Sequence-Independent, Single-Primer Amplification Next-Generation Sequencing of Hantaan Virus Cell Culture–Based Isolates

Song, Dong Hyun; Kim, Won Keun; Gu, Se Hun; Lee, Dae-Sang; Kim, Jeong Ah; No, Jin Sun; Lee, Seung Ho; Wiley, Michael R.; Palacios, Gustavo; Jeong, Seong Tae

doi:10.4269/ajtmh.16-0683

Cited by 15 publications

(12 citation statements)

References 32 publications

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“…The termini of 3’ and 5’ sequences were determined by RACE PCR. Both end sequences of HTNV L, M, and S segments contained a mismatch at 9 th and the noncanonical U-G pair at 10 th nucleotides, suggesting the incomplete complementarity as previously described [32]. The total length of HTNV L segment for Cp Humphreys and the expansion site, Pyeongtaek, was three nucleotides shorter (6,530nt) than that of HTNV 76–118, demonstrating the deletion of 5’-AUC-3’ at the 5’ end of the L segment.…”

Section: Discussionsupporting

confidence: 55%

Hantavirus surveillance and genetic diversity targeting small mammals at Camp Humphreys, a US military installation and new expansion site, Republic of Korea

Kim

Klein³

et al. 2017

PLoS ONE

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Small mammal surveillance was conducted (2008–2010, 2012) at Camp (Cp) Humphreys, a US Army installation and new expansion site, Republic of Korea (ROK), to identify hemorrhagic fever with renal syndrome health threats to US military/civilian populations during its ongoing expansion phase. Small mammals were collected using Sherman live capture traps and transported to Korea University where they were euthanized, tissues removed, and assayed to determine hantavirus IgG antibody-positive and hantavirus-positive rates by RT-PCR. A total of 2,364 small mammals were captured over 11,300 trap nights (capture rate = 20.92%). Apodemus agrarius was the most commonly collected (76.65%), with capture rates of 9.62% and 21.70% for Cp Humphreys and the expansion site, respectively. Overall, Hantaan virus (HTNV) IgG antibody-positive (Ab+) rate for A. agrarius was 2.15% (39/1,812). A total of 5.43% (10/184) Crocidura lasiura, 0.79% (2/254) Microtus fortis and 2.44% (1/41) Micromys minutus were serologically IgG Ab+ for hantaviruses. HTNV-specific RT-PCR demonstrated that 28.2% (11/39) HTNV Ab+ A. agrarius harbored the 328-nt sequence of the GC glycoprotein-encoding M segment of HTNV. Among them, the whole genome sequences of 3 HTNV strains were obtained by conventional RT-PCR and Rapid Amplification cDNA Ends PCR. Phylogenetic analyses of the HTNV strains from Cp Humphreys and the expansion site, Pyeongtaek, show a greater diversity of rodent-borne hantaviruses compared to HTNV previously identified in Gyeonggi province of the ROK. Thus, this study provides significant insights for raising HFRS threat awareness, analysis, and risk reduction strategies in southern Gyeonggi province.

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Section: Discussionsupporting

confidence: 55%

Hantavirus surveillance and genetic diversity targeting small mammals at Camp Humphreys, a US military installation and new expansion site, Republic of Korea

Kim

Klein³

et al. 2017

PLoS ONE

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“…These technologies are still scarcely used in the case of hantaviruses. Moreover, the rare studies that used HTS to analyze hantavirus diversity were often performed on patient, cell culture-based or wild reservoir samples [106,107,108,109]. To our knowledge, HTS has never been combined with experiments to assess hantavirus diversity and its evolution throughout the course of reservoir infection.…”

Section: Challenges and Future Directionsmentioning

confidence: 99%

The Needs for Developing Experiments on Reservoirs in Hantavirus Research: Accomplishments, Challenges and Promises for the Future

Madrières

Castel

Murri

et al. 2019

Viruses

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Due to their large geographic distribution and potential high mortality rates in human infections, hantaviruses constitute a worldwide threat to public health. As such, they have been the subject of a large array of clinical, virological and eco-evolutionary studies. Many experiments have been conducted in vitro or on animal models to identify the mechanisms leading to pathogenesis in humans and to develop treatments of hantavirus diseases. Experimental research has also been dedicated to the understanding of the relationship between hantaviruses and their reservoirs. However, these studies remain too scarce considering the diversity of hantavirus/reservoir pairs identified, and the wide range of issues that need to be addressed. In this review, we present a synthesis of the experimental studies that have been conducted on hantaviruses and their reservoirs. We aim at summarizing the knowledge gathered from this research, and to emphasize the gaps that need to be filled. Despite the many difficulties encountered to carry hantavirus experiments, we advocate for the need of such studies in the future, at the interface of evolutionary ecology and virology. They are critical to address emerging areas of research, including hantavirus evolution and the epidemiological consequences of individual variation in infection outcomes.

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“…Four distinct approaches have been reported for WGS of Old World hantavirus species Hantaan virus (HTNV) and Seoul virus from patient and rodent specimens (Kim et al, 2016 , 2018 , 2019 ; Song et al, 2017 ). These include sequence-independent, single primer amplification (SISPA), rapid amplification of cDNA ends (RACE), target-capture using virus-specific probes, and amplicon-based approaches.…”

Section: Introductionmentioning

confidence: 99%

Amplicon-Based, Next-Generation Sequencing Approaches to Characterize Single Nucleotide Polymorphisms of Orthohantavirus Species

Taylor

Williams

Wongsurawat

et al. 2020

Front. Cell. Infect. Microbiol.

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Whole-genome sequencing (WGS) of viruses from patient or environmental samples can provide tremendous insight into the epidemiology, drug resistance or evolution of a virus. However, we face two common hurdles in obtaining robust sequence information; the low copy number of viral genomes in specimens and the error introduced by WGS techniques. To optimize detection and minimize error in WGS of hantaviruses, we tested four amplification approaches and different amplicon pooling methods for library preparation and examined these preparations using two sequencing platforms, Illumina MiSeq and Oxford Nanopore Technologies MinION. First, we tested and optimized primers used for whole segment PCR or one kilobase amplicon amplification for even coverage using RNA isolated from the supernatant of virus-infected cells. Once optimized we assessed two sources of total RNA, virus-infected cells and supernatant from the virus-infected cells, with four variations of primer pooling for amplicons, and six different amplification approaches. We show that 99–100% genome coverage was obtained using a one-step RT-PCR reaction with one forward and reverse primer. Using a two-step RT-PCR with three distinct tiling approaches for the three genomic segments (vRNAs), we optimized primer pooling approaches for PCR amplification to achieve a greater number of aligned reads, average depth of genome, and genome coverage. The single nucleotide polymorphisms identified from MiSeq and MinION sequencing suggested intrinsic mutation frequencies of ~10 −5 -10 −7 per genome and 10 −4 -10 −5 per genome, respectively. We noted no difference in the coverage or accuracy when comparing WGS results with amplicons amplified from RNA extracted from infected cells or supernatant of these infected cells. Our results show that high-throughput diagnostics requiring the identification of hantavirus species or strains can be performed using MiSeq or MinION using a one-step approach. However, the two-step MiSeq approach outperformed the MinION in coverage depth and accuracy, and hence would be superior for assessment of genomes for epidemiology or evolutionary questions using the methods developed herein.

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Sequence-Independent, Single-Primer Amplification Next-Generation Sequencing of Hantaan Virus Cell Culture–Based Isolates

Cited by 15 publications

References 32 publications

Hantavirus surveillance and genetic diversity targeting small mammals at Camp Humphreys, a US military installation and new expansion site, Republic of Korea

Hantavirus surveillance and genetic diversity targeting small mammals at Camp Humphreys, a US military installation and new expansion site, Republic of Korea

The Needs for Developing Experiments on Reservoirs in Hantavirus Research: Accomplishments, Challenges and Promises for the Future

Amplicon-Based, Next-Generation Sequencing Approaches to Characterize Single Nucleotide Polymorphisms of Orthohantavirus Species

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