TaqMan real-time RT-PCR assay for detecting Japanese encephalitis virus in swine blood samples and mosquitoes.

Japanese encephalitis (JE) is an emerging mosquito-borne zoonotic flaviviral disease. The present study was undertaken with the objective to develop TaqMan real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for rapid detection and quantification of Japanese encephalitis virus (JEV) in swine blood and mosquito vectors. The amplification of envelope (E) gene was targeted by designing gene-specific MGB TaqMan fluorescent probe along with the primers. The best performance in terms of sensitivity was achieved by standardized TaqMan real-time RT-PCR with a detection limit of 2.8 copies/reaction and it was found to be 4-log more sensitive than conventional RT-PCR. The applicability of the standardized TaqMan assay was evaluated by screening representative sets of field swine blood samples and mosquito pools for JEV. The viral load ranged between 3.32 × 107-4.2 × 102 copies/ml of swine blood samples, and 5.7 × 109-1.3 × 102 copies/pool of mosquitoes. The standardized assay which is highly sensitive, specific and rapid would aid in screening sentinel swine and mosquitoes under JEV surveillance programs for effective prevention and control of disease in human beings.

Seasonal occurrence of Japanese encephalitis vectors in Bareilly district, Uttar Pradesh, India.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is one of the most common causes of acute encephalitis syndrome in many states of India. Uttar Pradesh state is well known for JE endemicity, contributing 75% of total cases during recent past. Several sporadic cases have been reported from Bareilly region of the state. The disease spread by bite of Culex mosquito. Survey of literature revealed no data on mosquito fauna with reference to JE in this region. Therefore, this study was planned to survey seasonal mosquito population and occurrence of JE vectors in Bareilly region. METHODS: Mosquitoes were sampled on monthly basis from organized pig farm from February 2016 to January 2017 and identified using mosquito identification keys. The meteorological parameters of the area were obtained monthly and standard statistical methods were used to assess the relationship between different weather variables and mosquito population. RESULTS: A total of 4337 mosquitoes belonging to five genera were collected. Mosquitoes of genus Culex were predominant and contributed 84.41% to the total catch. The most dominant species was Cx. tritaeniorhynchus (30.81%), followed by Cx. quinquefasciatus (28.50%), Cx. gelidus (17.24%), Cx. pseudovishnui (11.85%), Cx. vishnui (8.11%), Cx. fuscocephala (2.70%), Cx. infula (0.76%) and Cx. bitaeniorhynchus (0.03%). Pronounced seasonal variation was observed with majority of mosquitoes showing high density in monsoon and post-monsoon period. INTERPRETATION & CONCLUSION: The present study provides knowledge on distribution of JE vector in Bareilly which indicates that the area is at risk of JE outbreak. Abundance of Culex vector clearly demarcates possible threat of JE incidence in the study area. A long-term entomological study is needed to further evaluate the significant role of different weather variables in shaping mosquito densities.