scholarly journals Replication capacity of minority variants in viral populations can affect the assessment of resistance in HCV chimeric replicon phenotyping assays

2012 ◽  
Vol 67 (10) ◽  
pp. 2327-2337 ◽  
Author(s):  
T. Verbinnen ◽  
T. Jacobs ◽  
L. Vijgen ◽  
H. Ceulemans ◽  
J. Neyts ◽  
...  
Keyword(s):  
Replication Capacity ◽  
Chimeric Replicon ◽  
Minority Variants

A new system to measure and compare hepatitis C virus replication capacity using full-length, replication competent viruses

2013 ◽  
Vol 194 (1-2) ◽  
pp. 82-88
Author(s):  
Britta Lassmann ◽  
Vaithilingaraja Arumugaswami ◽  
Kara W. Chew ◽  
Martha J. Lewis
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Full Length ◽  
Replication Capacity ◽  
New System

scholarly journals Resistance Analysis of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir

2012 ◽  
Vol 56 (7) ◽  
pp. 3670-3681 ◽  
Author(s):  
Fiona McPhee ◽  
Jacques Friborg ◽  
Steven Levine ◽  
Chaoqun Chen ◽  
Paul Falk ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protease Inhibitor ◽  
Clinical Studies ◽  
Replication Capacity ◽  
Replication Complex ◽  
Hcv Genotype ◽  
Genotype 1A ◽  
Genotype 1B ◽  
Ns3 Protease

ABSTRACTAsunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensivein vitrogenotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.

scholarly journals Novel Single-Cell-Level Phenotypic Assay for Residual Drug Susceptibility and Reduced Replication Capacity of Drug-Resistant Human Immunodeficiency Virus Type 1

2004 ◽  
Vol 78 (4) ◽  
pp. 1718-1729 ◽  
Author(s):  
Haili Zhang ◽  
Yan Zhou ◽  
Cecily Alcock ◽  
Tara Kiefer ◽  
Daphne Monie ◽  
...  
Keyword(s):  
Drug Combination ◽  
Wild Type Virus ◽  
Replication Capacity ◽  
Drug Resistant ◽  
Type Virus ◽  
Wild Type ◽  
Cell Level ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-infected individuals who develop drug-resistant virus during antiretroviral therapy may derive benefit from continued treatment for two reasons. First, drug-resistant viruses can retain partial susceptibility to the drug combination. Second, therapy selects for drug-resistant viruses that may have reduced replication capacities relative to archived, drug-sensitive viruses. We developed a novel single-cell-level phenotypic assay that allows these two effects to be distinguished and compared quantitatively. Patient-derived gag-pol sequences were cloned into an HIV-1 reporter virus that expresses an endoplasmic reticulum-retained Env-green fluorescent protein fusion. Flow cytometric analysis of single-round infections allowed a quantitative analysis of viral replication over a 4-log dynamic range. The assay faithfully reproduced known in vivo drug interactions occurring at the level of target cells. Simultaneous analysis of single-round infections by wild-type and resistant viruses in the presence and absence of the relevant drug combination divided the benefit of continued nonsuppressive treatment into two additive components, residual virus susceptibility to the drug combination and selection for drug-resistant variants with diminished replication capacities. In some patients with drug resistance, the dominant circulating viruses retained significant susceptibility to the combination. However, in other cases, the dominant drug-resistant viruses showed no residual susceptibility to the combination but had a reduced replication capacity relative to the wild-type virus. In this case, simplification of the regimen might still allow adequate suppression of the wild-type virus. In a third pattern, the resistant viruses had no residual susceptibility to the relevant drug regimen but nevertheless had a replication capacity equivalent to that of wild-type virus. In such cases, there is no benefit to continued treatment. Thus, the ability to simultaneously analyze residual susceptibility and reduced replication capacity of drug-resistant viruses may provide a basis for rational therapeutic decisions in the setting of treatment failure.

scholarly journals Identification of Novel Mutations Responsible for Resistance to MK-2048, a Second-Generation HIV-1 Integrase Inhibitor

2010 ◽  
Vol 84 (18) ◽  
pp. 9210-9216 ◽  
Author(s):  
Tamara Bar-Magen ◽  
Richard D. Sloan ◽  
Daniel A. Donahue ◽  
Björn D. Kuhl ◽  
Alexandra Zabeida ◽  
...  
Keyword(s):  
Viral Replication ◽  
First Generation ◽  
Second Generation ◽  
Integrase Inhibitor ◽  
Replication Capacity ◽  
Strand Transfer ◽  
Viral Replication Capacity ◽  
Transfer Inhibitor ◽  
Potential Basis

ABSTRACT MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer “off” rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg2+ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.

scholarly journals Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Elizabeth Jaworski ◽  
Rose M Langsjoen ◽  
Brooke Mitchell ◽  
Barbara Judy ◽  
Patrick Newman ◽  
...  
Keyword(s):  
Full Range ◽  
Virus Genome ◽  
Clinical Samples ◽  
Rna Recombination ◽  
Single Nucleotide ◽  
Reverse Transcription Reaction ◽  
Minority Variants ◽  
Next Generation Sequencing Ngs ◽  
Unique Molecular Identifier ◽  
Generation Sequencing

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ‘Tiled-ClickSeq’, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5’UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

scholarly journals Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model

2020 ◽  
Author(s):  
Leo YY Lee ◽  
Jie Zhou ◽  
Paulina Koszalka ◽  
Rebecca Frise ◽  
Rubaiyea Farrukee ◽  
...  
Keyword(s):  
Influenza A ◽  
Relative Fitness ◽  
Replication Capacity ◽  
Wild Type ◽  
Influenza A Viruses ◽  
Widespread Occurrence ◽  
Host Fitness ◽  
Recombinant Viruses ◽  
Risk Patients ◽  
Virus Isolates

AbstractBaloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses.

scholarly journals Control of archetype BK polyomavirus miRNA expression

2020 ◽  
Author(s):  
Wei Zou ◽  
Gau Shoua Vue ◽  
Benedetta Assetta ◽  
Heather Manza ◽  
Walter J. Atwood ◽  
...  
Keyword(s):  
Hemorrhagic Cystitis ◽  
Antigen Expression ◽  
Marrow Transplant ◽  
T Antigen ◽  
Replication Capacity ◽  
Late Gene Expression ◽  
Cis Acting ◽  
Transplant Patients ◽  
Bk Polyomavirus ◽  
Dna Replication Origin

AbstractBK polyomavirus (BKPyV) is a ubiquitous human pathogen, with over 80% of adults worldwide persistently infected. BKPyV infection is usually asymptomatic in healthy people; however, it causes polyomavirus-associated nephropathy in renal transplant patients and hemorrhagic cystitis in bone marrow transplant patients. BKPyV has a circular, double-stranded DNA genome that is divided genetically into three parts: an early region, a late region, and a non-coding control region (NCCR). The NCCR contains the viral DNA replication origin and cis-acting elements regulating viral early and late gene expression. It was previously shown that a BKPyV miRNA expressed from the late strand regulates viral large T antigen expression and limits the replication capacity of archetype BKPyV. A major unanswered question in the field is how expression of the viral miRNA is regulated. Typically, miRNA is expressed from introns in cellular genes but there is no intron readily apparent in the BKPyV from which the miRNA could derive. Here we provide evidence for primary RNA transcripts that circle the genome more than once and include the NCCR. We identified splice junctions resulting from splicing of primary transcripts circling the genome more than once, and Sanger sequencing of RT-PCR products indicates that there are viral transcripts that circle the genome up to four times. Our data suggest that the miRNA is expressed from the intron of these greater-than-genome size primary transcripts.

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