State Key Laboratory of Marine Environmental Science, College of the Environment & Ecology, Xiamen University, Xiamen, China.
CSIRO Environment, Ecosciences Precinct, Dutton Park, Queensland, Australia.
Microbiol Spectr. 2024 Oct 3;12(10):e0136424. doi: 10.1128/spectrum.01364-24. Epub 2024 Aug 20.
The co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) has impacted human and animal health in multiple countries worldwide. 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%-100% (Cohen's 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.IMPORTANCEThe 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.
虫媒乙型脑炎病毒（JEV）、默里谷脑炎病毒（MVEV）和西尼罗河病毒（WNV）的共同循环在全球多个国家影响着人类和动物的健康。为了促进对这些病毒在环境中的存在进行早期预警和监测，开发了一种三重逆转录定量 PCR（RT-qPCR），用于同时定量猪舍和城市废水以及环境水样中的 JEV、MVEV 和 WNV。比较了开发的三重 RT-qPCR 与 simplex 对照的性能，所有对照均使用相同的引物和探针序列。可量化的结果表明，三重和 simplex 测定之间的一致性率为 93.9%-100%（Cohen's kappa）。在猪舍/城市废水中，三重和 simplex RT-qPCR 测定的 JEV、MVEV 和 WNV 的平均浓度非常相似和环境水样。然而，环境水样的基质效应（即样品组成和 PCR 抑制）对这些病毒的准确定量的影响需要考虑。总的来说，这种新开发的 JEV、MVEV 和 WNV 的三重 RT-qPCR 测定法将允许更快速和更具成本效益的样品分析和数据解释。三重测定在环境监测中的应用可能是一种有价值的工具，可以补充用于保护人类和动物健康的现有疾病和蚊子监测方法。
重要性：虫媒乙型脑炎病毒（JEV）、默里谷脑炎病毒（MVEV）和西尼罗河病毒（WNV）的共同循环对全球人类和动物健康构成重大威胁。在这项研究中，开发了一种三重 RT-qPCR 测定法，用于同时定量废水中的这些病毒和环境水样。结果表明，与 simplex 测定相比，新开发的三重 RT-qPCR 测定具有较高的一致性和灵敏度，表明其对环境监测的有效性。这种具有成本效益且快速的测定法为及时监测环境样本中的虫媒病毒提供了重要工具，增强了我们减轻潜在疫情爆发和保护公共健康的能力。
