The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the causal agent of the COVID-19 pandemic that emerged in late 2019. The outbreak of variants with mutations in the region ...encoding the spike protein S1 sub-unit that can make them more resistant to neutralizing or monoclonal antibodies is the main point of the current monitoring. This study examines the feasibility of predicting the variant lineage and monitoring the appearance of reported mutations by sequencing only the region encoding the S1 domain by Pacific Bioscience Single Molecule Real-Time sequencing (PacBio SMRT). Using the PacBio SMRT system, we successfully sequenced 186 of the 200 samples previously sequenced with the Illumina COVIDSeq (whole genome) system. PacBio SMRT detected mutations in the S1 domain that were missed by the COVIDseq system in 27/186 samples (14.5%), due to amplification failure. These missing positions included mutations that are decisive for lineage assignation, such as G142D (
= 11), N501Y (
= 6), or E484K (
= 2). The lineage of 172/186 (92.5%) samples was accurately determined by analyzing the region encoding the S1 domain with a pipeline that uses key positions in S1. Thus, the PacBio SMRT protocol is appropriate for determining virus lineages and detecting key mutations.
Fast, accurate sequencing methods are needed to identify new variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) genome. ...Single‐molecule real‐time (SMRT) Pacific Biosciences (PacBio) provides long, highly accurate sequences by circular consensus reads. This study compares the performance of a target capture SMRT PacBio protocol for whole‐genome sequencing (WGS) of SARS‐CoV‐2 to that of an amplicon PacBio SMRT sequencing protocol. The median genome coverage was higher (p < 0.05) with the target capture protocol (99.3% interquartile range, IQR: 96.3–99.5) than with the amplicon protocol (99.3% IQR: 69.9–99.3). The clades of 65 samples determined with both protocols were 100% concordant. After adjusting for Ct values, S gene coverage was higher with the target capture protocol than with the amplicon protocol. After stratification on Ct values, higher S gene coverage with the target capture protocol was observed only for samples with Ct > 17 (p < 0.01). PacBio SMRT sequencing protocols appear to be suitable for WGS, genotyping, and detecting mutations of SARS‐CoV‐2.
New variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) genome can only be identified using accurate sequencing methods. Single molecule real‐time ...(SMRT) sequencing has been used to characterize Alpha and Delta variants, but not Omicron variants harboring numerous mutations in the SARS‐CoV‐2 genome. This study assesses the performance of a target capture SMRT sequencing protocol for whole genome sequencing (WGS) of SARS‐CoV‐2 Omicron variants and compared it to that of an amplicon SMRT sequencing protocol optimized for Omicron variants. The failure rate of the target capture protocol (6%) was lower than that of the amplicon protocol (34%, p < 0.001) on our data set, and the median genome coverage with the target capture protocol (98.6% interquartile range (IQR): 86–99.4) was greater than that with the amplicon protocol (76.6% IQR: 66–89.6, p < 0.001). The percentages of samples with >95% whole genome coverage were 64% with the target capture protocol and 19% with the amplicon protocol (p < 0.05). The clades of 96 samples determined with both protocols were 93% concordant and the lineages of 59 samples were 100% concordant. Thus, target capture SMRT sequencing appears to be an efficient method for WGS, genotyping and detecting mutations of SARS‐CoV‐2 Omicron variants.
Diversity of hepatitis E virus genotype 3 Nicot, Florence; Jeanne, Nicolas; Roulet, Alain ...
Reviews in medical virology,
September 2018, Volume:
28, Issue:
5
Journal Article
Peer reviewed
Summary
Hepatitis E virus genotype 3 (HEV‐3) can lead to chronic infection in immunocompromised patients, and ribavirin is the treatment of choice. Recently, mutations in the polymerase gene have ...been associated with ribavirin failure but their frequency before treatment according to HEV‐3 subtypes has not been studied on a large data set. We used single‐molecule real‐time sequencing technology to sequence 115 new complete genomes of HEV‐3 infecting French patients. We analyzed phylogenetic relationships, the length of the polyproline region, and mutations in the HEV polymerase gene. Eighty‐five (74%) were in the clade HEV‐3efg, 28 (24%) in HEV‐3chi clade, and 2 (2%) in HEV‐3ra clade. Using automated partitioning of maximum likelihood phylogenetic trees, complete genomes were classified into subtypes. Polyproline region length differs within HEV‐3 clades (from 189 to 315 nt). Investigating mutations in the polymerase gene, distinct polymorphisms between HEV‐3 subtypes were found (G1634R in 95% of HEV‐3e, G1634K in 56% of HEV‐3ra, and V1479I in all HEV‐3efg, clade HEV‐3ra, and HEV‐3k strains). Subtype‐specific polymorphisms in the HEV‐3 polymerase have been identified. Our study provides new complete genome sequences of HEV‐3 that could be useful for comparing strains circulating in humans and the animal reservoir.
Hepatitis E virus (HEV) genotype 3 is the most common genotype linked to HEV infections in Europe and America. Three major clades (HEV-3.1, HEV-3.2, and HEV-3.3) have been identified but the overlaps ...between intra-subtype and inter-subtype p-distances make subtype classification inconsistent. Reference sequences have been proposed to facilitate communication between researchers and new putative subtypes have been identified recently. We have used the full or near full-length HEV-3 genome sequences available in the Genbank database (April 2020;
= 503) and distance analyses of clades HEV-3.1 and HEV-3.2 to determine a p-distance cut-off (0.093 nt substitutions/site) in order to define subtypes. This could help to harmonize HEV-3 genotyping, facilitate molecular epidemiology studies and investigations of the biological and clinical differences between HEV-3 subtypes.
Hepatitis E virus (HEV) can lead to chronic infection in solid-organ transplant patients. Ribavirin is efficient for treatment of chronically infected patients. Recently, the1634R mutation in the HEV ...polymerase has been associated with treatment failure. However, it is unclear if this mutation can be used as a prognostic marker of treatment outcome. We studied the prevalence of the 1634R mutation in the HEV polymerase of patients starting ribavirin therapy, the influence of the 1634R variants on the viral response, the frequency of the 1634R mutation in patients whose treatment failed, and its impact on ribavirin retreatment. We analyzed pretreatment samples from 63 solid-organ transplant patients with chronic hepatitis E using deep sequencing; 42 patients had a sustained virologic response (SVR), and 21 were non-SVR patients. We detected the 1634R variant by deep sequencing in 36.5% (23/63) of the patients (proportions, 1.3 to 100%). The 1634R variant was detected in 31.0% (13/42) of baseline plasma samples from patients with SVR and in 47.6% (10/21) in the other patients (P = 0.2). The presence of this mutation did not influence the initial decrease in viral RNA. Lastly, a second prolonged ribavirin treatment led to SVR in 70% of the patients who initially did not have SVR, despite the presence of the 1634R variant. We conclude that the presence of the 1634R variant at ribavirin initiation does not lead to absolute ribavirin resistance. Although its proportion increased in patients whose treatment failed, the presence of the 1634R variant did not compromise the response to a second ribavirin treatment.
The spread of SARS-CoV-2 and the resulting disease COVID-19 has killed over 2.6 million people as of 18 March 2021. We have used a modified susceptible, infected, recovered (SIR) epidemiological ...model to predict how the spread of the virus in regions of France will vary depending on the proportions of variants and on the public health strategies adopted, including anti-COVID-19 vaccination. The proportion of SARS-CoV-2 variant B.1.1.7, which was not detected in early January, increased to become 60% of the forms of SARS-CoV-2 circulating in the Toulouse urban area at the beginning of February 2021, but there was no increase in positive nucleic acid tests. Our prediction model indicates that maintaining public health measures and accelerating vaccination are efficient strategies for the sustained control of SARS-CoV-2.
Recombinant strains of hepatitis E virus (HEV) with insertions of human genomic fragments or HEV sequence duplications in the sequence encoding the polyproline region (PPR) were previously described ...in chronically infected patients. Such genomic rearrangements confer a replicative advantage
but little is known about their frequency, location, or origin. As the sequences of only a few virus genomes are available, we analyzed the complete genomes of 114 HEV genotype 3 strains from immunocompromised (
= 85) and immunocompetent (
= 29) patients using the single molecular real-time sequencing method to determine the frequency, location, and origin of inserted genomic fragments, plus the proportions of variants with genomic rearrangements in each virus quasispecies. We also examined the amino acid compositions and post-translational modifications conferred by these rearrangements by comparing them to sequences without human gene insertions or HEV gene duplications. We found genomic rearrangements in 7/114 (6.1%) complete genome sequences (4 HEV-3f, 1 HEV-3e, 1 HEV-3 h, and 1 HEV-3chi-new), all from immunocompromised patients, and 3/7 were found at the acute phase of infection. Six of the seven patients harbored virus-host recombinant variants, including one patient with two different recombinant variants. We also detected three recombinant variants with genome duplications of the PPR or PPR + X domains in a single patient. All the genomic rearrangements (seven human fragment insertions of varying origins and three HEV genome duplications) occurred in the PPR. The sequences with genomic rearrangements had specific characteristics: increased net load (
< 0.001) and more ubiquitination (
< 0.001), phosphorylation (
< 0.001), and acetylation (
< 0.001) sites. The human fragment insertions and HEV genome duplications had slightly different characteristics. We believe this is the first description of HEV strains with genomic rearrangements in patients at the acute phase of infection; perhaps these strains are directly transmitted. Clearly, genomic rearrangements produce a greater net load with duplications and insertions having different features. Further studies are needed to clarify the mechanisms by which such modifications influence HEV replication.
The present study compares the performances of an in‐house sequencing protocol developed on MiSeq, the Sanger method, and the 454 GS‐FLX for detecting and quantifying drug‐resistant mutations (DRMs) ...in the human immunodeficiency virus polymerase gene (reverse transcriptase RT and protease PR). MiSeq sequencing identified all the resistance mutations detected by bulk sequencing (n = 84). Both the MiSeq and 454 GS‐FLX platforms identified 67 DRMs in the RT and PR regions, but a further 25 DRMs were identified by only one or other of them. Pearson’s analysis showed good concordance between the percentage of drug‐resistant variants determined by MiSeq and 454 GS‐FLX sequencing (ρ = .77, P < .0001). The MiSeq platform is as accurate as the 454 GS‐FLX Roche system for determining RT and PR DRMs and could be used for monitoring human immunodeficiency virus type 1 drug resistance.
•HIV‐1 RT or PR mutations detected by Sanger sequencing (n = 84) were all observed by MiSeq sequencing.
•Both MiSeq and 454 GS‐FLX sequencing methods identified 67 RT or PR mutations in the 24 samples tested.
•The percentage of DRMs determined by both methods were in agreement.
Abstract
Motivation
The circulating recombinant form of HIV-1 CRF02-AG is the most frequent non-B subtype in Europe. Anti-HIV therapy and pathophysiological studies on the impact of HIV-1 tropism ...require genotypic determination of HIV-1 tropism for non-B subtypes. But genotypic approaches based on analysis of the V3 envelope region perform poorly when used to determine the tropism of CRF02-AG. We, therefore, designed an algorithm based on information from the gp120 and gp41 ectodomain that better predicts the tropism of HIV-1 subtype CRF02-AG.
Results
We used a bio-statistical method to identify the genotypic determinants of CRF02-AG coreceptor use. Toulouse HIV Extended Tropism Algorithm (THETA), based on a Least Absolute Shrinkage and Selection Operator method, uses HIV envelope sequence from phenotypically characterized clones. Prediction of R5X4/X4 viruses was 86% sensitive and that of R5 viruses was 89% specific with our model. The overall accuracy of THETA was 88%, making it sufficiently reliable for predicting the tropism of subtype CRF02-AG sequences.
Availability and implementation
Binaries are freely available for download at https://github.com/viro-tls/THETA. It was implemented in Matlab and supported on MS Windows platform. The sequence data used in this work are available from GenBank under the accession numbers MK618182-MK618417.
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