Genotyping and Molecular Diagnosis of Hepatitis A Virus in Human Clinical Samples Using Multiplex PCR-Based Next-Generation Sequencing

Lee, Geum-Young; Kim, Won-Keun; Cho, Seungchan; Park, Kyungmin; Kim, Jong-Woo; Lee, Seung Ho; Lee, Jingyeong; Lee, Young‐Sun; Kim, Ji H.; Byun, Kwan Soo; Song, Jin-Won

doi:10.3390/microorganisms10010100

Cited by 12 publications

(16 citation statements)

References 66 publications

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“…Rapid genome‐based diagnostic assays play a crucial role in clinical diagnosis, precision patient management, and control of pathogen outbreaks 32 . Various molecular methods have been established for the diagnosis of viral infections, including loop‐mediated isothermal amplification, qPCR, RT‐PCR, and NGS 33–37 . NGS‐based diagnostics have been used to mitigate emerging viral infections through transmission epidemiology for viruses 38 .…”

Section: Discussionmentioning

confidence: 99%

High‐resolution phylogeographical surveillance of Hantaan orthohantavirus using rapid amplicon‐based Flongle sequencing, Republic of Korea

Kim,

Park,

Kim

et al. 2024

Journal of Medical Virology

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Orthohantaviruses, etiological agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome, pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) outbreaks are particularly endemic in Gyeonggi Province in northern area of the Republic of Korea (ROK). Small mammals were collected from three regions in the Gyeonggi Province during 2017 and 2018. Serological and molecular prevalence of HTNV was 25/201 (12.4%) and 10/25 (40%), respectively. A novel nanopore‐based diagnostic assay using a cost‐efficient Flongle chip was developed to rapidly and sensitively detect HTNV infection in rodent specimens within 3 h. A rapid phylogeographical surveillance of HTNV at high‐resolution phylogeny was established using the amplicon‐based Flongle sequencing. In total, seven whole‐genome sequences of HTNV were newly obtained from wild rodents collected in Paju‐si (Gaekhyeon‐ri) and Yeoncheon‐gun (Hyeonga‐ri and Wangnim‐ri), Gyeonggi Province. Phylogenetic analyses revealed well‐supported evolutionary divergence and genetic diversity, enhancing the resolution of the phylogeographic map of orthohantaviruses in the ROK. Incongruences in phylogenetic patterns were identified among HTNV tripartite genomes, suggesting differential evolution for each segment. These findings provide crucial insights into on‐site diagnostics, genome‐based surveillance, and the evolutionary dynamics of orthohantaviruses to mitigate hantaviral outbreaks in HFRS‐endemic areas in the ROK.

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Section: Discussionmentioning

confidence: 99%

High‐resolution phylogeographical surveillance of Hantaan orthohantavirus using rapid amplicon‐based Flongle sequencing, Republic of Korea

Kim,

Park,

Kim

et al. 2024

Journal of Medical Virology

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“…Real‐time RT–PCR methods with higher sensitivity can detect HAV RNA for longer periods of time and more accuracy 76,77 . New methods with higher sensitivity have been developed for detection of HAV RNA 78–81 . RT droplet digital PCR (RT–ddPCR) has greater precision than real‐time RT–PCR 80 .…”

Section: Consensus Statements and Recommendationsmentioning

confidence: 99%

Recent advances in hepatitis A virus research and clinical practice guidelines for hepatitis A virus infection in Japan

Kanda,

Sasaki‐Tanaka,

Ishii

et al. 2023

Hepatology Research

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In 2018, there was a hepatitis A outbreak in Japan, and hepatitis A virus (HAV) infection is considered a sexually transmitted disease. In general, patients with hepatitis A should be given attention, and this disease should be prevented more than ever. The Japan Agency for Medical Research and Development (AMED) Hepatitis A and E viruses (HAV and HEV) Study Group has worked on the project to create “Recent Advances in Hepatitis A Virus (HAV) Research and Clinical Practice Guidelines for HAV Infection in Japan”. The group consists of expert hepatologists and virologists who gathered at virtual meeting on August 5, 2023. Data about the pathogenesis, infection routes, diagnosis, complications, several factors for the severities, vaccination, and current and future treatments for hepatitis A were discussed and debated for a draft version. The participants assessed the quality of cited studies. The finalized recommendations are presented in this review. The recent advances in HAV research and clinical practice for HAV infection in Japan, have been reviewed by the AMED HAV and HEV Study Group.

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“…The virus is transmitted by the fecal-oral route, mainly by contaminated food and water, as well as through person-to-person contact (World Health Organization (WHO), 2023). HAV is a non-enveloped virus with a single stranded 7.5 kb genome that contains 5’ and 3’ noncoding regions, one open reading frame (ORF), and a poly(A) tract (Batista et al, 2020; Lee et al, 2022). Genetic variability has led to the identification of seven HAV genotypes (I-VII), with genotypes I to III and VII infecting humans, including seven sub-genotypes (IA, IB, IC, IIA, IIB, IIIA, and IIIB) (Lee et al, 2022; Vaughan et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Hepatitis A virus whole genome sequencing strategy using NGS/Illumina technology

Sicilia,

Fantilli,

Cuba

et al. 2024

Preprint

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Background: Molecular epidemiology of hepatitis A virus (HAV) plays a critical role in identifying outbreak origin and conducting surveillance. Although it is mostly carried out using short partial VP1/2A genomic sequences, utilizing whole-genome sequences (WGS) provides more accurate and robust information. Objective: we aimed to develop an amplicon-based next-generation sequencing (NGS) strategy to obtain complete HAV genomes utilizing the COVIDSeq Test (Illumina), available in surveillance laboratories after the COVID19 pandemic. Study design: 25 primer pairs were designed and used to amplify partial genomic fragments (400bp) that comprise the entire HAV genome sequence from 16 HAV previously positive serum and stool samples from Argentina. The DNA library was prepared using the Illumina COVIDSeq Test and sequenced in a MiSeq equipment. Phylogenetic analyses were performed with IQ-Tree using WGS and VP1/2A partial sequences of 1084pb and 422pb. Results: 11 samples could be successfully amplified and sequenced, with horizontal coverage between 79.3%-100% (>90% in 9 samples). Individual RT-PCRs and Sanger sequencing with specific primers had to be performed in 6 samples to cover gaps of Ns. Phylogenetic analyses of WGS and VP1/2A partial sequences yielded similar results (clustering with genotype IA), although 422pb fragments showed low accurate grouping definition. Conclusion: the amplicon-based NGS whole genome sequencing tool developed by adapting the COVIDSeq test to HAV, proved to be efficient in generating new complete and near-complete viral sequences. The study of the 1084bp fragment of the VP1/2A region would constitute a useful alternative option for outbreak investigation with public health impact.

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Genotyping and Molecular Diagnosis of Hepatitis A Virus in Human Clinical Samples Using Multiplex PCR-Based Next-Generation Sequencing

Cited by 12 publications

References 66 publications

High‐resolution phylogeographical surveillance of Hantaan orthohantavirus using rapid amplicon‐based Flongle sequencing, Republic of Korea

High‐resolution phylogeographical surveillance of Hantaan orthohantavirus using rapid amplicon‐based Flongle sequencing, Republic of Korea

Recent advances in hepatitis A virus research and clinical practice guidelines for hepatitis A virus infection in Japan

Hepatitis A virus whole genome sequencing strategy using NGS/Illumina technology

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