Sequencing kit (Applied Biosystems).
The nucleotide sequence of the S27 strain (GenBank accession no. AF369024
[GenBank]
) (Khan et al., 2002) was used for primer designing. Supplementary Table S2, available in JGV Online, provides a list of primers used for PCR/sequencing.
Sequence and phylogenetic analyses.
Phylogenetic analysis based on the available full-genome and E1 gene (1044 nt) sequences of CHIK viruses was performed by using MEGA version 3.1 (Kumar et al., 2004). CLUSTAL_X version 1.83 (Thompson et al., 1997) was used to perform multiple nucleotide and amino acid sequence alignments. For the construction of phylogenetic trees, the neighbour-joining algorithm and the Kimura two-parameter distance model were utilized. The reliability of the analysis was evaluated by a bootstrap test with 1000 replications.
Recombination analysis.
To search for recombination events between strains, within or between genotypes, SimPlot (Salminen et al., 1995) was used. The structural and non-structural genes of the CHIK virus strains for which full-genome sequences were available, and the E1 gene sequences of several other strains, were used as datasets. For graphical detection of conflicting phylogenetic signals, SimPlot was used, wherein isolates were examined by using sliding-window diversity and bootscan plots. The pairwise percentage difference between the query sequences and other sequences in the alignment was determined by sliding a window of 400 and 200 bp along the alignment in 3 and 10 bp increments. The diversity profiles were used to determine which of the other sequences were related most closely to the putative recombinant and could therefore be used as parental sequences. Putative recombination break points detected were assessed for significance by reconstructing a separate maximum-likelihood (ML) tree for each region.
Molecular sequence evolution and diversifying selection analyses.
The modified method of Nei and Gojobori (Nei & Gojobori, 1989; Suzuki & Gojobori, 1999) as implemented in the MEGA package was used to calculate the synonymous (dS) and non-synonymous (dN) substitution rates and the dN/dS ratio (
) across all amino acid sites in pairwise comparisons between nucleotide sequences. This ratio, if >1, is used as evidence for positive, diversifying selection or adaptive evolution. On the other hand, if <1, it is inferred as negative, deleterious or purifying selection, whilst =1 in the case of neutral substitutions. To test whether the sequences are under selection pressure and also whether there are specific amino acids affected by diversifying selection, ML models of codon substitution that allow for heterogeneous selection pressures among sites were implemented (Yang et al., 2000), using the CODEML program in the PAML package (Yang, 1997). Among the various codon-substitution models, only M1, M2, M7 and M8 were employed in the present study, as comparisons of M1 (neutral) with M2 (selection) and M7 (
) with M8 ( and ) are specific tests for positive selection. The likelihood-ratio test (LRT) was applied to compare the null models (M1 and M7) with alternative ones (M2 and M8, respectively) that account for sites under positive selection. M1 divides codons into two categories, representing the proportion (p0) of conserved sites with =0 and the proportion of neutral sites (p1) with =1. M2 accounts for positive selection by including a third category of codons (p2) with 2 that can take any value, including >1, as estimated from the data. M7 and M8 are more complex models; M7 uses a discrete distribution (0<<1), whilst M8 also uses a distribution, where >1 is incorporated.
The F3x4 model, which computes equilibrium codon frequencies from the nucleotide frequencies at the three codon positions, was used to account for codon-usage bias. Branch lengths of the phylogeny (measured as the expected number of nucleotide substitutions per codon along a branch) and the transition-to-transversion ratio (
) were estimated by using ML. Further, the Bayes theorem (Yang et al., 2005) as implemented in CODEML was used to calculate the posterior probability that a particular amino acid site belongs to a given selection class (neutral, deleterious or advantageous). Sites with a high posterior probability of being from the class with >1 were deemed more likely to be under diversifying selection.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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provides the lengths of genomic RNA and 3'/5'-NTR fragments of all eight Indian CHIK virus isolates with reference to the S27 strain (genomic RNA, 11 805 nt; 5'-NTR, 76 nt; 3'-NTR, 526 nt). The sequences from the earlier cases of the outbreak were from the states of Karnataka (11 729 nt), AP (11 779 nt) and Maharashtra (11 800 nt), whereas sequences corresponding to the cases occurring 3 months later were from Tamilnadu (11 750 nt) and Rajasthan (11 767 nt). In addition, the earliest isolated strain from the 1963 Kolkata epidemic (11 784 nt), the last strain isolated during the 1973 Barsi epidemic (11 805 nt) and a strain incidentally isolated from mosquitoes in 2000 (Yawat, 11 814 nt) were also sequenced.
Phylogenetic analyses
Fig. 1(a) depicts the phylogenetic tree based on full-genome analysis. All Indian isolates from the 2005–2006 (IND-06) resurgence representing the five affected states, the isolate from Yawat in 2000 (Yawat-2000), all Reunion isolates of 2005–2006 (RU) and the S27 and Ross isolates (1952) clustered together into the ECSA genotype. The earlier Indian isolates (1963 and 1973) belonged to the Asian genotype, whereas the Senegal strain formed a distinct branch (west African genotype). Similar results were obtained when the structural and non-structural regions were analysed separately (data not shown).
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). This analysis revealed that all of the RU and IND-06 isolates grouped together, whereas the Yawat-2000 and Uganda-1982 isolates, sharing 99 % nucleotide identity, constituted a separate branch. Similarly, the isolates from the DRC (2000) formed a separate cluster within the same genotype, whereas the 1996 isolate from the Central African Republic remained as a separate branch. Isolates from Thailand recovered in 1975 and 1996 and the 1965 Indian and 1985 Indonesian isolates clustered in the Asian genotype. Both Senegal isolates (1966, 1983) and a Nigerian isolate remained in the west African genotype.
Sequence comparisons
Irrespective of the place of isolation (Reunion Island or India), the 2005–2006 isolates were related very closely (99.9 % identity). These isolates differed from the S27 and Yawat-2000 isolates by 2.7 and 1.7–1.8 %, respectively. The Asian genotype differed from the ECSA and west African genotypes by 4.4–5.3 and 15.4–15.5 %, respectively. The African genotypes (ECSA versus west African) were 14.5–14.8 % divergent. Amino acid identities across the three genotypes varied from 95.2 to 99.8 % (see Supplementary Table S3, available in JGV Online).
Sequence analysis of the ECSA genotype
Non-structural region.
In this region, the recent Indian and RU strains exhibited 99.85±0.06 % identitiy at the amino acid level. Compared with the S27 prototype, nine identical substitutions were present in both groups of isolates: Q488R, S589N, A1328V, Y1550H, T1670I, L1794P, P1804S, T1938A and T2117A. There were seven substitutions, i.e. L507R, H909Y, V1508I, V1664A, I1709T, S1795N and Q2363L, that were shared between the Yawat-2000, IND-06 and RU isolates. The Yawat-2000 strain also showed six unique substitutions: V326M, Q1661P, S1691P, C1768R, V1771A and M1782T. The S1691P substitution in Yawat-2000 was also observed in one of the RU isolates, RU05-209 (Table 2).
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Structural region.
The IND-06 and RU isolates shared six substitutions in the structural region: E2-I536T, T637M, S700T and V711A, 6K-V756I and E1-D1093E. Further, the two groups had four substitutions (K63R, I284T, A489T and M1078V) identical to Yawat-2000. The unique mutations in the Yawat-2000 strain were A487V, V643M, I702V (in the E2 region), V828I and A1186T (in the E1 region). The mutation I702V (E2), although different from all of the IND-06 and RU isolates, was similar to the IND-63-WB1 and IND-73-MH5 strains, belonging to the Asian genotype (Table 3).
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Sequence analysis of the Asian genotype
CHIK viruses of the Asian genotype isolated in 1963 and 1973 showed 99.4 (99.72) and 99.39 (99.44) % nucleotide (amino acid) identities in the non-structural and structural regions, respectively. These strains exhibited several amino acid substitutions compared with the S27 strain.
Non-structural region.
There were eight, six, 17 and eight unique substitutions in the Nsp1, Nsp2, Nsp3 and Nsp4 regions, respectively, in the two strains of the Asian genotype (Table 3) compared with the S27 strain. In addition, unique amino acid substitutions were recorded for the IND-63-WB1 strain (Nsp1, V451M, and Nsp3, V1402A). The IND-73-MH5 strain displayed several unique substitutions, such as S765P, K1028T (Nsp2), S1571N (Nsp3) and D2427E (Nsp4).
Thirteen mutations were shared between the IND-06, RU, Yawat-2000 and Asian isolates. These included L172V, E234K, M383L, I384L (only in IND-73-MH5), T481I, C1177Y, S1178N, P1659S, K1685E, A1715T, I2377T, V2418I and V2467I.
Structural region.
As shown in Table 3, the capsid, E3, E2 and E1 regions exhibited three, four, ten and seven amino acid replacements, respectively, with reference to the S27 strain. In addition, the IND-63-WB1 strain exhibited a single substitution in the capsid (K89T) and 6K (A795T) regions, whereas the IND-73-MH5 strain showed four substitutions: capsid-P32L, E2-I609T, 6K-A795I and E1-I864T. Eleven mutations were shared between the IND-06, RU, Yawat-2000 and Asian isolates: G382K, I399M, G404E, N485T, L506M, S519G, M592R, S624N, A669T, I802V and V1131A.
5'- and 3'-NTRs.
The 5'-NTR was highly conserved, whereas the 3'-NTR showed maximum divergence (10.1–17.4 % between different genotypes). Within the 3'-NTR, the Asian genotype was characterized by an insertion of 10 nt between positions 11377 and 11378, another of 11 nt between positions 11514 and 11515 and one insertion at position 11425 with respect to S27. Similarly, several deletions compared with S27 were reported in the Asian genotype. These included two deletions at positions 11465–11466, one deletion at position 11595 and another deletion at position 11629. The IND-73-MH5 strain exhibited unique deletions at positions 11436, 11743 and 11744 compared with the S27 strain. A stretch of 19 ‘A’ nucleotides, a possible I-poly(A) site in S27 (Khan et al., 2002), showed six substitutions in the Asian genotype (Fig. 2). Deletion of a stretch of 14 of the 19 ‘A’ nucleotides reported for the RU isolates (Schuffenecker et al., 2006) was maintained in all Indian isolates belonging to the ECSA genotype. In addition, the AP strain showed one insertion between positions 11579 and 11580 and two deletions at positions 11629 and 11800.
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Molecular evolution
All IND-06 isolates exhibited three unique substitutions (T128K and T376M in Nsp1 and P23S in capsid). Another substitution, V27I (capsid), was displayed by all IND-06 isolates except the isolate from the AP state, which reported cases earlier. Additional substitutions were seen in the Karnataka (E1, K211N) and Tamilnadu (capsid, N80D) isolates. The isolate from the state of Rajasthan, reporting cases later, exhibited two additional substitutions (Nsp1, A101V; Nsp2, T1210M).
None of the values, calculated by the modified method of Nei & Gojobori (1989) for all of the non-structural as well as the structural genes, exceeded 1 (data not shown). The results of applying the tests for positive selection on all of the genes are presented in Table 4.
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8.3 under both M2 and M8 models. The Bayesian method assigned two sites (384I and 507L) to the positively selected class with 81 % probability under M8. In the Nsp2 gene, M8 identified 0.2 % of the sites to be under positive selection pressure with =10.9. Here, site 642C was identified with a posterior probability of 82 %. It was noted that, for all of the non-structural proteins, neither of the LRTs, between M2 (selection) and M1 (neutral) or between the more complex models M8 and M7, allowed the model of neutrality to be rejected in favour of positively selected sites (Table 4).
Among the structural proteins, for the capsid gene, M8 identified 5.6 % of sites with >1. A single site, 27V, was identified by Bayesian analysis to be under positive selection with a probability of 79 %. In the E3 gene, 5.2 % of sites were identified to be under positive selection with 3.2 under both the M2 and M8 models. Site 23I was detected to be in the positively selected class with a probability of 82 % under M8. The strength of positive selection in the case of the E2 gene under model M2, as well as under M8, was low, with 1.3, and the proportion of positively selected sites was estimated to be about 11.1 %. Of the five sites, i.e. 57, 194, 211, 318 and 377, identified in the E2 gene by using Bayesian analysis, one, 318V, was found with a probability of 88 % under M8. On the other hand, both the M2 and M8 models estimated a higher strength of positive selection (8.8) in the 6K gene, with the proportion of positively selected sites estimated as being 7.6 %. The 6K region was found to have two sites, 8V and 47A, under positive selection, with a higher probability of 86 % under M8. Again, for all of the structural genes, the models for neutrality (M1 and M7) fitted the data better than those for positive selection (M2 and M8).
Among the positively selected sites with a posterior probability of >75 %, Nsp1-507L, Nsp3-358S, E2-318V, 6K-8V, E1-211K and capsid-27V represent non-conserved sites (Tables 2 and 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In Asia, although epidemic re-emergence of CHIK was noted after 20 years during 2001–2003 in Indonesia (Laras et al., 2005), no sequence-based phylogenetic analysis was performed and hence evidence, if any, of a genotypic shift from Asian to African is not available. These data would be crucial to understanding the mode of genotypic shift in the continent. As the ECSA genotype was implicated in the current epidemics in the RU islands, it was envisaged that the same genotype was introduced into India from the Indian Ocean islands.
The key issues are understanding of (i) the origin and spread of the current CHIK virus strain and (ii) the association of mutations in the viral genome with increased transmissibility/virulence of the virus. As far as the first issue is concerned, sequence similarity of 99.9 % between the RU and Indian strains at the full-genome level implies circulation of the same strains in both countries. It also indicates a possibility of spread of the current strain from Indian Ocean islands to India, leading to an explosive epidemic of the ECSA genotype and not the Asian genotype that circulated earlier. However, the presence of the ECSA genotype in India in the year 2000 without any epidemic of the disease is noteworthy. The origin of this strain is not clear. As is evident from Fig. 1(b), the Yawat-2000 strain had highest nucleotide identity (99.62 %) with a strain from Uganda isolated in 1982. This strain was related more closely (98.2 %) to the IND-06 strains than to the S27 strain (97.3 %). It is pertinent to note here that, in the year 2000, the DRC experienced epidemic resurgence after 39 years. However, the IND-06 strains are related more closely to Yawat-2000 than to the DRC strains. Further studies would be required to understand the genesis of IND-06 strains with special reference to the Yawat-2000 strain. Sequence comparison (Table 2) of Yawat-2000 with the IND-06 and RU isolates showed that, with reference to the S27 strain, seven and four mutations in the non-structural and structural polyprotein, respectively, were present in all of these isolates. The Yawat-2000 strain exhibited six and five unique substitutions in the non-structural and structural regions, respectively. Association of these mutations with the increased transmissibility of the virus needs to be determined.
Taking into consideration the co-circulation of the Asian and ECSA genotypes in India, the possibility of generation of the current strain as a result of recombination events between the two genotypes was examined. However, no evidence for this was noted. Thus, the current strain is not a recombination product of the ECSA genotype with the Asian (present study) or west African (Schuffenecker et al., 2006) genotype. Further, no statistically significant evidence for positive selection was obtained and all of the genes in the CHIK virus genome were under purifying selection. It was further noted that the positively selected sites with a posterior probabability of >75 % represented some of the non-conserved sites.
An interesting observation of the evolution of CHIK viruses with the progression of the outbreak in Reunion includes a shift, A226V, during the latter period (beyond September 2005). However, among all of the Indian isolates, including Yawat-2000, 226A was maintained. The relationship of this change with the increase in the rate of transmission postulated for RU isolates does not hold true in the Indian scenario. In the present study, a few additional mutations at different locations were recorded in viruses isolated later during the epidemic and no definite pattern emerged. Of the seven amino acid substitutions (A164T, T312M, S375Tand V386A in E2, V8I in 6K, M269V and D284E in E1) in the structural polyprotein recorded as being unique for the RU isolates (Schuffenecker et al., 2006), all were present in the IND-06 isolates. Significantly, Yawat-2000 showed two of these seven substitutions: A164T in the E2 ectodomain and M269V in the E1 protein, suggesting importance of the other five mutations to the increased transmissibility of the virus.
Similar to the RU isolates (Schuffenecker et al., 2006), we confirm the presence of an opal stop codon at position 1857 in all of the IND-06 isolates. However, an arginine residue (as in S27) was maintained in Yawat-2000 and in both isolates belonging to the Asian genotype. As seen from Table 1, none of these isolates have been passaged extensively and, hence, this substitution does not seem to be related to the number of in vitro passages, as postulated for the S27 strain.
Similar to the RU isolates, we confirm the deletion of 14 of the 19 ‘A’ nucleotides in the I-poly(A) site in all of the IND-06 isolates and the Yawat-2000 isolate. All of the above isolates were sequenced at very early passage levels (Table 1). As speculated by Khan et al. (2002), the possibility of introduction of the I-poly(A) site during passaging (>50 in the C6/36 cell line) needs to be confirmed by repeated passaging of virus(es) lacking the same. Sequences of two strains of the Asian genotype isolated 10 years apart showed distinct insertions/deletions in the 3'-NTR (Fig. 2). The I-poly(A) stretch of 19 nt reported for the S27 isolate showed several mutations, leading to three stretches of four ‘A’ nucleotides interspersed with seven substitutions. Insertions of 10 nt (after position 11377 with respect to S27) and 11 nt (after 11514) are other characteristic features of this genotype. Considering 99.4 % nucleotide identity at the full-genome level over a period of 10 years between the two Asian strains sequenced, it may be concluded that the virus is relatively stable.
Understanding the association of genotypic and epidemiological changes is particularly important. The earlier outbreaks (1963–1973, Asian genotype) affected urban areas, whereas the current resurgence (African genotype) affected mainly rural areas for almost 8–10 months and later penetrated some of the large cities. Interestingly, although the African genotype was responsible for the resurgence of CHIK in the DRC in 2000, urban areas were affected for the first time, compared with earlier observations of the disease being essentially endemic to rural areas of tropical Africa and caused by virus belonging to the same genotype.
The state of Maharashtra represents a unique situation. This state reported epidemics of the disease in 1965 (Nagpur, Asian genotype), 1973 (Barsi, Asian genotype) and 2006 (almost all districts of the state, African genotype), as well as isolation of the African genotype from mosquitoes in 2000. It would be interesting to study the age-stratified prevalence of anti-CHIK virus antibodies in this state over a period of time as a measure of CHIK virus activity.
Overall, the full-genome analysis documented that an enormous number of cases in Reunion and India are caused by the same strain and are not the result of recombination of Asian and ECSA genotypes. The diversifying selection analysis suggests that the CHIK virus genome is under purifying selection, molecular divergence in the species being driven by random fixation of selectively neutral and few non-synonymous mutations. Future studies should endeavour to characterize transmissibility/virulence of various isolates of different genotypes at the molecular level.
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ACKNOWLEDGEMENTS |
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Received 14 November 2006;
accepted 19 March 2007.
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