Molecular characterization of Chikungunya virus isolates from clinical samples and adult Aedes albopictus mosquitoes emerged from larvae from Kerala, South India
© Niyas et al. 2010
Received: 18 March 2010
Accepted: 13 August 2010
Published: 13 August 2010
Chikungunya virus (CHIKV), an arthritogenic alphavirus, is transmitted to humans by infected Aedes (Ae.) aegypti and Ae.albopictus mosquitoes. In the study, reverse-transcription PCR (RT PCR) and virus isolation detected CHIKV in patient samples and also in adult Ae.albopictus mosquitoes that was derived from larvae collected during a chikungunya (CHIK) outbreak in Kerala in 2009. The CHIKV strains involved in the outbreak were the East, Central and South African (ECSA) genotype that had the E1 A226V mutation. The viral strains from the mosquitoes and CHIK patients from the same area showed a close relationship based on phylogenetic analysis. Genetic characterization by partial sequencing of non-structural protein 2 (nsP2; 378 bp), envelope E1 (505 bp) and E2 (428 bp) identified one critical mutation in the E2 protein coding region of these CHIKV strains. This novel, non-conservative mutation, L210Q, consistently present in both human and mosquito-derived samples studied, was within the region of the E2 protein (amino acids E2 200-220) that determines mosquito cell infectivity in many alpha viruses. Our results show the involvement of Ae. albopictus in this outbreak in Kerala and appearance of CHIKV with novel genetic changes. Detection of virus in adult mosquitoes, emerged in the laboratory from larvae, also points to the possibility of transovarial transmission (TOT) of mutant CHIKV strains in mosquitoes.
Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family and is an important re-emerging pathogen. It has been responsible for major fever epidemics in many parts of the world [1, 2]. The disease, chikungunya (CHIK), is characterized by high fever, headache, myalgia, severe and prolonged arthralgia, and erythematous skin rashes . In general, it is considered as a self-limiting illness. However, recent outbreaks of CHIK exhibited unusual severity, neurological complications and suspected mortality [3–6]. The disease is transmitted by the bite of Aedes ( Ae.) aegypti and Ae. albopictus mosquitoes. Studies have shown that Ae. albopictus facilitates rapid transmission of the new strains of CHIKV that had adaptive mutations in the viral genome [7, 8].
CHIK epidemic has caused considerable morbidity in recent years in India [9, 10]. Kerala, in South India, was one among the worst affected states [11–14]. Abundance of Ae.albopictus in many parts of the state was implicated for the rapid spread of the infection . Recent studies carried out in CHIKV from Kerala [11, 12, 14] have revealed novel genetic changes in the virus isolates from 2006-2008 outbreaks. Reports on virus isolation from mosquito vectors from the region are currently not available. The aim of the present work was to look for novel genetic changes in the isolates from 2009 by sequence analysis of selected genomic regions, and also to look for CHIKV in Ae. albopictus mosquitoes
For virus detection in mosquitoes, households of CHIK patients, whose serum samples were confirmed in the laboratory by RT-PCR, were subsequently visited and larval sampling was done. Stagnant water collected in discarded articles such as coconut shells, broken earthern-wares, plastic bottles and damaged drains were searched for Ae. albopictus larvae. Third and fourth instar larvae and pupae were phenotypically identified in situ using standard keys and these were collected and transferred to containers with fresh water. Four households each in Olavanna and Chaliyum, and three households in Beypore were surveyed. Larvae and pupae collected from each location were made into a single pool. In the laboratory, these three pools were independently reared in bowls with water, kept in mosquito cages at an ambient temperature of 25-30°C and a relative humidity 60-70%. The newly emerged adult mosquitoes were collected and frozen at -20°C for 30 minutes. Whole-mosquito tissue extracts were prepared by homogenizing pools of adult mosquitoes [each pool with 30 individual mosquitoes (both males and females) representing a single location]. Frozen mosquitoes were homogenized in 700 μl of Dulbecos Modified Eagle's Medium (DMEM) using a micropestle. These were then clarified by centrifugation at 800 × g at 4°C and sterilized by filtering through 0.2 μM membrane filter (Millex GV, Millipore) and used for RNA isolation.
Details of the primers used for PCR amplification in the study.
Sequence (5'→3'); location with respect to S27 sequence (GenBank Accession AF369024)
RT PCR for CHIKV detection in patient and adult mosquitoes derived from larvae
RT PCR of partial sequences CHIKV genes for sequencing and phylogenetic analysis
Analysis of the partial nucleotide sequences of nsP2 (378 bp; position 3246-3623), E1 (position 10427-10931) and E2 (position 8893- 9320) revealed a few random nucleotide changes in the CHKV isolates studied (Additional File 1) with respect to the corresponding sequences of the previous isolates from Kerala . The nucleotide change T3297C observed in the 2007 & 2008 Kerala isolates, causing an L539 S mutation in the nsP2 protein, was absent in CHIKV strains of the present outbreak. A novel substitution (T3296C) was consistently observed in a few strains from patients (RGCB711, RGCB730, and RGCB755) and in all the three isolates from mosquito samples. However, this was a synonymous substitution. The E1 sequence of all the strains had the C10670T substitution resulting in the A226V mutation identified in the recent isolates of CHIKV [3, 14]. Another new substitution (E1 G10864A) detected consistently in all the mosquito-derived strains and two of the clinical isolates (RGCB711 & RGCB755) can result in an amino acid change of V291I. Two nucleotide substitutions (A9114G and T9170A) were observed in the E2 coding region of all the strains studied from the outbreak. The latter substitution resulted in an amino acid change L210Q in the predicted sequence of amino acids of the E2 protein. Phylogenetic analysis revealed that the strains involved in the outbreak were closely related to the East-Central South African genotype of the CHIKV (Figure 3b). The gene sequences of CHIKV obtained from mosquito and patient samples formed a close cluster, distinct from the strains isolated previously from Kerala , rest of India and other parts of the world. This show a common genetic origin of the virus strains from patients and mosquitoes in this outbreak.
The results from this study, along with the previous observations [11, 12, 14], indicate a constant genomic evolution of the CHIKV strains circulating in Kerala. The availability of large numbers of Ae.albopictus vector mosquitoes  and an immunologically naïve human population unexposed to CHIK in different parts of the state might facilitate recurrent infections and viral evolution. Emergence of newer strains with altered virulence and transmission potential is a possible out come of the long term viral persistence in the community. Further entomological and virological studies with these new CHIKV strains would help to understand the changing epidemiology of this re-emerging virus.
The authors are thankful to the medical staff of the primary health centres (PHC) in Olavanna, Beypore and Chaliyum for the help extended for the patient sample collection. The financial assistance by Department of Biotechnology, Government of India as intramural funding and the encouragement and support by the Director, RGCB, are gratefully acknowledged.
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