Computational protocol: Persistence of Hepatitis C Virus during and after Otherwise Clinically Successful Treatment of Chronic Hepatitis C with Standard Pegylated Interferon α-2b and Ribavirin Therapy

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Protocol publication

[…] To assess possible sequence variations and compartmentalization of HCV, 5′-UTR amplicons were cloned using the TOPO-TA cloning kit (Invitrogen). The highly conserved 5′-UTR was chosen because it would allow for reliable identification of most unwavering sequence variants. Ten or 20 randomly selected clones were analyzed from each PCR product derived from 7 pairs of plasma and PBMC samples collected at the same time points of follow-up (with one exception) prior to, during or after completion of PegIFN/R treatment from 2 patients (5/M and 8/F) from whom sufficient amounts of sample material were available. The sequence was determined in both directions using universal forward and reverse M13 primers, and the ABI-Prism 7000 Sequence Detection System (Applied Biosystems, Streetsville, Canada). The resulting sequences were aligned using Sequencher software version 4.7 (Gene Codes Corp., Ann Arbor, MI). The HCV subgenotype-specific sequences D00944 and AF169005 for 2a genotype, and D10988 and AF238486 for 2b genotype from GenBank were used as the references. The phylogenetic relationships of the variant 5′-UTR sequences identified in plasma and PBMC of 5/M and 8/F patients were examined by the maximum-likelihood method . [...] Results were analyzed by Chi-Square test using SPSS Statistics software version 19.0 (IBM, Armonk, New York State, USA). Differences between sample groups were considered to be significant when P values were below or equal to 0.05. […]

Pipeline specifications

Software tools Sequencher, SPSS
Applications Miscellaneous, Phylogenetics
Organisms Classical swine fever virus, Homo sapiens
Diseases Hepatitis C, Hepatitis C, Chronic
Chemicals Ribavirin