A pan-genotypic Hepatitis C Virus NS5A amplification method for reliable genotyping and resistance testing

Walker, Andreas; Ennker, Kim Sophie; Kaiser, Rolf; Lübke, Nadine; Timm, Joerg

doi:10.1016/j.jcv.2019.01.012

Cited by 10 publications

(10 citation statements)

References 39 publications

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“…RNA extraction and reverse transcription were carried out as previously described [2]. DNA amplification and sequencing on the Oxford Nanopore platform were carried out according to the Artic protocol [3,4] (Supplementary Text), yielding between 31 and 582 Mb of raw sequencing data per sample (Supplementary Table S1).…”

Section: Severe Acute Respiratory Syndrome Coronavirus 2 Genome Sequencingmentioning

confidence: 99%

Genetic structure of SARS-CoV-2 reflects clonal superspreading and multiple independent introduction events, North-Rhine Westphalia, Germany, February and March 2020

et al. 2020

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We whole-genome sequenced 55 SARS-CoV-2 isolates from Germany to investigate SARS-CoV-2 outbreaks in 2020 in the Heinsberg district and Düsseldorf. While the genetic structure of the Heinsberg outbreak indicates a clonal origin, reflecting superspreading dynamics from mid-February during the carnival season, distinct viral strains were circulating in Düsseldorf in March, reflecting the city’s international links. Limited detection of Heinsberg strains in the Düsseldorf area despite geographical proximity may reflect efficient containment and contact-tracing efforts.

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Section: Severe Acute Respiratory Syndrome Coronavirus 2 Genome Sequencingmentioning

confidence: 99%

Genetic structure of SARS-CoV-2 reflects clonal superspreading and multiple independent introduction events, North-Rhine Westphalia, Germany, February and March 2020

et al. 2020

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“…55 SARS-CoV-2 isolate samples, 10 directly linked to the Heinsberg outbreak (obtained from medical practices in the Heinsberg district or from residents of Heinsberg district patients treated at Düsseldorf University Hospital) and 45 from the city of Düsseldorf and surrounding districts, were acquired from diagnostic swabs sent to the Institute of Virology at Düsseldorf University Hospital. RNA extraction and reverse transcription were carried out as previously described 4 . DNA amplification and sequencing on the Oxford Nanopore platform were carried out according to the Artic protocol 5,6 (Supplementary Text), yielding between 31 and 582Mb of raw sequencing data per sample (Supplementary Table S1).…”

Section: Sars-cov-2 Genome Sequencingmentioning

confidence: 99%

Genetic structure of SARS-CoV-2 in Western Germany reflects clonal superspreading and multiple independent introduction events

Walker

Houwaart

Wienemann

et al. 2020

Preprint

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We whole-genome sequenced 55 SARS-CoV-2 isolates from Western Germany and investigated the genetic structure of SARS-CoV-2 outbreaks in the Heinsberg district and Düsseldorf. While the genetic structure of the Heinsberg outbreak indicates a clonal origin, reflective of superspreading dynamics during the carnival season, distinct viral strains are circulating in Düsseldorf, reflecting the city's international links. Limited detection of Heinsberg strains in the Düsseldorf area despite geographical proximity may reflect efficient containment and contact tracing efforts.

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“…Of the NS5B positive samples, the vast majority were successfully amplified using in-house genotype specific NS5a primer sets. Recent studies independently arrived at comparable pan-genotypic NS5A primer sets that target similar conserved genomic regions, but also employing additional measures to improve amplification success, such as inosine degeneracy and utilising higher input serum and RNA [45,46]. However, the additional nested strategy employed in one method would necessitate an additional level of expertise in molecular contamination control, potentially adding further logistical challenges in most settings [45].…”

Section: Discussionmentioning

confidence: 99%

“…Recent studies independently arrived at comparable pan-genotypic NS5A primer sets that target similar conserved genomic regions, but also employing additional measures to improve amplification success, such as inosine degeneracy and utilising higher input serum and RNA [45,46]. However, the additional nested strategy employed in one method would necessitate an additional level of expertise in molecular contamination control, potentially adding further logistical challenges in most settings [45]. In general, these simple Sanger-based sequencing and subtyping pipelines should be easily deployable in most resource-poor settings [44].…”

Section: Discussionmentioning

confidence: 99%

Hepatitis C subtyping assay failure in UK patients born in Sub-Saharan Africa: implications for global treatment and elimination

Adeboyejo

King

Tsoleridis

et al. 2021

Preprint

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The newly developed direct-acting antivirals (DAAs) have revolutionised the treatment of chronic hepatitis C virus (HCV), where cohort studies have shown that cure rates as high as 98% can be achieved. Whilst genome sequencing has demonstrated that some subtypes of HCV naturally harbour drug resistance associated substitutions (RAS), these have not been considered important as previous molecular epidemiological studies have suggested that such difficult-to-treat subtypes are rare. Therefore, to optimise and streamline molecular detection and sequence-based typing of diverse RAS-containing subtypes, a novel panel of single round PCR assays was applied to HCV derived from 146 individuals, whose likely source of infection was from regions of sub-Saharan Africa (SSA). Partial NS5A and NS5B sequences were obtained from 135 HCV-positive patients born in 19 different countries from SSA but attending clinics in the UK.Virus subtype assignments were determined by pairwise-distance analysis and compared to both diagnostic laboratory assignments and free-to-use online typing tools. We determined that routine clinical diagnostic methods incorrectly subtyped 59.0% of samples, with a further 6.8% incorrectly genotyped. Of five commonly used online tools, Geno2Pheno performed most effectively in determining a subtype in agreement with pairwise distance analysis. Considering the estimated number of HCV infections to have occurred in across Africa, this study provides a simple low-cost pathway to guide regional therapeutic choice and assist global eradication programmes.

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A pan-genotypic Hepatitis C Virus NS5A amplification method for reliable genotyping and resistance testing

Cited by 10 publications

References 39 publications

Genetic structure of SARS-CoV-2 reflects clonal superspreading and multiple independent introduction events, North-Rhine Westphalia, Germany, February and March 2020

Genetic structure of SARS-CoV-2 reflects clonal superspreading and multiple independent introduction events, North-Rhine Westphalia, Germany, February and March 2020

Genetic structure of SARS-CoV-2 in Western Germany reflects clonal superspreading and multiple independent introduction events

Hepatitis C subtyping assay failure in UK patients born in Sub-Saharan Africa: implications for global treatment and elimination

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