Isolation and full-genome sequences of Japanese encephalitis virus genotype I strains from Cambodian human patients, mosquitoes and pigs

Abstract

Japanese encephalitis remains the most important cause of viral encephalitis in humans in several southeast Asian countries, including Cambodia, causing at least 65 000 cases of encephalitis per year. This vector-borne viral zoonosis - caused by Japanese encephalitis virus (JEV) - is considered to be a rural disease and is transmitted by mosquitoes, with birds and pigs being the natural reservoirs, while humans are accidental hosts. In this study we report the first two JEV isolations in Cambodia from human encephalitis cases from two studies on the aetiology of central nervous system disease, conducted at the two major paediatric hospitals in the country. We also report JEV isolation from Culextritaeniorhynchus mosquitoes and from pig samples collected in two farms, located in peri-urban and rural areas. Out of 11 reverse-transcription polymerase chain reaction-positive original samples, we generated full-genome sequences from 5 JEV isolates. Five additional partial sequences of the JEV NS3 gene from viruses detected in five pigs and one complete coding sequence of the envelope gene of a strain identified in a pig were generated. Phylogenetic analyses revealed that JEV detected in Cambodia belonged to genotype I and clustered in two clades: genotype I-a, mainly comprising strains from Thailand, and genotype I-b, comprising strains from Vietnam that dispersed northwards to China. Finally, in this study, we provide proof that the sequenced JEV strains circulate between pigs, Culex tritaeniorhynchus and humans in the Phnom Penh vicinity.

Comparison of the dynamics of Japanese encephalitis virus circulation in sentinel pigs between a rural and a peri-urban setting in Cambodia

“…This corresponds to our detection rate of 6/29. By contrast, during the previous study, only two out of the twenty-nine pigs monitored tested positive by real time RT-PCR [ 31 ]. Since the same detection methods were used in 2014 and 2015, the difference in the detection rates is difficult to explain and the lower detection rate in 2014 might only be due to chance.…”

“…Briefly, full JEV genome sequences were generated from two pigs (C14 and D03), partial sequences of the NS3 gene were generated from four pigs (C04, C12, D08, and D15), and one complete coding sequence of the E (envelope) gene was generated from one pig (D08). All JEV strains from 2015 detected in both the peri-urban and rural farms belonged to genotype I clade b [ 31 ].…”

“…Methods used for RNA extraction, amplification of JEV RNA using conventional and real-time RT-PCR, JEV isolation on cells, amplicon sequencing, and phylogenetic analyses are detailed in Duong et al . [ 31 ]. Briefly, for each pig serum sample, the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) was used according to the manufacturer’s instructions to extract viral RNA.…”

“…Following the manufacturer’s instructions, total RNA was extracted from the original samples or supernatant previously detected positive for JEV using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). cDNA was synthetized and then amplified by PCR for full genome, complete envelope or partial NS3 sequencing by Sanger method at a commercial facility (Macrogen, Seoul, South Korea) [ 31 ]. CLC Main Workbench 5.5 package (CLC bio A/S, Aarhus, Denmark) was used to analyze and assemble the sequences generated from the PCR products.…”

Zika Virus, Microcephaly and its Possible Global Spread

“…The other important arboviruses that damage the nervous system include the Japanese encephalitis virus (JEV) from the family Flaviviridae [63]. The vector for JEV is Culex mosquito species that feeds mostly on birds and livestock blood while humans are the accidental host of the virus [63,64]. JEV causes birth defects and certain neurological complications [63].…”