Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses.
Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses.
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DOI：
10.3390/ani11102976
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发表时间：
2021-10-15
期刊：
影响因子：
--
通讯作者：
Misinzo G
中科院分区：
文献类型：
--
作者：
Kinimi E;Mahapatra M;Kgotlele T;Makange MR;Tennakoon C;Njeumi F;Odongo S;Muyldermans S;Kock R;Parida S;Rweyemamu M;Misinzo G
小反刍兽疫病毒 (PPRV) 在绵羊和山羊中引起一种高度破坏性的疾病，即小反刍兽疫 (PPR)，该病是全球控制和根除的目标。然而，在许多发展中国家，获得昂贵的测序技术的机会有限，并且由于跨境运输临床样本的困难而雪上加霜。 Oxford nanopore MinION 是一种相对便宜的测序技术，使用便携式设备，需要最少的支持实验室基础设施或样品制备和快速测序的技术专业知识。在这项研究中，通过牛津纳米孔 MinION 测序，从分别于 2016 年和 2018 年坦桑尼亚恩戈罗恩戈罗和蒙巴地区山羊 PPR 疫情中收集的 PPRV 阳性存档样本生成了完整的 PPRV 基因组。测序后四小时内生成了 15,948 个核苷酸长的 PPRV 完整基因组。完整基因组的系统发育分析显示，与目前在东非流行的 III 系 PPR 病毒具有很高的核苷酸一致性（96.19-99.24%），表明有共同的起源。牛津纳米孔 MinION 测序仪可用于克服发展中国家小反刍兽疫全球控制和根除计划中的诊断和监测挑战。然而，整个基因组的覆盖深度不均匀，并且在基质（M）和融合（F）基因之间观察到扩增子丢失。因此，需要进行更大规模的现场研究，以收集足够的数据来评估纳米孔测序技术的稳健性。小反刍兽疫病毒 (PPRV) 会引起绵羊和山羊的高度毁灭性疾病，威胁粮食安全、小反刍动物生产和易受影响的濒危野生反刍动物。鉴于政策旨在实现全球消灭小反刍兽疫，在小反刍兽疫流行的地区，建立具有成本效益的基因组监测工具至关重要。基因组数据可以提供足够深入的信息来识别导致 PPRV 持续存在和病毒进化的流行区域，并指导适当的疫苗接种反应。然而，在资源有限的环境中，获得所需测序技术的机会很少，而且由于《濒危野生动植物种国际贸易公约》(CITES) 和《名古屋议定书》的规定，跨境运输野生动物临床样本的难度加大了这一难度。牛津纳米孔 MinION 测序技术最近在 PPRV 测序中表现出了非凡的性能，因为与其他全基因组 (WGS) 测序平台相比，其速度快、在流行国家中实用且每个样本的成本相对较低。在本研究中，利用牛津纳米孔 MinION 测序生成了 PPRV 分离株的完整基因组，这些分离株分别于 2016 年和 2018 年从坦桑尼亚北部和南部高地的恩戈罗恩戈罗和蒙巴地区的感染山羊中收集。使用二十五对长读长引物进行平铺多重聚合酶链反应（PCR）。所得 PCR 扩增子用于纳米孔文库制备和测序。输出数据的分析是 PPRV 的完整基因组，在测序后四小时内产生（登录号：MW960272 和 MZ322753）。完整基因组的系统发育分析显示，与目前在东非流行的谱系 III PPRV 具有高达 96.19% 至 99.24% 的核苷酸同一性，表明有共同的起源。牛津纳米孔 MinION 测序仪可用于克服小反刍兽疫全球控制和根除计划中的诊断和监测挑战。然而，整个基因组的覆盖深度不均匀，并且主要在 PPRV 的基质（M）和融合（F）基因之间的富含 GC 的区域观察到扩增子丢失。因此，需要进行更大规模的现场研究，以收集足够的数据来评估纳米孔测序技术的稳健性。
Peste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR), in sheep and goats, which is targeted for global control and eradication. However, in many developing countries, access to expensive sequencing technologies is limited and is compounded by difficulties in transporting clinical samples across international borders. Oxford nanopore MinION is a relatively cheap sequencing technology using portable devices that require minimal supporting laboratory infrastructure or technical expertise for sample preparation and rapid sequencing. In this study, Oxford nanopore MinION sequencing was carried out to generate complete genomes of PPRV from archived PPRV-positive samples collected from PPR outbreaks in goats in Ngorongoro and Momba districts in Tanzania during 2016 and 2018, respectively. Complete genomes of PPRV of 15,948 nucleotides long were generated within four hours of sequencing. The phylogenetic analysis of the complete genomes revealed a high nucleotide identity (96.19–99.24%) with lineage III PPR viruses currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in developing countries in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed between the matrix (M) and fusion (F) genes. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology. Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.
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