College of Public Health, Medical and Veterinary Sciences, James Cook University, Smithfield, Queensland, Australia
Australian Institute of Tropical Health and Medicine, Smithfield, Queensland, Australia.
mSphere. 2020 Sep 9;5(5):e00587-20. doi: 10.1128/mSphere.00587-20.
Traditional screening for arboviruses in mosquitoes requires knowledge and the utilization of appropriate assays for their detection. Mosquitoes can also provide other valuable information, including unexpected or novel arboviruses, nonarboviral pathogens ingested from hosts they feed on, and their own genetic material. Metagenomic analysis using next-generation sequencing (NGS) is a rapidly advancing technology that allows us to potentially obtain all this information from a mosquito sample without any prior knowledge of virus, host, or vector. Moreover, it has been recently demonstrated that pathogens, including arboviruses and parasites, can be detected in mosquito excreta by molecular methods. In this study, we investigated whether RNA viruses could be detected in mosquito excreta by NGS. Excreta samples were collected from and experimentally exposed to either Ross River or West Nile viruses and from field mosquitoes collected across Queensland, Australia. Total RNA was extracted from the excreta samples, reverse transcribed to cDNA, and sequenced using the Illumina NextSeq 500 platform. Bioinformatic analyses from the generated reads demonstrate that mosquito excreta provide sufficient RNA for NGS, allowing the assembly of near-full-length viral genomes. We detected Australian Anopheles totivirus, Wuhan insect virus 33, and Hubei odonate virus 5 and identified seven potentially novel viruses closely related to members of the order (2/7) and to previously described, but unclassified, RNA viruses (5/7). Our results suggest that metagenomic analysis of mosquito excreta has great potential for virus discovery and for unbiased arbovirus surveillance in the near future. When a mosquito feeds on a host, it ingests not only its blood meal but also an assortment of microorganisms that are present in the blood, thus acting as an environmental sampler. By using specific tests, it is possible to detect arthropod-borne viruses (arboviruses) like dengue and West Nile viruses in mosquito excreta. Here, we explored the use of next-generation sequencing (NGS) for unbiased detection of RNA viruses present in excreta from experimentally infected and field-collected mosquitoes. We have demonstrated that mosquito excreta provide a suitable template for NGS and that it is possible to recover and assemble near-full-length genomes of both arboviruses and insect-borne viruses, including potentially novel ones. These results importantly show the direct practicality of the use of mosquito excreta for NGS, which in the future could be used for virus discovery, environmental virome sampling, and arbovirus surveillance.
传统的蚊虫中虫媒病毒筛查需要具备相关知识,并运用适当的方法进行检测。此外,蚊子还可以提供其他有价值的信息,包括意想不到或新型的虫媒病毒、从其取食的宿主中摄入的非虫媒病毒病原体,以及它们自身的遗传物质。基于下一代测序(NGS)的宏基因组分析是一项快速发展的技术,它使我们能够在不了解病毒、宿主或媒介的情况下,从蚊子样本中获取所有这些信息。此外,最近的研究表明,包括虫媒病毒和寄生虫在内的病原体可以通过分子方法在蚊子粪便中检测到。在这项研究中,我们研究了 NGS 是否可以检测蚊子粪便中的 RNA 病毒。从实验感染罗斯河病毒或西尼罗河病毒的蚊子和从澳大利亚昆士兰州采集的野外蚊子中收集粪便样本。从粪便样本中提取总 RNA,反转录为 cDNA,然后使用 Illumina NextSeq 500 平台进行测序。从生成的读取序列进行的生物信息学分析表明,蚊子粪便提供了足够的 RNA 用于 NGS,从而可以组装接近全长的病毒基因组。我们在澳大利亚伊蚊 totivirus、武汉昆虫病毒 33 和湖北蜻蜓病毒 5 中检测到 RNA 病毒,并鉴定了 7 种可能的新型病毒,它们与目(2/7)的成员密切相关,与以前描述但未分类的 RNA 病毒(5/7)也密切相关。我们的结果表明,蚊子粪便的宏基因组分析在病毒发现和未来不久的无偏 arbovirus 监测方面具有巨大潜力。当蚊子吸食宿主的血液时,它不仅摄入了血液餐,还摄入了血液中存在的各种微生物,因此充当了环境采样器。通过使用特定的测试,可以在蚊子粪便中检测到登革热和西尼罗河等虫媒病毒。在这里,我们探索了使用下一代测序(NGS)来检测实验感染和野外采集的蚊子粪便中存在的 RNA 病毒的方法。我们已经证明,蚊子粪便为 NGS 提供了一个合适的模板,并且可以恢复和组装包括潜在新型病毒在内的 arbovirus 和昆虫传播病毒的近全长基因组。这些结果重要地表明了直接使用蚊子粪便进行 NGS 的实际可行性,未来它可以用于病毒发现、环境病毒组采样和 arbovirus 监测。
mSphere. 2020-9-9
Viruses. 2018-1-12
Front Immunol. 2025-3-14
Sci One Health. 2024-4-2
Microb Genom. 2024-7
Arch Microbiol. 2024-3-1
Annu Rev Virol. 2019-5-17
Virology. 2018-8-8