Detection of Japanese Encephalitis Virus in Mosquitoes Collected from Selected Areas of Laguna, Philippines Using an Optimized One-Step RT-PCR Protocol

Japanese encephalitis virus is a leading cause of encephalitis in humans and agricultural animals across Southern and Eastern Asia. It is primarily transmitted by mosquitoes of the Culex genus, notably Culex tritaeniorhynchus. In the Philippines, JEV is reported to account for 7% to 18% of clinical cases of meningitis and encephalitis. Therefore, updated and comprehensive data on circulating JEV strains in the country are crucial for developing effective mitigation strategies. This study aimed to optimize an assay for JEV detection using one-step reverse transcription polymerase chain reaction (RT-PCR). Additionally, the presence of JEV in mosquitoes collected from eleven cities and municipalities in Laguna was assessed, and the mosquito carriers were identified based on morphological characteristics. Results demonstrated that the optimized one-step RT-PCR assay successfully detected JEV at various dilutions, with a limit of detection (LoD) of 0.115 ng/L. The assay exhibited 100% specificity, with no amplification observed for any viruses other than JEV. Sequence analysis of the positive control, JEV SA14-14-2 RNA, revealed 96.96% to 98.41% similarity to JEV reference sequences in the NCBI database. Among the 5,522 female mosquitoes collected, Culex tritaeniorhynchus and Culex gelidus were the most prevalent species. The highest mosquito species diversity and the most even distribution were found in the three barangays of Paete, but Siniloan, Laguna had the highest vector density for the three Culex species, Culex tritaeniorhynchus, Culex gelidus, and Culex fuscocephala. RT-PCR analysis of 189 pooled mosquito samples revealed no detectable JEV genetic material. This study is likely the first investigation of JEV presence in mosquito samples from Laguna, Philippines. These findings contribute to proactive JEV surveillance, enhancing our understanding of the virus's presence and distribution. The results underscore the importance of targeted prevention and control measures to mitigate JEV transmission in high-risk areas.