Development of a chimeric replicon system for phenotypic analysis of NS3 protease sequences from HCV clinical isolates

2011 ◽  
Vol 16 (5) ◽  
pp. 705-718 ◽  
Author(s):  
Amy K Sheaffer ◽  
Min S Lee ◽  
Dennis Hernandez ◽  
Susan Chaniewski ◽  
Fei Yu ◽  
...  
Keyword(s):  
Clinical Isolates ◽  
Phenotypic Analysis ◽  
Chimeric Replicon ◽  
Ns3 Protease

scholarly journals Identification of Clinical Isolates of Candida albicans with Increased Fitness in Colonization of the Murine Gut

2021 ◽  
Vol 7 (9) ◽  
pp. 695
Author(s):  
Rebeca Alonso-Monge ◽  
Daniel Prieto ◽  
Ioana Coman ◽  
Sara Rochas ◽  
David M. Arana ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Opportunistic Pathogen ◽  
Clinical Isolates ◽  
Fungal Colonization ◽  
Phenotypic Analysis ◽  
Bacterial Microbiota ◽  
Specific Trait ◽  
Strain Sc5314 ◽  
Previously Treated

The commensal and opportunistic pathogen Candida albicans is an important cause of fungal diseases in humans, with the gastrointestinal tract being an important reservoir for its infections. The study of the mechanisms promoting the C. albicans commensal state has attracted considerable attention over the last few years, and several studies have focused on the identification of the intestinal human mycobiota and the characterization of Candida genes involved in its establishment as a commensal. In this work, we have barcoded 114 clinical C. albicans isolates to identify strains with an enhanced fitness in a murine gastrointestinal commensalism model. The 114 barcoded clinical isolates were pooled in four groups of 28 to 30 strains that were inoculated by gavage in mice previously treated with antibacterial therapy. Eight strains that either exhibited higher colonization load and/or remained in the gut after antibiotic removal were selected. The phenotypic analysis of these strains compared to an RFP-tagged SC5314 wild type strain did not reveal any specific trait associated with its increased colonization; all strains were able to filament and six of the eight strains displayed invasive growth on Spider medium. Analysis of one of these strains, CaORAL3, revealed that although mice required previous bacterial microbiota reduction with antibiotics to be able to be colonized, removal of this procedure could take place the same day (or even before) Candida inoculation. This strain was able to colonize the intestine of mice already colonized with Candida without antibiotic treatment in co-housing experiments. CaORAL3 was also able to be established as a commensal in mice previously colonized by another (CaHG43) or the same (CaORAL3) C. albicans strain. Therefore, we have identified C. albicans isolates that display higher colonization load than the standard strain SC5314 which will surely facilitate the analysis of the factors that regulate fungal colonization.

scholarly journals Potency and Resistance Analysis of Hepatitis C Virus NS5B Polymerase Inhibitor BMS-791325 on All Major Genotypes

2014 ◽  
Vol 58 (12) ◽  
pp. 7416-7423 ◽  
Author(s):  
Mengping Liu ◽  
Maria Tuttle ◽  
Min Gao ◽  
Julie A. Lemm
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Chronic Infections ◽  
Thumb Domain ◽  
Ns5b Polymerase Inhibitor ◽  
Chimeric Replicon ◽  
Polymerase Inhibitor ◽  
Ns3 Protease ◽  
Hybrid Clones ◽  
Ns5b Polymerase

ABSTRACTBMS-791325 is a hepatitis C virus (HCV) inhibitor binding to the thumb domain of the NS5B RNA-dependent RNA polymerase. BMS-791325 is well characterized in genotype 1 (GT1) and exhibits good inhibitory activity (50% effective concentration [EC50], <10 nM) against hybrid replicons containing patient NS5B sequences from GT3a, -4a, and -5a while potency against GT2 is significantly reduced (J. A. Lemm et al., Antimicrob. Agents Chemother. 58:3485–3495, 2014, doi:http://dx.doi.org/10.1128/AAC.02495-13). BMS-791325 potency against GT6a hybrid replicons is more variable, with two of three hybrid clones having EC50s similar to that for GT1 while a third patient clone was ∼10 times less susceptible to BMS-791325. To characterize the resistance profile of BMS-791325 beyond GT1, curing studies were performed across GT1a and -3a to -6a and demonstrated that GT1a has the highest resistance barrier versus BMS-791325 while GT6a has the lowest. Selection of GT3 to -6 NS5B chimeric replicon cells at different concentrations of BMS-791325 revealed substitutions in the thumb domain of NS5B at residues 494 and 495 that conferred different levels of resistance to BMS-791325 but remained susceptible to NS5A or NS3 protease inhibitors. In addition, we demonstrate that the reduced potency of BMS-791325 against one GT6a patient is due to an A494 polymorphism present in ∼21% of sequences in the European HCV database. The results from this report suggest that BMS-791325 is a candidate for combination treatment of HCV GT3 to -6 chronic infections, and the resistance profiles identified will provide useful information for future clinical development.

scholarly journals Susceptibility of Treatment-Naive Hepatitis C Virus (HCV) Clinical Isolates to HCV Protease Inhibitors

2010 ◽  
Vol 54 (12) ◽  
pp. 5288-5297 ◽  
Author(s):  
Andrew Bae ◽  
Siu-Chi Sun ◽  
Xiaoping Qi ◽  
Xiaowu Chen ◽  
Karin Ku ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protease Inhibitors ◽  
Natural Variation ◽  
Clinical Isolates ◽  
Replication Assay ◽  
Treatment Naïve ◽  
Transient Replication Assay ◽  
Ns3 Protease

ABSTRACT In order to assess the natural variation in susceptibility to hepatitis C virus (HCV) NS3 protease inhibitors (PIs) among untreated HCV patient samples, the susceptibilities of 39 baseline clinical isolates were determined using a transient-replication assay on a panel of HCV PIs, including two α-ketoamides (VX-950 and SCH-503034) and three macrocyclic inhibitors (MK-7009, ITMN-191, and TMC-435350). Some natural variation in susceptibility to all HCV PIs tested was observed among the baseline clinical isolates. The susceptibility to VX-950 correlated strongly with the susceptibility to SCH-503034. A moderate correlation was observed between the susceptibilities to ITMN-191 and MK-7009. In contrast, the phenotypic correlations between the α-ketoamides and macrocyclic inhibitors were significantly lower. This difference is partly attributable to reduced susceptibility of the HCV variants containing the NS3 polymorphism Q80K (existing in 47% of genotype 1a isolates) to the macrocyclic compounds but no change in the sensitivity of the same variants to the α-ketoamides tested. Our results suggest that the natural variation in baseline susceptibility may contribute to different degrees of antiviral response among patients in vivo, particularly at lower doses.

Development of a replicon-based phenotypic assay for assessing the drug susceptibilities of HCV NS3 protease genes from clinical isolates

2009 ◽  
Vol 81 (2) ◽  
pp. 166-173 ◽  
Author(s):  
Xiaoping Qi ◽  
Andrew Bae ◽  
Shan Liu ◽  
Huiling Yang ◽  
Siu-Chi Sun ◽  
...  
Keyword(s):  
Clinical Isolates ◽  
Phenotypic Assay ◽  
Ns3 Protease

Klonale Analyse von Mutationen der HCV NS3 Protease bei Genotyp 1 Non-Respondern, die mit Boceprevir (SCH503034) behandelt wurden

2008 ◽  
Vol 46 (09) ◽  
Author(s):  
S Susser ◽  
MW Welker ◽  
M Zettler ◽  
A Dragan ◽  
E Hughes ◽  
...  
Keyword(s):  
Ns3 Protease

Protein S mRNA in Patients with Protein S Deficiency

1995 ◽  
Vol 73 (05) ◽  
pp. 746-749 ◽  
Author(s):  
E Sacchi ◽  
M Pinotti ◽  
G Marchetti ◽  
G Merati ◽  
L Tagliabue ◽  
...  
Keyword(s):  
Gene Polymorphism ◽  
Total Protein ◽  
Restriction Analysis ◽  
Protein S ◽  
Type I ◽  
Protein S Deficiency ◽  
Phenotypic Analysis ◽  
S Deficiency ◽  
Deficiency Type ◽  
S Genes

SummaryA protein S gene polymorphism, detectable by restriction analysis (BstXI) of amplified exonic sequences (exon 15), was studied in seven Italian families with protein S deficiency. In the 17 individuals heterozygous for the polymorphism the study was extended to platelet mRNA through reverse transcription, amplification and densitometric analysis. mRNA produced by the putative defective protein S genes was absent in three families and reduced to a different extent (as expressed by altered allelic ratios) in four families. The allelic ratios helped to distinguish total protein S deficiency (type I) from free protein S deficiency (type IIa) in families with equivocal phenotypes. This study indicates that the study of platelet mRNA, in association with phenotypic analysis based upon protein S assays in plasma, helps to classify patients with protein S deficiency.

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