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

2022 ◽  
Vol 10 (1) ◽  
pp. 100
Author(s):  
Geum-Young Lee ◽  
Won-Keun Kim ◽  
Seungchan Cho ◽  
Kyungmin Park ◽  
Jongwoo Kim ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Multiplex Pcr ◽  
Molecular Diagnosis ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Severe Illness ◽  
Whole Genome ◽  
Next Generation ◽  
Genome Sequences ◽  
Generation Sequencing

Hepatitis A virus (HAV) is a serious threat to public health worldwide. We used multiplex polymerase chain reaction (PCR)-based next-generation sequencing (NGS) to derive information on viral genetic diversity and conduct precise phylogenetic analysis. Four HAV genome sequences were obtained using multiplex PCR-based NGS. HAV whole-genome sequence of one sample was obtained by conventional Sanger sequencing. The HAV strains demonstrated a geographic cluster with sub-genotype IA strains in the Republic of Korea. The phylogenetic pattern of HAV viral protein (VP) 3 region showed no phylogenetic conflict between the whole-genome and partial-genome sequences. The VP3 region in serum and stool samples showed sensitive detection of HAV with differences of quantification that did not exceed <10 copies/μL than the consensus VP4 region using quantitative PCR (qPCR). In conclusion, multiplex PCR-based NGS was implemented to define HAV genotypes using nearly whole-genome sequences obtained directly from hepatitis A patients. The VP3 region might be a potential candidate for tracking the genotypic origin of emerging HAV outbreaks. VP3-specific qPCR was developed for the molecular diagnosis of HAV infection. This study may be useful to predict for the disease management and subsequent development of hepatitis A infection at high risk of severe illness.

scholarly journals Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 619 ◽  
Author(s):  
Zhihui Yang ◽  
Mark Mammel ◽  
Chris Whitehouse ◽  
Diana Ngo ◽  
Michael Kulka
Keyword(s):  
Next Generation Sequencing ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rna Viruses ◽  
Sequence Variant ◽  
Single Individual ◽  
Next Generation ◽  
Source Attribution ◽  
Clinical Specimens ◽  
Generation Sequencing

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

scholarly journals Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing

2018 ◽  
Author(s):  
Zhihui Yang ◽  
Mark Mammel ◽  
Chris A. Whitehouse ◽  
Diana Ngo ◽  
Michael Kulka
Keyword(s):  
Next Generation Sequencing ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rna Viruses ◽  
Sequence Variant ◽  
Single Individual ◽  
Next Generation ◽  
Source Attribution ◽  
Clinical Specimens ◽  
Generation Sequencing

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as Hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

scholarly journals Urinary genome detection and tracking of Hantaan virus from hemorrhagic fever with renal syndrome patients using multiplex PCR-based next-generation sequencing

2021 ◽  
Vol 15 (9) ◽  
pp. e0009707
Author(s):  
Seungchan Cho ◽  
Won-Keun Kim ◽  
Jin Sun No ◽  
Seung-Ho Lee ◽  
Jaehun Jung ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Multiplex Pcr ◽  
Phylogenetic Analyses ◽  
Hemorrhagic Fever ◽  
Hantaan Virus ◽  
Hantavirus Infection ◽  
Next Generation ◽  
Genome Sequences ◽  
Generation Sequencing ◽  
Urine Specimens

Background Hantavirus infection occurs through the inhalation of aerosolized excreta, including urine, feces, and saliva of infected rodents. The presence of Hantaan virus (HTNV) RNA or infectious particles in urine specimens of patient with hemorrhagic fever with renal syndrome (HFRS) remains to be investigated. Methodology/Principal findings We collected four urine and serum specimens of Republic of Korea Army (ROKA) patients with HFRS. We performed multiplex PCR-based next-generation sequencing (NGS) to obtain the genome sequences of clinical HTNV in urine specimens containing ultra-low amounts of viral genomes. The epidemiological and phylogenetic analyses of HTNV demonstrated geographically homogenous clustering with those in Apodemus agrarius captured in highly endemic areas, indicating that phylogeographic tracing of HTNV genomes reveals the potential infection sites of patients with HFRS. Genetic exchange analyses showed a genetic configuration compatible with HTNV L segment exchange in nature. Conclusion/Significance Our results suggest that whole or partial genome sequences of HTNV from the urine enabled to track the putative infection sites of patients with HFRS by phylogeographically linking to the zoonotic HTNV from the reservoir host captured at endemic regions. This report raises awareness among physicians for the presence of HTNV in the urine of patients with HFRS.

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