Agriculture Victoria, AgriBio, Centre for AgriBioscience, Bundoora, VIC, 3083, Australia.
School of Applied Systems Biology, La Trobe University, Bundoora, VIC, 3083, Australia.
Parasit Vectors. 2023 Jun 6;16(1):186. doi: 10.1186/s13071-023-05734-z.
Ross River virus (RRV) is Australia's most common and widespread mosquito-transmitted arbovirus and is of significant public health concern. With increasing anthropogenic impacts on wildlife and mosquito populations, it is important that we understand how RRV circulates in its endemic hotspots to determine where public health efforts should be directed. Current surveillance methods are effective in locating the virus but do not provide data on the circulation of the virus and its strains within the environment. This study examined the ability to identify single nucleotide polymorphisms (SNPs) within the variable E2/E3 region by generating full-length haplotypes from a range of mosquito trap-derived samples.
A novel tiled primer amplification workflow for amplifying RRV was developed with analysis using Oxford Nanopore Technology's MinION and a custom ARTIC/InterARTIC bioinformatic protocol. By creating a range of amplicons across the whole genome, fine-scale SNP analysis was enabled by specifically targeting the variable region that was amplified as a single fragment and established haplotypes that informed spatial-temporal variation of RRV in the study site in Victoria.
A bioinformatic and laboratory pipeline was successfully designed and implemented on mosquito whole trap homogenates. Resulting data showed that genotyping could be conducted in real time and that whole trap consensus of the viruses (with major SNPs) could be determined in a timely manner. Minor variants were successfully detected from the variable E2/E3 region of RRV, which allowed haplotype determination within complex mosquito homogenate samples.
The novel bioinformatic and wet laboratory methods developed here will enable fast detection and characterisation of RRV isolates. The concepts presented in this body of work are transferable to other viruses that exist as quasispecies in samples. The ability to detect minor SNPs, and thus haplotype strains, is critically important for understanding the epidemiology of viruses their natural environment.
罗斯河病毒（RRV）是澳大利亚最常见和分布最广的蚊媒传播的虫媒病毒，对公共卫生具有重大意义。随着人为活动对野生动物和蚊虫种群的影响不断增加，了解 RRV 在其地方性热点地区的传播方式对于确定公共卫生工作的重点非常重要。目前的监测方法能够有效地定位病毒，但不能提供病毒及其在环境中的菌株循环的数据。本研究通过从一系列蚊虫诱捕样本中生成全长单倍型，检查了在可变 E2/E3 区域中识别单核苷酸多态性（SNP）的能力。
开发了一种新颖的瓷砖引物扩增工作流程，用于扩增 RRV，分析采用牛津纳米孔技术的 MinION 和定制的 ARTIC/InterARTIC 生物信息学协议。通过在整个基因组上创建一系列扩增子，可以通过专门针对作为单个片段扩增的可变区域以及建立在维多利亚研究地点告知 RRV 时空变异的单倍型来实现精细的 SNP 分析。
成功地在蚊虫整体诱捕匀浆物上设计和实施了生物信息学和实验室工作流程。结果表明，可以实时进行基因分型，并且可以及时确定病毒的整体诱捕共识（具有主要 SNP）。从小型 E2/E3 区域成功检测到 RRV 的变体，从而允许在复杂的蚊虫匀浆样本中确定单倍型。
这里开发的新颖的生物信息学和湿实验室方法将能够快速检测和表征 RRV 分离株。本研究工作中提出的概念可转移到以准种形式存在于样本中的其他病毒。检测次要 SNP 的能力，因此是确定单倍型株的能力，对于理解病毒及其自然环境的流行病学至关重要。
