Molecular detection of Indian Ocean Lineage Chikungunya virus RNA in field collected Culex quinquefasciatusSay from Bangkok, Thailand but no evidence of virus replication.

Atchara Phumee,Akanitt Jittmittraphap,Sriwatapron Sor-Suwan,Proawpilart Intayot,Padet Siriyasatien,Pierre Roques

doi:10.1371/journal.pone.0246026

Atchara Phumee, Akanitt Jittmittraphap + Show 4 more

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https://doi.org/10.1371/journal.pone.0246026

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Abstract

Following an outbreak of chikungunya virus (CHIKV) infections in Thailand in 2019, numerous cases of CHIKV infection have been diagnosed in Bangkok, the capital of the country. In our previous investigation of the vectors for disease transmission, we found natural infection of CHIKV in both male and female Aedes aegypti mosquitoes collected from the outbreak areas in Bangkok. Some reports mentioned the detection of CHIKV in Culex mosquitoes. In Thailand, the Culex quinquefasciatus Say mosquito is a common species found in urban and rural settings that coexists with Ae. aegypti. However, the role of Cx. quinquefasciatus mosquitoes in the spread of the Indian Ocean Lineage (IOL) of CHIKV in Thailand has never been investigated. In this study, Cx. quinquefasciatus were collected (16 males and 27 females) from an outbreak area in Bangkok. Eight of the 27 in field-caught female Cx. quinquefasciatus were positive for IOL CHIKV RNA, and 99–100% identity and full 100% coverage of sequences similar to CHIKV isolated from female Ae. aegypti in Bangkok, Thailand, whereas viral RNA was not detected in male samples using nested-RT-PCR. To determine whether CHIKV is able to replicate in Cx. quinquefasciatus, the laboratory strain of Cx. quinquefasciatus was allowed to feed on blood containing IOL CHIKV isolated from patient serum. The nested-RT-PCR, virus isolation, and immunofluorescence assay (IFA) were performed for CHIKV detection and replication. The results showed that CHIKV RNA was detected in Cx. quinquefasciatus until day 4 post infection. CHIKV did not produce any remarkable signs of infection, dissemination, or transmission in Cx. quinquefasciatus, and cytopathic effect (CPE) was not observed in C6/36 cells when infected with supernatant obtained from Cx. quinquefasciatus at days 7, 10, 14, and 21 post infection when compared to Ae. aegypti. The data from this study infer that CHIKV may be detected in Cx. quinquefasciatus but that the mosquito is not able to transmit CHIKV in Thailand.

Highlights

Chikungunya virus (CHIKV) is an emerging and re-emerging infection that has occurred in many countries over several continents, including the Americas, eastern Africa, the eastern Indian Ocean islands, the western Indian Ocean islands, France, Italy, India, Singapore, Malaysia, Indonesia, and Thailand [1, 2]
The BLAST result showed that the sequence similar to CHIKV isolated from female Ae. aegypti in Bangkok, Thailand with 99–100% identity and full 100% coverage of the nucleotide sequences
The result of phylogenetic tree revealed that these CHIKV sequences were classed into the Indian Ocean clade, which belonged to the ECSA genotype (Fig 1A); no presence of the E1: A226V in Indian Ocean clade of CHIKV showed in present study (Fig 1B)

Summary

Introduction

Chikungunya virus (CHIKV) is an emerging and re-emerging infection that has occurred in many countries over several continents, including the Americas, eastern Africa, the eastern Indian Ocean islands, the western Indian Ocean islands, France, Italy, India, Singapore, Malaysia, Indonesia, and Thailand [1, 2]. In Thailand, the reported of CHIKV outbreaks in the southern part of the country from the Bureau of Epidemiology, Department of Disease Control, Ministry of Public Health in 2008–2009 were affected more than 54,000 cases and the genetic studies found a characteristic alanine-to-valine mutation at amino acid residue 226 (A226V) of the E1 envelope glycoprotein of an East/Central/South/Africa (ECSA) genotype [3]. Chansaenroj et al (2020) demonstrated that the complete genome sequences of CHIKV isolated from patients and an Aedes aegypti mosquito from this outbreak in southwest Bangkok belonged to an East/Central/South African (ECSA) lineage, with a mutation at A226V. The replication of CHIKV was studied in laboratory strains of Cx. quinquefasciatus by using nested-RT-PCR, virus isolation in C6/36 cells, and immunofluorescence assay

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