Amplification of Sequences Suggestive of Old Port Virus from a Pool of Culex tritaeniorhynchus from Malaysia

Abstract

Purpose: A previously completed project on the surveillance of West Nile virus (WNV) among migratory birds in Malaysia found that the local WNV isolates were clustered with the WNV lineage 2. This project was conducted at the Kuala Gula Bird Sanctuary, a site which is home to some 200 species of birds and a popular tourist destination. Since human-vector-animal interactions can only intensify in the years to come due to increased eco-tourism and economic activities, we sought to assess the possibility of local mosquitoes carrying viruses belonging to the Alphavirus and Flavivirus genera through a mosquito-surveillance program. Methods & Materials: The CDC miniature light trap was placed around possible landing areas of migratory birds. Sampling of mosquitoes was carried out during the period of migratory birds’ stopover in Malaysia in 2017. After morphological identification, the captured mosquitoes were sorted pools with each pool containing ten individuals. Following that, they were homogenized and subjected to RNA extraction. Pan-Alphavirus and Flavivirus primers were used for RT-PCR amplification of the extracted RNA samples and, the amplified product was sequenced and subsequently analysed. Results: A total of 137 pools (n=1370 mosquitoes) containing Culex gelidus, Culex pseudovishnui, Culex tritaeniorhynchus and Culex vishnui were studied. From that, a PCR product was successfully amplified using the Pan-Flavivirus primers on one Cx. tritaeniorhynchus pool. BLAST analysis revealed that the amplified sequences had 83.52% similarity to Old Port virus (OPV). Next Generation Sequencing is currently ongoing to obtain the full or near-full-length OPV genome sequences. Conclusion: Our results suggest that the mosquito-surveillance program has to be maintained and magnified as it signalled a potential uncovering of greater novel mosquito-borne viruses. Even though still unclassified, the OPV could possibly cause disease in humans and animals, hence justifying further investigation.