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Characterisation of human astrovirus in a diarrhoea outbreak using nanopore and Sanger sequencing protocols.

ABSTRACT: Human astroviruses (HAstV) are etiologic agents of acute gastroenteritis that most often afflict young children and elderly adults. Most studies of HAstV have focused on epidemiology. In this study, we collected 10 stool samples from a diarrhea outbreak from a diarrhea sentinel surveillance hospital in Beijing. Samples were evaluated immediately using parallel multiplex RT-qPCR and nanopore sequencing, and were then amplified by designed primers and Sanger sequencing to obtain whole genome sequences. Six isolates were categorized as HAstV-5 and subjected to whole genome analysis to characterize their genetic variation and evolution. Full genome analysis revealed low genetic variation (99.38-100% identity) among isolates. Phylogenetic analysis showed that all isolates were closely related to domestic strains Yu/1-CHN and 2013/Fuzhou/85. The recombination breakpoint of the six isolates was located at 2741 bp in the overlap region of ORF1a and ORF1b, similar to those of Yu/1-CHN and 2013/Fuzhou/85. Overall, our study highlights the combined use of RT-qPCR and sequencing as an important tool in rapid diagnosis and acquisition of whole genome sequences of HAstV.

SUBMITTER: Li J

PROVIDER: S-EPMC10647158 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Characterisation of human astrovirus in a diarrhoea outbreak using nanopore and Sanger sequencing protocols.

Li Jinhui J Yang Lang L Wang Kaiying K Gao Zhiyong Z Li Peihan P Lin Yanfeng Y Jia Leili L Wang Quanyi Q Song Hongbin H Li Peng P

Virology journal 20231114 1

Human astroviruses (HAstV) are etiologic agents of acute gastroenteritis that most often afflict young children and elderly adults. Most studies of HAstV have focused on epidemiology. In this study, we collected 10 stool samples from a diarrhea outbreak from a diarrhea sentinel surveillance hospital in Beijing. Samples were evaluated immediately using parallel multiplex RT-qPCR and nanopore sequencing, and were then amplified by designed primers and Sanger sequencing to obtain whole genome seque ...[more]

PMID: 37964283

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Project description:BackgroundBovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. The unknown mechanism behind the unique variety in clinical presentation on the one hand and the host dependent clinical outcome of BPV-1 infection on the other hand indicate the involvement of additional factors. Earlier studies have reported the potential functional significance of intratypic sequence variants, along with the existence of sarcoid-sourced BPV variants. Therefore, intratypic sequence variation seems to be an important emerging viral factor. This study aimed to give a broad insight in sarcoid-sourced BPV variation and explore its potential association with disease presentation.MethodsIn order to do this, a nanopore sequencing approach was successfully optimized for screening a wide spectrum of clinical samples. Specimens of each tumour were initially screened for BPV-1/-2 by quantitative real-time PCR. A custom-designed primer set was used on BPV-positive samples to amplify the complete viral genome in two multiplex PCR reactions, resulting in a set of overlapping amplicons. For phylogenetic analysis, separate alignments were made of all available complete genome sequences for BPV-1/-2. The resulting alignments were used to infer Bayesian phylogenetic trees.ResultsWe found substantial genetic variation among sarcoid-derived BPV-1, although this variation could not be linked to disease severity. Several of the BPV-1 genomes had multiple major deletions. Remarkably, the majority of them cluster within the region coding for late viral genes. Together with the extensiveness (up to 603 nucleotides) of the described deletions, this suggests an altered function of L1/L2 in disease pathogenesis.ConclusionsBy generating a significant amount of complete-length BPV genomes, we succeeded to introduce next-generation sequencing into veterinary research focusing on the equine sarcoid, thus facilitating the first report of both nanopore-based sequencing of complete sarcoid-sourced BPV-1/-2 and the simultaneous nanopore sequencing of multiple complete genomes originating from a single clinical sample.

Dataset Information

Characterisation of human astrovirus in a diarrhoea outbreak using nanopore and Sanger sequencing protocols.

Publications

Characterisation of human astrovirus in a diarrhoea outbreak using nanopore and Sanger sequencing protocols.

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