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To facilitate early warnings and surveillance of the presence of these viral infectious agents in the environment, a triplex reverse transcription-quantitative PCR (RT-qPCR) was developed for simultaneous quantification of JEV, MVEV, and WNV in potential hotspots such as piggery and urban wastewater and environmental water samples. The performance of the developed triplex RT-qPCR assay was compared with that of simplex counterparts, all using the same primer and probe sequences. The quantifiable results showed a concordance rate of 93.9%\u2013100% (Cohen\u2019s kappa) between the triplex and simplex assays. The mean concentrations of exogenous JEV, MVEV, and WNV using the triplex and simplex RT-qPCR assays were remarkably similar in piggery\/urban wastewater and environmental water samples. However, the impacts of the matrix effects (i.e., sample composition and PCR inhibition) of environmental water samples on the accurate quantification of these viruses need to be considered. Taken together, this newly developed triplex RT-qPCR assay of JEV, MVEV, and WNV will allow for a more rapid and cost-efficient sample analysis and data interpretation. The application of the triplex assay for environmental surveillance may be a valuable tool to complement the existing disease and mosquito surveillance approaches used to safeguard the health of both humans and animals.<\/jats:p>\n            <jats:sec>\n              <jats:title>IMPORTANCE<\/jats:title>\n              <jats:p>The co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) poses significant threats to human and animal health globally. In this study, a triplex RT-qPCR assay was developed for simultaneous quantification of these viruses in wastewater and environmental water samples. Results demonstrated high concordance and sensitivity of the newly developed triplex RT-qPCR assay compared to simplex assays, indicating its efficacy for environmental surveillance. This cost-effective and rapid assay offers a vital tool for timely monitoring of mosquito-borne viruses in environmental samples, enhancing our ability to mitigate potential outbreaks and safeguard public health.<\/jats:p>\n            <\/jats:sec>\n          <\/jats:sec>","DOI":"10.1128\/spectrum.01364-24","type":"journal-article","created":{"date-parts":[[2024,8,20]],"date-time":"2024-08-20T13:05:01Z","timestamp":1724159101000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":6,"title":["Development of a triplex RT-qPCR assay for simultaneous quantification of Japanese encephalitis, Murray Valley encephalitis, and West Nile viruses for environmental surveillance"],"prefix":"10.1128","volume":"12","author":[{"given":"Yawen","family":"Liu","sequence":"first","affiliation":[{"name":"State Key Laboratory of Marine Environmental Science, College of the Environment &amp; Ecology, Xiamen University, Xiamen, China"},{"name":"CSIRO Environment, Ecosciences Precinct, Dutton Park, Queensland, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wendy J","family":"Smith","sequence":"additional","affiliation":[{"name":"CSIRO Environment, Ecosciences Precinct, Dutton Park, Queensland, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Metasebia","family":"Gebrewold","sequence":"additional","affiliation":[{"name":"CSIRO Environment, Ecosciences Precinct, Dutton Park, Queensland, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stuart L.","family":"Simpson","sequence":"additional","affiliation":[{"name":"CSIRO Environment, Ecosciences Precinct, Dutton Park, Queensland, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinhong","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Marine Environmental Science, College of the Environment &amp; Ecology, Xiamen University, Xiamen, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1351-8963","authenticated-orcid":true,"given":"Warish","family":"Ahmed","sequence":"additional","affiliation":[{"name":"CSIRO Environment, Ecosciences Precinct, Dutton Park, Queensland, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.2147\/TCRM.S51168"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.3390\/v14061208"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.3390\/pathogens11050575"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.3390\/v14102213"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1098\/rstb.2013.0561"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.5694\/j.1326-5377.1993.tb137623.x"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1445-5994.1998.tb00653.x"},{"key":"e_1_3_3_9_2","first-page":"368","article-title":"Murray Valley encephalitis in Western Australia in 2000, with evidence of southerly spread","volume":"24","author":"Cordova SP","year":"2000","unstructured":"Cordova SP, Smith DW, Broom AK, Lindsay MD, Dowse GK, Beers MY. 2000. 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