Laboratory of Viral Diseases, Wadsworth Center, New York State Department of Health, Albany, New York 12208, USA.
Laboratory of Viral Diseases, Wadsworth Center, New York State Department of Health, Albany, New York 12208, USA.
J Virol Methods. 2023 Feb;312:114649. doi: 10.1016/j.jviromet.2022.114649. Epub 2022 Nov 12.
Since 2016, the United States has experienced a resurgence in the number of hepatitis A virus (HAV) cases and outbreaks. These outbreaks have been sustained by person-to-person transmission with cases occurring predominantly in high-risk populations including intravenous drug users, individuals experiencing homelessness, and men who have sex with men. To investigate HAV transmission, a molecular-surveillance system consisting of real-time RT-PCR (rRT-PCR) for detection, and a conventional RT-PCR assay for genotyping of HAV, was established in New York State (NYS) in 2019. Since then, a total of 271 HAV-positive serum samples collected from cases across NYS between 2019 and 2021 were identified by rRT-PCR. To rapidly and efficiently generate HAV whole-genome sequences, a custom AmpliSeq™ panel was designed in collaboration with Thermo Fisher. To streamline the process, sample preparation was performed on an Ion Chef and sequencing on an Ion S5XL. Of the 271 HAV-positive samples, the whole-genome sequencing (WGS) assay successfully generated 134 near-complete, high-quality HAV sequences. Phylogenetic analysis of the VP1-2A region identified 216 IB, 48 IA, and 2 IIIA genotypes, while 5 were unable to be typed due to poor sequence in this key region. The HAV whole-genome sequencing approach provided a more efficient and streamlined approach for genotyping HAV compared to previous methods and resulted in phylogenetic trees with enhanced resolution compared to the HAV VP1-2A region alone.
自 2016 年以来，美国甲型肝炎病毒（HAV）病例和疫情数量再次出现增长。这些疫情主要通过人际传播，病例主要发生在高危人群中，包括静脉吸毒者、无家可归者和男男性行为者。为了调查 HAV 传播情况，2019 年在纽约州（NYS）建立了一个由实时 RT-PCR（rRT-PCR）检测和 HAV 基因分型的常规 RT-PCR 检测组成的分子监测系统。自那时以来，通过 rRT-PCR 从 2019 年至 2021 年期间 NYS 的病例中总共鉴定出 271 份 HAV 阳性血清样本。为了快速有效地生成 HAV 全基因组序列，与 Thermo Fisher 合作设计了一个定制的 AmpliSeq™ 面板。为了简化流程，在 Ion Chef 上进行样品制备，在 Ion S5XL 上进行测序。在 271 份 HAV 阳性样本中，全基因组测序（WGS）检测成功生成了 134 条近乎完整的高质量 HAV 序列。VP1-2A 区的系统发育分析鉴定出 216 个 IB、48 个 IA 和 2 个 IIIA 基因型，而由于该关键区域的序列较差，有 5 个无法进行分型。与之前的方法相比，HAV 全基因组测序方法为 HAV 基因分型提供了一种更高效、更精简的方法，并产生了与仅 HAV VP1-2A 区相比具有增强分辨率的系统发育树。
