Abstract：
:Measuring the in vitro replication capacity of viruses is an important tool for assessing the effects of selective pressure of immune responses and drug therapy. Measuring hepatitis C virus (HCV) replication capacity utilizing primarily sub-genomic reporter constructs is limited. To overcome some of these limitations a quantitative reverse transcriptase PCR (RT-qPCR) was designed to measure simultaneously the growth rate of 2 whole genome HCV variants under identical culture conditions. The assay demonstrates 100% specificity of detection of each variant and a linear detection range from 200 to 2×10(8) copies. The system was validated using a panel of HCV mutants, including the NS3 protease inhibitor drug resistance mutants R155K and T54A. The creation of a unique sequence tag results in highly sensitive and specific discrimination of parental JFH-FNX and modified clones using distinct probes in a RT-qPCR allowing for comparison of the effect of drug resistance or immune escape mutations on HCV replication. This system has advantages over existing methods both by permitting direct comparison of the replication capacity of fully replication-competent HCV mutants under identical culture conditions and by measuring effects on replication capacity due to mutations affecting all stages of the viral life cycle including entry and egress.
journal_name
J Virol Methodsjournal_title
Journal of virological methodsauthors
Lassmann B,Arumugaswami V,Chew KW,Lewis MJdoi
10.1016/j.jviromet.2013.08.009subject
Has Abstractpub_date
2013-12-01 00:00:00pages
82-8issue
1-2eissn
0166-0934issn
1879-0984pii
S0166-0934(13)00341-8journal_volume
194pub_type
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