We aimed to evaluate the correct assignment of HCV genotypes by three commercial methods—Trugene HCV genotyping kit (Siemens), VERSANT HCV Genotype 2.0 assay (Siemens), and Real-Time HCV genotype II (Abbott)—compared to NS5B sequencing. We studied 327 clinical samples that carried representative HCV genotypes of the most frequent geno/subtypes in Spain. After commercial genotyping, the sequencing of a 367 bp fragment in the NS5B gene was used to assign genotypes. Major discrepancies were defined, e.g. differences in the assigned genotype by one of the three methods and NS5B sequencing, including misclassification of subtypes 1a and 1b. Minor discrepancies were considered when differences at subtype levels, other than 1a and 1b, were observed. The overall discordance with the reference method was 34% for Trugene and 15% for VERSANT HCV2.0. The Abbott assay correctly identified all 1a and 1b subtypes, but did not subtype all the 2, 3, 4 and 5 (34%) genotypes. Major discordances were found in 16% of cases for Trugene HCV, and the majority were 1b- to 1a-related discordances; major discordances were found for VERSANT HCV 2.0 in 6% of cases, which were all but one 1b to 1a cases. These results indicated that the Trugene assay especially, and to a lesser extent, Versant HCV 2.0, can fail to differentiate HCV subtypes 1a and 1b, and lead to critical errors in clinical practice for correctly using directly acting antiviral agents.
Antibody linear epitopes of the glycoprotein G (gpG) of the viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus of salrnonids, were mapped by pepscan using overlapping 15-mer peptides covering the entire gpG sequence and ELISA with polyclonal and monoclonal murine and polyclonal trout antibodies. Among the regions recognized in the pepscan by the polyclonal antibodies (PAbs) were the previously identified phosphatidylserine binding heptad-repeats (Estepa & Col1 1996; Virology 216:60-70) and leucocyte stimulating peptides (Lorenzo et al. 1995; Virology 212:348-355). Among 17 monoclonal antibodies (MAbs), only 2 non-neutralizing MAbs, 110 (aa 139-153) and IPlH3 (aa 399-413), could b e mapped to specific peptides in the pepscan of the gpG. Mapping of these MAbs was confirmed by immunoblotting with recombinant proteins and/or other synthetic peptides covering those sequences.None of the neutralizing MAbs tested reacted with any of the gpG peptides. Previously mapped MAb resistant mutants in the gpG did not coincide with any of the linear epitopes defined by the pepscan strategy, suggesting the complementarity of the 2 methods for the identification of antibody recogn~tion sites.
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