Sequences of primers used in this study.

Being a tropical country with a conducive environment for mosquitoes, mosquito-borne illnesses such as dengue, chikungunya, lymphatic filariasis, malaria, and Japanese encephalitis are prevalent in Malaysia. Recent studies reported asymptomatic infection of West Nile virus (WNV) in animals and humans, but none of the studies included mosquitoes, except for one report made half a century ago. Considering the scarcity of information, our study sampled mosquitoes near migratory bird stopover wetland areas of West Coast Malaysia located in the Kuala Gula Bird Sanctuary and Kapar Energy Venture, during the southward migration period in October 2017 and September 2018. Our previous publication reported that migratory birds were positive for WNV antibody and RNA. Using a nested RT-PCR analysis, WNV RNA was detected in 35 (12.8%) out of 285 mosquito pools consisting of 2,635 mosquitoes, most of which were Culex spp. (species). Sanger sequencing and phylogenetic analysis revealed that the sequences grouped within lineage 2 and shared 90.12%–97.01% similarity with sequences found locally as well as those from Africa, Germany, Romania, Italy, and Israel. Evidence of WNV in the mosquitoes substantiates the need for continued surveillance of WNV in Malaysia.马来西亚作为热带国家，拥有适宜蚊虫孳生的环境，登革热、基孔肯雅热、淋巴丝虫病、疟疾及日本脑炎等虫媒传染病在该国广泛流行。近期有研究报道在动物与人类体内检出西尼罗病毒（West Nile virus, WNV）的无症状感染病例，但此前仅在半个世纪前有一项研究涉及蚊虫样本。鉴于相关研究资料较为匮乏，本研究于2017年10月及2018年9月的南迁候鸟季期间，在马来西亚西海岸瓜拉古拉鸟类保护区（Kuala Gula Bird Sanctuary）与巴生能源企业园区（Kapar Energy Venture）的候鸟停歇湿地周边采集蚊虫样本。本团队此前已发表研究成果，证实该区域候鸟体内可检出西尼罗病毒抗体与RNA。本研究通过巢式逆转录聚合酶链反应（nested RT-PCR）检测，在285份蚊虫混合样本（共涵盖2635只蚊虫）中，有35份（占比12.8%）检出西尼罗病毒RNA，其中绝大多数为库蚊属（Culex spp.）蚊虫。桑格测序（Sanger sequencing）与系统发育分析结果显示，本次检出的病毒序列归属于第2谱系，与本地及非洲、德国、罗马尼亚、意大利、以色列等地的已知序列相似度介于90.12%至97.01%之间。蚊虫体内检出西尼罗病毒的证据表明，马来西亚有必要持续开展西尼罗病毒监测工作。