Department of Pathology and Center for Tropical Diseases, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, USA1
Author for correspondence: Ann M. Powers. Fax +1 409 747 2415. e-mail ampowers{at}culex.utmb.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Africa, CHIK virus appears to be maintained in a sylvatic cycle involving wild primates and forest-dwelling Aedes spp. mosquitoes. Serological studies have repeatedly demonstrated the presence of antibodies in humans and wild primates throughout the moist forests and semi-arid savannas of Africa (Adesina & Odelola, 1991 ; Jupp & McIntosh, 1988
; Rodhain et al., 1989
; Salim & Porterfield, 1973
; Karabatsos, 1975
). To date, a vertebrate reservoir or sylvan transmission cycle has not been identified outside Africa, supporting the historical evidence (Carey, 1971
) that CHIK virus originated in Africa and was subsequently introduced into Asia, where it is now typically associated with Ae. aegypti mosquitoes. Strains from Africa and Asia are reported to differ biologically (Jupp & McIntosh, 1988
), indicating that distinct lineages may exist.
In 1996, a closely related alphavirus, onyong-nyong (ONN) virus, caused a major epidemic in southern Uganda (Lanciotti et al., 1998 ). This was the first epidemic of ONN virus infection since 1959, when a large epidemic swept across East Africa involving over 2 million reported cases (Johnson, 1988
). Unlike CHIK and all other alphaviruses, ONN virus is unique in its transmission patterns: the virus is not transmitted by culicine mosquitoes, but rather by anophelines, typically Anopheles funestus and An. gambiae. A vertebrate reservoir for ONN virus has not yet been identified. The transmission of ONN virus by two common vectors that inhabit much of tropical Africa and that live in close association with humans may be a factor in the rapid spread of the virus during epidemics.
With the exception of information derived from a limited number of serosurveys, little is known about the relationships of CHIK and ONN viruses (Chanas et al., 1979 ; Karabatsos, 1975
; Porterfield, 1961
). ONN is considered to be a subtype of CHIK virus: serological tests reveal a one-way antigenic cross-reactivity between the two agents. Antibody to CHIK virus reacts almost equally with both CHIK and ONN viral antigens while ONN virus antibodies react weakly against CHIK virus antigen (Blackburn et al., 1995
; Chanas et al., 1979
; Lee et al., 1997
; Karabatsos, 1985
). It was once postulated that mutations in CHIK virus led to the emergence of ONN virus and its ability to be transmitted by anopheline mosquitoes (Johnson, 1988
). However, genetic studies by Lanciotti et al. (1998)
as well as the phylogenetic analyses presented here clearly demonstrate that ONN and CHIK viruses are genetically distinct. The phylogenetic and serological studies presented here were designed to help elucidate the evolutionary relationships of these viruses and to aid in understanding their epidemic and maintenance transmission patterns.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Sequencing/phylogenetic analyses.
PCR products ranging from 1·2 to 1·7 kb were isolated from 1% agarose gels. The cleaned DNA fragments were cloned into the pCR2.1 TA cloning vector (Invitrogen) and white bacterial colonies screened for plasmids containing inserts of the correct size. Selected clones were sequenced using the plasmid-specific T7 promoter and m13 reverse primers combined with internal, CHIK virus-specific primers (C3205, 5' GCRACAAACCCSGTAAG 3'; C3152, 5' ACTGGCTRAAAGAACGAGG 3'). Sequencing was performed using an Applied Biosystems Prism 377 sequencer and automated DNA sequencing kit. The deduced amino acid sequences were aligned by using the PILEUP program in the Wisconsin Package (Genetics Computer Group) with default parameters, and the nucleotide sequences were aligned manually based on codon homology. Phylogenetic analyses were performed using maximum parsimony, neighbour joining and maximum likelihood programs implemented in the PAUP 4.0 software (Swofford, 1998 ). Distance analyses used the Kimura 2-parameter formula to correct for multiple substitutions of the same nucleotides. Unordered and ordered characters (transition/transversion ratio of 4:1; based on previous alphavirus estimates) were used in the parsimony analysis. Alphaviruses in the Venezuelan equine encephalitis, Barmah Forest and eastern equine encephalitis antigenic complexes were used as an outgroup. Bootstrap analysis (Felsenstein, 1985
) was performed with 1000 replicates to determine confidence values on the clades within trees.
Estimation of divergence times.
An average divergence rate for CHIK and ONN virus lineages was estimated by identification of sister-sequence pairs that were robust (bootstrap values >>90%), closely related and isolated at least 7 years apart in the same geographical region. The number of differences in synonymous changes depicted in branch lengths separating each sister sequence from the predicted common ancestors sequence was divided by the number of years between isolations to yield rates expressed as changes per nucleotide per year, and several estimates were compared to provide an estimated mean and standard deviation. Synonymous nucleotide divergence estimates for pair-wise sequence comparisons were generated using the formula of Li et al. (1985) to correct for multiple substitutions of the same nucleotides.
Production of immune sera.
Syrian golden hamsters and BALB/C mice were used to generate immune sera to three strains of CHIK virus (37997, Ross and 1455/75) and one strain of ONN virus (Igbo Ora, IbH12628). Animals received a single injection of virus (~105 p.f.u./ml), either intraperitoneally (i.p.) alone or subcutaneously with a mixture of virus and an Ae. aegypti mosquito salivary gland suspension to enhance the infection. Approximately 4 weeks post-inoculation, blood was obtained from the rodents from the retroorbital sinus and tested for antibody to CHIK virus by an immunofluorescent antibody assay (IFA) or by neutralization test (NT). Mice that were positive for CHIK virus antibody by IFA were injected i.p. with sarcoma 180 cells to produce hyperimmune ascitic fluid. Abdominal fluid was removed between 1 and 2 weeks after injection of the sarcoma cells and was used in IFA and plaque reduction neutralization tests (PRNT) to determine homologous titres.
Titration of neutralizing antibody.
Three of the four viruses (37997, Ross and 1455/75) generated a detectable homologous antibody response as determined by IFA (in mice) or NT (in hamsters). Only two of these, 37997 and Ross, had IFA titres sufficient to perform additional serological analyses. These two viruses were used in 80% PRNTs to determine both the homologous and heterologous neutralizing antibody titres.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Initial parsimony analyses revealed that several isolates, previously designated as CHIK virus, were genetically quite distinct from the prototype strain and from all other isolates examined. Inclusion of representative members of the Semliki Forest, Venezuelan equine encephalitis, Barmah Forest and eastern equine encephalitis virus antigenic complexes showed that these viruses were actually ONN (strain IPD A234), Semliki Forest (DAK ArB16878) and Sindbis-like (ArMg812 and B448) viruses. Additionally, CHIK virus strains 3412/78 and C-0392/95 were isolated from patients in Thailand suspected of having dengue virus infection, reinforcing the uncertainties of viral diagnosis based upon clinical presentation.
All of the CHIK and ONN virus isolates examined formed a monophyletic group within the Semliki Forest virus antigenic complex (Fig. 1), supported by a 100% bootstrap value. The ONN virus isolates formed a robust, distinct clade (100% bootstrap support) apart from all isolates of CHIK virus. ONN and CHIK virus sequences were approximately 28% and 13% divergent at the nucleotide and amino acid levels, respectively, underscoring the distinct nature of the two virus groups. Igbo Ora virus (strain IbH12628) grouped closely with the other strains of ONN, supporting previous reports that this is indeed an antigenic variant of ONN virus (Lanciotti et al., 1998
).
|
Estimated divergence times
An attempt was made to estimate the average rate of evolution of the CHIK and ONN viruses by comparison of sequences of sister taxa from the same geographical areas. Analysis of individual lineages was not possible because too few strains were available. Sister pairs were chosen that had bootstrap values >>90% and were isolated at least 7 years apart. Using these sequences, an estimated rate of evolution was determined to be 6x10-4 substitutions per nucleotide per year with a standard deviation of 4x10-4. The same estimate was obtained when pair-wise comparisons included distance corrections using the Kimura two-parameter formula or maximum likelihood (see below). The synonymous rate was 5x10-4 (standard deviation 3x10-4) and the nonsynonymous rate was 6x10-5 (standard deviation 5x10-5). Although these estimates were based on only six sister-pair sequences and therefore had a high degree of error, they are similar to those previously determined for neotropical alphaviruses (Weaver et al., 1993 , 1997
; Powers et al., 1997
). Using the synonymous rate and the Ks values computed for CHIK strain comparisons (ranging from 0·12 to 0·25 with standard deviations of 0·03), the Asian genotype evolved from a hypothetical African ancestor (node A, Fig. 1
) an estimated 50 to 430 (±1 standard deviation) years ago. Ks values for strains from the West African vs East African/Asian genotypes ranged from 0·75 to 0·86 with standard deviations of 0·10 to 0·12. Using these values, the ancestor of all of the CHIK virus strains is estimated to have emerged between 150 and 1350 years ago. Although the divergence time of ONN virus from CHIK virus could not be estimated reliably due to excessive variance in the Ks values resulting from near saturation of synonymous changes, divergence of CHIK and ONN viruses probably occurred at least thousands of years ago.
A potential flaw in these time estimates is that substitution rates may vary across nucleotide sites, including synonymous sites, as has been reported for human immunodeficiency virus (Leitner et al., 1997 ). Unequal substitution rates across sites could result in an underestimation of true sequence divergence because the sites undergoing more change may accumulate more sequential mutations than are estimated by traditional formulas that assume equal rates. Therefore, we estimated the gamma distribution shape parameter for unequal rates using maximum likelihood analysis applied to all equally parsimonious tree topologies as well as the topology generated by neighbour joining. The gamma distribution shape parameter estimate was 0·42, and the transition/transversion ratio estimate was 4·3, similar to previous alphavirus estimates using substitution data from parsimony analyses (Cilnis et al., 1996
; Weaver et al., 1994
, 1997
). We used these values to generate trees with maximum likelihood branch lengths applied to tree topologies generated using maximum parsimony and neighbour joining methods. Using this approach, similar divergence time estimates were obtained, with the Asian CHIK virus genotype emerging between 50 and 310 years ago, and the West and East African genotypes diverging 100 to 840 years ago.
Antigenic analysis
To determine the antigenic relatedness of viruses in the CHIK and ONN virus clades, one virus from each CHIK and ONN genotype was selected and used to generate antibodies in hamsters and mice (Table 2). Four weeks after a single injection of virus, animals were bled, and their sera tested for antibodies by IFA. The three CHIK viruses all produced specific antibodies while the ONN virus-infected mice and hamsters produced no detectable antibody response. The homologous IFA titre of CHIK virus strain 1455/75 was too low to be useful in neutralization assays; however, strains 37997 and Ross produced adequate antibody titres and were tested by 80% PRNT (Table 3
). Results indicated that these viruses have a greater than 4-fold difference in one direction suggesting that they are distinct enough to be classified as antigenic subtypes (Calisher & Karabatsos, 1988
; Calisher et al., 1980
). While no antibody against ONN virus was generated here, eliminating the possibility of performing two-way cross neutralization tests between CHIK and ONN viruses, it would be reasonable to assume that because distinct genotypes of CHIK virus are sufficiently different antigenically to be considered subtypes, the ONN virus lineage would be more likely to be considered a distinct group of viruses within this antigenic complex.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast to the numerous species involved in maintenance of CHIK virus infection in Africa, Ae. aegypti and Ae. albopictus are the only vector species known to transmit CHIK virus in Asia. These are urban and peridomestic, anthropophilic mosquitoes that maintain close associations with humans. It is therefore not surprising that outbreaks of CHIK virus infection are noted more frequently in Asia than in Africa. Several studies have demonstrated the varying susceptibility of different Asian mosquito strains for CHIK viruses (Banerjee et al., 1988 ; Mourya & Banerjee, 1987
; Mourya et al., 1987
; Tesh et al., 1976
). Because CHIK and dengue viruses are transmitted by the same mosquito species in Asia and because the clinical symptoms of the two viral diseases are similar, the two diseases are difficult to differentiate. Furthermore, there have been documented cases of simultaneous coinfection with CHIK and dengue viruses (Halstead, 1966
; Myers & Carey, 1967
), further complicating the characterization of CHIK virus maintenance, evolution and emergence in Asia.
Another question concerning the transmission of CHIK virus in Asia relates to the high degree of genetic similarity among Asian genotype viruses. Although our sampling of the Asian virus was limited, sequences from viruses spanning a wide geographical range and isolated over a period of almost 35 years showed less than 3% nucleotide sequence divergence (Fig. 1). This genetic conservation in Asia is intriguing for a virus that is known to be maintained only between humans and peridomestic mosquitoes. A similar, high degree of sequence conservation is observed within several other groups of alphaviruses: the North American eastern equine encephalitis viruses (Weaver et al., 1994
; Brault et al., 1999
), Highlands J virus from North America (Cilnis et al., 1996
), western equine encephalitis viruses (Weaver et al., 1997
) and the Sindbis-like viruses distributed throughout Australia (Sammels et al., 1999
). As an example, North American eastern equine encephalitis viruses are maintained by an avian reservoir host; therefore, the increased movement of the virus due to migration of the birds may effectively increase the virus population size and decrease founder effects and genetic drift. This may explain their sequence conservation (Weaver, 1995
; Weaver et al., 1992
; Brault et al., 1999
). It is unknown whether such an avian transmission cycle exists for CHIK viruses in Asia. Migratory patterns of both passerines and shorebirds do encompass much of Southeast Asia ranging from the Yellow Sea and South China Sea across the Philippines and Indonesia to Australia. Additionally, migration routes from India across the Indian Ocean to East Africa have been documented (Williams & Williams, 1990
). Serological testing of passerines and shorebirds in Southeast Asia could reveal if this is a plausible means of virus dispersal. Alternatively, dispersal of the virus by travel of humans could account for the presence of virtually identical viruses in areas as distant as Indonesia and the Philippines to Barsi in central India, as well as the introduction of the virus into Asia from Africa.
The phylogenetic results presented here clearly demonstrate that ONN virus did not emerge via a recent mutation of CHIK virus as was once postulated (Johnson, 1988 ). This hypothesis was based on serological evidence indicating that the viruses could only be distinguished by two-way specific antigenic tests (i.e. neutralization assay) or the use of monoclonal antibodies (Karabatsos, 1975
; Porterfield, 1961
). Antiserum raised against CHIK virus reacted with ONN virus but the reciprocal was not true, leading to the hypothesis that mutations in CHIK virus generated altered structural configurations in ONN virus affecting seroassay results (Johnson, 1988
; Williams & Woodall, 1961
; Williams et al., 1962
). It was suggested that these same mutations were responsible for the novel ability of ONN virus to replicate in and be transmitted by anopheline mosquitoes. However, if ONN virus undergoes periodic emergence from CHIK virus progenitors, ONN virus isolates from the outbreak in Uganda in 1996 would be predicted to group phylogenetically with CHIK virus isolates rather than with the other strains of ONN virus as seen in our analysis (Fig. 1
). For example, repeated emergence from a common progenitor has been found with epidemic/epizootic Venezuelan equine encephalitis viruses, which emerge periodically from enzootic viruses and occupy clades nested within the enzootic ID lineage (Kinney et al., 1992
; Powers et al., 1997
; Weaver et al., 1996
).
In addition to the antigenic and sequence differences between CHIK and ONN viruses, differences in several other biological patterns exist. Studies examining the relative ability of various strains of CHIK and ONN to replicate in different cell types have shown clear distinctions between these two viruses. CHIK viruses can replicate in both Ae. aegypti cell lines and numerous Aedes spp. mosquitoes (Chanas et al., 1979 ; Jupp & McIntosh, 1988
; Mourya et al., 1987
) while ONN does not appear to replicate in Ae. aegypti cells (Chanas et al., 1979
). Interestingly, both CHIK and ONN viruses can replicate in An. gambiae cells; however, only ONN replicates in and is believed to be transmitted primarily by An. gambiae or An. funestus mosquitoes under natural conditions (Corbet et al., 1961
; Williams et al., 1965
). Differences between the plaque sizes of CHIK and ONN viruses on mammalian cells have also been described (Chanas et al., 1979
; Tesh et al., 1976
); however, among CHIK and ONN viruses, plaque size may be strain specific (Chanas et al., 1979
).
A putative explanation for the varying biological properties among CHIK and ONN viruses is differences in the 3'NCR. All alphaviruses sequenced have repeat sequence elements in the 3'NCR that vary in length and number, often according to serogroup (Pfeffer et al., 1998 ; reviewed in Strauss & Strauss, 1994). Within some virus groups (e.g. the Sindbis-like viruses) very little sequence heterogeneity is detected in the 3'NCR (Shirako et al., 1991
) while other alphaviruses including Ross River virus, Venezuelan equine encephalitis and Semliki Forest complex viruses show high degrees of 3'NCR variation in both length and nucleotide composition (Faragher & Dalgarno, 1986
; Pfeffer et al., 1998
). Kuhn et al. (1991)
have shown that changes in the repeat sequence elements can affect virus replication in different cell types, suggesting that this region may be important in binding cellular proteins utilized during virus replication. Our sequences from 22 strains of CHIK and ONN viruses from a diverse geographical and temporal range demonstrated such a high degree of variability in the 3'NCR that nucleotide sequence alignments in this area were unreliable. This information, combined with the knowledge that the replicative ability of a given CHIK or ONN viral strain varies tremendously with different strains of mosquitoes, may support Kuhns hypothesis.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Banerjee, K., Mourja, D. T. & Malunjkar, A. S. (1988). Susceptibility and transmissibility of different geographical strains of Aedes aegypti mosquitoes to Chikungunya virus. Indian Journal of Medical Research 87, 134-138.[Medline]
Blackburn, N. K., Besselaar, T. G. & Gibson, G. (1995). Antigenic relationship between Chikungunya virus strains and onyong nyong virus using monoclonal antibodies. Research in Virology 146, 69-73.[Medline]
Brault, A. C., Powers, A. M., Chavez, S. L. V., Lopez, R. N., Cachon, M. F., Gutierrez, L. F. L., Kang, W., Tesh, R. B., Shope, R. E. & Weaver, S. C. (1999). Genetic and antigenic diversity among eastern equine encephalitis viruses from North, Central, and South America. American Journal of Tropical Medicine and Hygiene 61, 579-586.
Calisher, C. H. & Karabatsos, N. (1988). Arbovirus serogroups: definition and geographic distribution. In The Arboviruses: Epidemiology and Ecology, pp. 19-57. Edited by T. P. Monath. Boca Raton, FL: CRC Press.
Calisher, C. H., Shope, R. E., Brandt, W., Casals, J., Karabatsos, N., Murphy, F. A., Tesh, R. B. & Wiebe, M. E. (1980). Proposed antigenic classification of registered arboviruses. Intervirology 14, 229-232.[Medline]
Carey, D. E. (1971). Chikungunya and dengue: a case of mistaken identity? Journal of the History of Medicine and Allied Sciences 26, 243-262.[Medline]
Chanas, A. C., Hubalek, Z., Johnson, B. K. & Simpson, D. I. (1979). A comparative study of onyong nyong virus with Chikungunya virus and plaque variants. Archives of Virology 59, 231-238.[Medline]
Cilnis, M. J., Kang, W. & Weaver, S. C. (1996). Genetic conservation of Highlands J viruses. Virology 218, 343-351.[Medline]
Corbet, P. S., Williams, M. C. & Gillett, J. D. (1961). Onyong-nyong fever: an epidemic virus disease in East Africa. IV. Vector studies at epidemic sites. Transactions of the Royal Society of Tropical Medicine and Hygiene 55, 463-000.[Medline]
Diallo, M., Thonnon, J., Traore-Lamizana, M. & Fontenille, D. (1999). Vectors of Chikungunya virus in Senegal: current data and transmission cycles. American Journal of Tropical Medicine and Hygiene 60, 281-286.
Faragher, S. G. & Dalgarno, L. (1986). Regions of conservation and divergence in the 3' untranslated sequences of genomic RNA from Ross River virus isolates. Journal of Molecular Biology 190, 141-148.[Medline]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783-791.
Halstead, S. B. (1966). Mosquito-borne haemorrhagic fevers of South and South-East Asia. Bulletin of the World Health Organization 35, 3-15.[Medline]
Halstead, S. B., Scanlon, J. E., Umpaivit, P. & Udomsakdi, S. (1969a). Dengue and Chikungunya virus infection in man in Thailand, 19621964. IV. Epidemiologic studies in the Bangkok metropolitan area. American Journal of Tropical Medicine and Hygiene 18, 997-1021.[Medline]
Halstead, S. B., Udomsakdi, S., Scanlon, J. E. & Rohitayodhin, S. (1969b). Dengue and Chikungunya virus infection in man in Thailand, 19621964. V. Epidemiologic observations outside Bangkok. American Journal of Tropical Medicine and Hygiene 18, 1022-1033.[Medline]
Johnson, B. K. (1988). Onyong-nyong virus disease. In The Arboviruses: Epidemiology and Ecology, pp. 217-223. Edited by T. P. Monath. Boca Raton, FL: CRC Press.
Jupp, P. G. & McIntosh, B. M. (1988). Chikungunya virus disease. In The Arboviruses: Epidemiology and Ecology, pp. 137-157. Edited by T. P. Monath. Boca Raton, FL: CRC Press.
Jupp, P. G. & McIntosh, B. M. (1990). Aedes furcifer and other mosquitoes as vectors of Chikungunya virus at Mica, northeastern Transvaal, South Africa. Journal of the American Mosquito Control Association 6, 415-420.[Medline]
Karabatsos, N. (1975). Antigenic relationships of group A arboviruses by plaque reduction neutralization testing. American Journal of Tropical Medicine and Hygiene 24, 527-532.[Medline]
Karabatsos, N. (1985). International Catalogue of Arthropod-borne Viruses, 3rd edn, pp. 327328, 767768. San Antonio, TX: American Society of Tropical Medicine and Hygiene.
Killington, R. A., Stokes, A. & Hierholzer, J. C. (1996). Virus purification. In Virology Methods Manual, pp. 71-89. Edited by B. W. J. Mahy & H. O. Kangro. San Diego, CA: Academic Press.
Kinney, R. M., Tsuchiya, K. R., Sneider, J. M. & Trent, D. W. (1992). Genetic evidence that epizootic Venezuelan equine encephalitis (VEE) viruses may have evolved from enzootic VEE subtype I-D virus. Virology 191, 569-580.[Medline]
Kuhn, R. J., Niesters, H. G., Hong, Z. & Strauss, J. H. (1991). Infectious RNA transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis virus. Virology 182, 430-441.[Medline]
Lanciotti, R. S., Ludwig, M. L., Rwaguma, E. B., Lutwama, J. J., Kram, T. M., Karabatsos, N., Cropp, B. C. & Miller, B. R. (1998). Emergence of epidemic onyong-nyong fever in Uganda after a 35-year absence: genetic characterization of the virus. Virology 252, 258-268.[Medline]
Lee, E., Stocks, C., Lobigs, P., Hislop, A., Straub, J., Marshall, I., Weir, R. & Dalgarno, L. (1997). Nucleotide sequence of the Barmah Forest virus genome. Virology 227, 509-514.[Medline]
Leitner, T., Kumar, S. & Albert, J. (1997). Tempo and mode of nucleotide substitutions in gag and env gene fragments in human immunodeficiency virus type 1 populations with a known transmission history. Journal of Virology 71, 4761-4770.[Abstract]
Li, W.-H., Wu, C.-I. & Luo, C.-C. (1985). A new method for estimating synonymous and nonsynonymous rates of nucleotide substitution considering the relative likelihood of nucleotide and codon changes. Molecular Biology and Evolution 2, 150-174.[Abstract]
McCarthy, M. C., Haberberger, R. L., Salib, A. W., Soliman, B. A., El-Tigani, A., Khalid, I. O. & Watts, D. M. (1996). Evaluation of arthropod-borne viruses and other infectious disease pathogens as the causes of febrile illnesses in the Khartoum Province of Sudan. Journal of Medical Virology 48, 141-146.[Medline]
Mourya, D. T. & Banerjee, K. (1987). Experimental transmission of Chikungunya virus by Aedes vittatus mosquitoes. Indian Journal of Medical Research 86, 269-271.[Medline]
Mourya, D. T., Malunjkar, A. S. & Banerjee, K. (1987). Susceptibility and transmissibility of Aedes aegypti to four strains of Chikungunya virus. Indian Journal of Medical Research 86, 185-190.[Medline]
Myers, R. M. & Carey, D. E. (1967). Concurrent isolation from patient of two arboviruses, chikungunya and dengue type 2. Science 157, 1307-1308.[Medline]
Pfeffer, M., Kinney, R. M. & Kaaden, O. R. (1998). The alphavirus 3'-nontranslated region: size heterogeneity and arrangement of repeated sequence elements. Virology 240, 100-108.[Medline]
Porterfield, J. S. (1961). Cross-neutralization studies with group A arthropod-borne viruses. Bulletin of the World Health Organization 24, 735-741.[Medline]
Powers, A. M., Oberste, M. S., Brault, A. C., Rico-Hesse, R., Schmura, S. M., Smith, J. F., Kang, W., Sweeney, W. P. & Weaver, S. C. (1997). Repeated emergence of epidemic/epizootic Venezuelan equine encephalitis from a single genotype of enzootic subtype ID virus. Journal of Virology 71, 6697-6705.[Abstract]
Rao, T. R. (1966). Recent epidemics caused by Chikungunya virus in India, 19631965. Scientific Culture 32, 215.
Rodhain, F., Gonzalez, J. P., Mercier, E., Helynck, B., Larouze, B. & Hannoun, C. (1989). Arbovirus infections and viral haemorrhagic fevers in Uganda: a serological survey in Karamoja district, 1984. Transactions of the Royal Society of Tropical Medicine and Hygiene 83, 851-854.[Medline]
Salim, A. R. & Porterfield, J. S. (1973). A serological survey on arbovirus antibodies in the Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene 67, 206-210.[Medline]
Sammels, L. M., Lindsay, M. D., Poidinger, M., Coelen, R. J. & Mackenzie, J. S. (1999). Geographic distribution and evolution of Sindbis virus in Australia. Journal of General Virology 80, 739-748.[Abstract]
Shirako, Y., Niklasson, B., Dalrymple, J. M., Strauss, E. G. & Strauss, J. H. (1991). Structure of the Ockelbo virus genome and its relationship to other Sindbis viruses. Virology 182, 753-764.[Medline]
Swofford, D. L. (1998). PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sunderland, MA: Sinauer Associates.
Tesh, R. B., Gubler, D. J. & Rosen, L. (1976). Variation among geographic strains of Aedes albopictus in susceptibility to infection with Chikungunya virus. American Journal of Tropical Medicine and Hygiene 25, 326-335.[Medline]
Traore-Lamizana, M., Fontenille, D., Zeller, H. G., Mondo, M., Diallo, M., Adam, F., Eyraud, M., Maiga, A. & Digoutte, J. P. (1996). Surveillance for yellow fever virus in eastern Senegal during 1993. Journal of Medical Entomology 33, 760-765.[Medline]
Weaver, S. C. (1995). Evolution of alphaviruses. In Molecular Basis of Virus Evolution, pp. 501-530. Edited by A. J. Gibbs, C. H. Calisher & F. Garcia-Arenal. Cambridge: Cambridge University Press.
Weaver, S. C., Rico-Hesse, R. & Scott, T. W. (1992). Genetic diversity and slow rates of evolution in New World alphaviruses. Current Topics in Microbiology and Immunology 176, 99-117.[Medline]
Weaver, S. C., Hagenbaugh, A., Bellew, L. A., Netesov, S. V., Volchkov, V. E., Chang, G. J., Clarke, D. K., Gousset, L., Scott, T. W., Trent, D. W. and others (1993). A comparison of the nucleotide sequences of eastern and western equine encephalomyelitis viruses with those of other alphaviruses and related RNA viruses Virology 197, 375390; erratum 202, 1083.[Medline]
Weaver, S. C., Hagenbaugh, A., Bellew, L. A., Gousset, L., Mallampalli, V., Holland, J. J. & Scott, T. W. (1994). Evolution of alphaviruses in the eastern equine encephalomyelitis complex. Journal of Virology 68, 158-169.[Abstract]
Weaver, S. C., Salas, R., Rico-Hesse, R., Ludwig, G. V., Oberste, M. S., Boshell, J. & Tesh, R. B. (1996). Re-emergence of epidemic Venezuelan equine encephalomyelitis in South America. VEE Study Group. Lancet 348, 436-440.[Medline]
Weaver, S. C., Kang, W., Shirako, Y., Rumenapf, T., Strauss, E. G. & Strauss, J. H. (1997). Recombinational history and molecular evolution of western equine encephalomyelitis complex alphaviruses. Journal of Virology 71, 613-623.[Abstract]
Williams, T. C. & Williams, J. M. (1990). The orientation of transoceanic migrants. In Bird Migration: Physiology and Ecophysiology, pp. 7-21. Edited by E. Gwinner. New York: Springer-Verlag.
Williams, M. C. & Woodall, J. P. (1961). Onyong-nyong fever: an epidemic virus disease in East Africa. II. Isolation and some properties of the virus. Transactions of the Royal Society of Tropical Medicine and Hygiene 55, 135-141.
Williams, M. C., Woodall, J. P. & Porterfield, J. S. (1962). Onyong-nyong fever: an epidemic virus disease in East Africa. V. Human antibody studies by plaque inhibition and other serological tests. Transactions of the Royal Society of Tropical Medicine and Hygiene 56, 166-172.[Medline]
Williams, M. C., Woodall, J. P., Corbet, P. S. & Gillett, J. D. (1965). Onyong-nyong fever: an epidemic virus disease in East Africa. VIII. Virus isolations from Anopheles mosquitoes. Transactions of the Royal Society of Tropical Medicine and Hygiene 59, 300-306.[Medline]
Received 9 June 1999;
accepted 21 October 1999.