The feasibility of field collected pig oronasal secretions as specimens for the virologic surveillance of Japanese encephalitis virus

Virologic surveillance of Japanese encephalitis virus (JEV) relies on collecting pig blood specimens and adult mosquitoes in the past. Viral RNAs extracted from pig blood specimens suffer from low detecting positivity by reverse transcription PCR (RT-PCR). The oronasal transmission of the virus has been demonstrated in experimentally infected pigs. This observation suggested oronasal specimens could be useful source in the virus surveillance. However, the role of this unusual route of transmission remains unproven in the operational pig farm. In this study, we explore the feasibility of using pig oronasal secretions collected by chewing ropes to improve the positivity of detection in commercial pig farms. The multiplex genotype-specific RT-PCR was used in this study to determine and compare the positivity of detecting JEV viral RNAs in pig's oronasal secretions and blood specimens, and the primary mosquito vector. Oronasal specimens had the overall positive rate of 6.0% (95% CI 1.3%-16.6%) (3/50) to 10.0% (95% CI 2.1%-26.5%) (3/30) for JEV during transmission period despite the negative results of all blood-derived specimens (n = 2442). Interestingly, pig oronasal secretions and female Culex tritaeniorhynchus mosquito samples collected from the same pig farm showed similar viral RNA positive rates, 10.0% (95% CI 2.1%-26.5%) (3/30) and 8.9% (95% CI 2.5%-21.2%) (4/45), respectively (p> 0.05). Pig oronasal secretion-based surveillance revealed the seasonality of viral activity and identified closely related genotype I virus derived from the mosquito isolates. This finding indicates oronasal secretion-based RT-PCR assay can be a non-invasive, alternative method of implementing JEV surveillance in the epidemic area prior to the circulation of virus-positive mosquitoes. Author summaryMosquito-borne Japanese encephalitis virus (JEV) has either endemic or seasonal patterns of transmission in Asia and Australia. Most hosts infected by the virus remains asymptomatic but can result in severe encephalitis in humans and horses, and abortion or stillbirth in pregnant sows. Isolation of virus in adult mosquitoes or pig seroconversion has been used as an early indicator of upcoming JE outbreak in humans. Genotype identification of the virus is important since current human and domestic animal vaccines are all genotype III (GIII) specific. GIII vaccine elicited immunity has reduced cross-protections to genotypes other than GIII. Our virologic surveillance using pig's oronasal secretion detected higher prevalence and earlier genotype I virus activity than using pig's blood and mosquitoes, respectively. This proposed surveillance tool might be more effective that will allow the public health agency to properly implement the preventive measures, such as implementing mosquito control, encouraging booster vaccination, and encouraging the use of mosquito repellent, to reduce the impact of upcoming outbreak. Collection of pig's oronasal secretion is non-invasive to pigs and less technically demanding to operators. Thus we propose the use of pig's oronasal secretions as the novel source of specimens for virologic surveillance to replace the traditional pig blood or adult mosquito specimens to monitor and control JE outbreak/epidemic in the future.