Abstract
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.
Highlights
Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne flavivirus
We carried out a longitudinal study to monitor JEV infection in 18 piglets raised in the operational pig farm at two-week interval during March to August 2009
We tested all the specimens with multiplex genotype-specific reverse transcription PCR (RT-PCR) [29] and found negative results for all. These pigs were seroconverted in June, 2009 (S1 Fig) and a part of this observation was described in the previous study when JEV-positive mosquitoes were identified in the same farm [28]
Summary
Most infection by the virus remains asymptomatic but can result in severe encephalitis in dead-end hosts, such as humans and horses, and abortion or stillbirth in pregnant sows [1]. The virus circulates primarily between pigs and Culex tritaeniorhynchus mosquitoes in June-July, and the circulation decreases after spread to humans and horses [1]. Porcine JEV infection rate associated with human cases was generally accepted for the epidemic regions of Asia [8,9]. JEV-positive mosquitoes appeared earlier or the same month as the first reported human case [12,16]. Current surveillance uses pig seroconversion or JEV-positive mosquitoes as an early indicator for the coming epidemic season in human [1]
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