Molecular characterization of Chikungunya virus isolates from clinical samples and adult Aedes albopictus mosquitoes emerged from larvae from Kerala, South India
© Niyas et al; licensee BioMed Central Ltd. 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.
- Powers AM, Logue CH: Changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus. J Gen Virol. 2007, 88: 2363-2377. 10.1099/vir.0.82858-0.PubMedView ArticleGoogle Scholar
- Staples JE, Breiman RF, Powers AM: Chikungunya fever: an epidemiological review of a re-emerging infectious disease. Clin Infect Dis. 2009, 49: 942-948. 10.1086/605496.PubMedView ArticleGoogle Scholar
- Schuffenecker I, Iteman I, Michault A, Murri S, Frangeul L, Vaney MC, Lavenir R, Pardigon N, Reynes JM, Pettinelli F, et al: Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak. PLoS Med. 2006, 3: e263-10.1371/journal.pmed.0030263.PubMedPubMed CentralView ArticleGoogle Scholar
- Rezza G, Nicoletti L, Angelini R, Romi R, Finarelli AC, Panning M, Cordioli P, Fortuna C, Boros S, Magurano F, et al: Infection with chikungunya virus in Italy: an outbreak in a temperate region. Lancet. 2007, 370: 1840-1846. 10.1016/S0140-6736(07)61779-6.PubMedView ArticleGoogle Scholar
- Chandak NH, Kashyap RS, Kabra D, Karandikar P, Saha SS, Morey SH, Purohit HJ, Taori GM, Daginawala HF: Neurological complications of Chikungunya virus infection. Neurol India. 2009, 57: 177-180. 10.4103/0028-3886.51289.PubMedView ArticleGoogle Scholar
- Das T, Jaffar-Bandjee MC, Hoarau JJ, Krejbich Trotot P, Denizot M, Lee-Pat-Yuen G, Sahoo R, Guiraud P, Ramful D, Robin S, et al: Chikungunya fever: CNS infection and pathologies of a re-emerging arbovirus. Prog Neurobiol. 2009, 91: 121-9. 10.1016/j.pneurobio.2009.12.006.PubMedView ArticleGoogle Scholar
- Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S: A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog. 2007, 3: e201-10.1371/journal.ppat.0030201.PubMedPubMed CentralView ArticleGoogle Scholar
- Vazeille M, Moutailler S, Coudrier D, Rousseaux C, Khun H, Huerre M, Thiria J, Dehecq JS, Fontenille D, Schuffenecker I, et al: Two Chikungunya isolates from the outbreak of La Reunion (Indian Ocean) exhibit different patterns of infection in the mosquito, Aedes albopictus. PLoS One. 2007, 2: e1168-10.1371/journal.pone.0001168.PubMedPubMed CentralView ArticleGoogle Scholar
- Yergolkar PN, Tandale BV, Arankalle VA, Sathe PS, Sudeep AB, Gandhe SS, Gokhle MD, Jacob GP, Hundekar SL, Mishra AC: Chikungunya outbreaks caused by African genotype, India. Emerg Infect Dis. 2006, 12: 1580-1583.PubMedPubMed CentralView ArticleGoogle Scholar
- Arankalle VA, Shrivastava S, Cherian S, Gunjikar RS, Walimbe AM, Jadhav SM, Sudeep AB, Mishra AC: Genetic divergence of Chikungunya viruses in India (1963-2006) with special reference to the 2005-2006 explosive epidemic. J Gen Virol. 2007, 88: 1967-76. 10.1099/vir.0.82714-0.PubMedView ArticleGoogle Scholar
- Kumar NP, Joseph R, Kamaraj T, Jambulingam P: A226V mutation in virus during the 2007 chikungunya outbreak in Kerala, India. J Gen Virol. 2008, 89: 1945-1948. 10.1099/vir.0.83628-0.PubMedView ArticleGoogle Scholar
- Santhosh SR, Dash PK, Parida MM, Khan M, Tiwari M, Lakshmana Rao PV: Comparative full genome analysis revealed E1: A226V shift in 2007 Indian Chikungunya virus isolates. Virus Res. 2008, 135: 36-41. 10.1016/j.virusres.2008.02.004.PubMedView ArticleGoogle Scholar
- Kannan M, Rajendran R, Sunish IP, Balasubramaniam R, Arunachalam N, Paramsivan R, Tewari SC, Samuel PP, Tyagi BK: A study on chikungunya outbreak during 2007 in Kerala, south India. Indian J Med Res. 2009, 129: 311-315.PubMedGoogle Scholar
- Sreekumar E, Issac A, Nair S, Hariharan R, Janki MB, Arathy DS, Regu R, Mathew T, Anoop M, Niyas KP, Pillai MR: Genetic characterization of 2006-2008 isolates of Chikungunya virus from Kerala, South India, by whole genome sequence analysis. Virus Genes. 2010, 40: 14-27. 10.1007/s11262-009-0411-9.PubMedView ArticleGoogle Scholar
- WHO/SEARO (2008) Chikungunya Fever, a re-emerging disease in Asia. WHO South East Asia Regional Office. [http://www.searo.who.int/en/Section10/Section2246.htm]
- Hasebe F, Parquet MC, Pandey BD, Mathenge EG, Morita K, Balasubramaniam V, Saat Z, Yusop A, Sinniah M, Natkunam S, Igarashi A: Combined detection and genotyping of Chikungunya virus by a specific reverse transcription-polymerase chain reaction. J Med Virol. 2002, 67: 370-374. 10.1002/jmv.10085.PubMedView ArticleGoogle Scholar
- Kumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform. 2004, 5: 150-163. 10.1093/bib/5.2.150.PubMedView ArticleGoogle Scholar
- Escobar-Herrera J, Medina-Ramirez FJ, Gutierrez-Escolano AL: A carboxymethyl-cellulose plaque assay for feline calicivirus. J Virol Methods. 2007, 146: 393-396. 10.1016/j.jviromet.2007.07.013.PubMedView ArticleGoogle Scholar
- Rosen L, Shroyer DA, Tesh RB, Freier JE, Lien JC: Transovarial transmission of dengue viruses by mosquitoes Aedes albopictus and Aedes aegypti. Am J Trop Med Hyg. 1983, 32: 1108-1119.PubMedGoogle Scholar
- Gunther J, Martinez-Munoz JP, Perez-Ishiwara DG, Salas-Benito J: Evidence of vertical transmission of dengue virus in two endemic localities in the state of Oaxaca, Mexico. Intervirology. 2007, 50: 347-352. 10.1159/000107272.PubMedView ArticleGoogle Scholar
- Mishra AC, Mourya DT: Transovarial transmission of West Nile virus in Culex vishnui mosquito. Indian J Med Res. 2001, 114: 212-214.PubMedGoogle Scholar
- Rosen L, Shroyer DA, Lien JC: Transovarial transmission of Japanese encephalitis virus by Culex tritaeniorhynchus mosquitoes. Am J Trop Med Hyg. 1980, 29: 711-712.PubMedGoogle Scholar
- Aitken TH, Tesh RB, Beaty BJ, Rosen L: Transovarial transmission of yellow fever virus by mosquitoes (Aedes aegypti). Am J Trop Med Hyg. 1979, 28: 119-121.PubMedGoogle Scholar
- Mourya DT: Absence of transovarial transmission of Chikungunya virus in Aedes aegypti &Ae. albopictus mosquitoes. Indian J Med Res. 1987, 85: 593-595.PubMedGoogle Scholar
- Vazeille M, Mousson L, Failloux AB: Failure to demonstrate experimental vertical transmission of the epidemic strain of Chikungunya virus in Aedes albopictus from La Reunion Island, Indian Ocean. Mem Inst Oswaldo Cruz. 2009, 104: 632-635. 10.1590/S0074-02762009000400017.PubMedView ArticleGoogle Scholar
- Zytoon EM, el-Belbasi HI, Matsumura T: Transovarial transmission of chikungunya virus by Aedes albopictus mosquitoes ingesting microfilariae of Dirofilaria immitis under laboratory conditions. Microbiol Immunol. 1993, 37: 419-421.PubMedView ArticleGoogle Scholar
- Thavara U, Tawatsin A, Pengsakul T, Bhakdeenuan P, Chanama S, Anantapreecha S, Molito C, Chompoosri J, Thammapalo S, Sawanpanyalert P, Siriyasatien P: Outbreak of chikungunya fever in Thailand and virus detection in field population of vector mosquitoes, Aedes aegypti (L.) and Aedes albopictus Skuse (Diptera: Culicidae). Southeast Asian J Trop Med Public Health. 2009, 40: 951-962.PubMedGoogle Scholar
- Myles KM, Pierro DJ, Olson KE: Deletions in the putative cell receptor-binding domain of Sindbis virus strain MRE16 E2 glycoprotein reduce midgut infectivity in Aedes aegypti. J Virol. 2003, 77: 8872-8881. 10.1128/JVI.77.16.8872-8881.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Pierro DJ, Powers EL, Olson KE: Genetic determinants of Sindbis virus strain TR339 affecting midgut infection in the mosquito Aedes aegypti. J Gen Virol. 2007, 88: 1545-1554. 10.1099/vir.0.82577-0.PubMedView ArticleGoogle Scholar
- Pierro DJ, Powers EL, Olson KE: Genetic determinants of Sindbis virus mosquito infection are associated with a highly conserved alphavirus and flavivirus envelope sequence. J Virol. 2008, 82: 2966-2974. 10.1128/JVI.02060-07.PubMedPubMed CentralView ArticleGoogle Scholar
- Tsetsarkin KA, McGee CE, Volk SM, Vanlandingham DL, Weaver SC, Higgs S: Epistatic roles of E2 glycoprotein mutations in adaption of chikungunya virus to Aedes albopictus and Ae. aegypti mosquitoes. PLoS One. 2009, 4: e6835-10.1371/journal.pone.0006835.PubMedPubMed CentralView ArticleGoogle Scholar
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