Rapid genotyping of hepatitis C virus by primer-specific extension analysis.

Quick and accurate genotyping of hepatitis C virus (HCV) is becoming increasingly important for clinical management of chronic infection and as an epidemiological marker. Furthermore, the incidence of HCV infection with mixed genotypes has clinical significance that is not addressed by most genotyping methods. We have developed a fluorescence-based genotyping assay called primer-specific extension analysis (PSEA) for the most prevalent HCV genotypes and have demonstrated the capacity of PSEA-HCV for detecting mixed-genotype HCV infections. PSEA-HCV detects genotype-specific sequence differences in the 5' untranslated region of HCV in products amplified by the COBAS AMPLICOR HCV Test, v2.0. Simulated mixed HCV infection of plasma with RNase-resistant RNA controls demonstrates that PSEA-HCV can detect as many as five genotypes in one specimen. Furthermore, in dual-genotype simulations, PSEA-HCV can unequivocally detect both genotypes, with one genotype representing only 3.1% of the mixture (313/10,000 IU in starting plasma). Compared to INNO-LiPA HCV II, both assays determined the same genotype for 191/199 (96%) patient specimens (175 subtype and 16 genotype-only identifications). Following the initial evaluation, PSEA-HCV was used routinely to genotype HCV from patient specimens submitted to our laboratory (n=312). Seventeen (5.4%) mixed infections were identified. The distribution of single-infection HCV genotypes in our population was 60.9% type 1 (n=190), 12.8% type 2 (n=40), 20.2% type 3 (n=63), 0.3% type 4 (n=1), and 0.3% other (n=1). In conclusion, PSEA-HCV provides an inexpensive, high-throughput screening tool for rapid genotyping of HCV while reliably identifying mixed HCV infections.

Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer.

Aeromonas hydrophila secretes protein toxins via the type II pathway, involving the products of at least two operons, exeAB (gspAB) and exeC-N (gspC-N). In the studies reported here, aerolysin secretion was restored to C5.84, an exeA::Tn5-751 mutant, by overexpression of exeD alone in trans. Expression studies indicated that these results did not reflect a role of ExeAB in the regulation of the exeC-N operon. Instead, immunoblot analysis showed that ExeD did not multimerize in C5.84, and fractionation of the membranes showed that the monomeric ExeD remained in the inner membrane. Expression of ExeAB, but not either protein alone, from a plasmid in C5.84 resulted in increases in the amount of multimeric ExeD, which correlated with increases in aerolysin secretion. Pulse-chase analysis also suggested that the induction of ExeAB allowed multimerization of previously accumulated monomer ExeD. In C5.84 cells overproducing ExeD, it multimerized even in the absence of ExeAB and, although most remained in the inner membrane, an amount similar to that in wild-type outer membranes fractionated with the outer membrane of the overproducing cells. These results indicate that the secretion defect of exeAB mutants is a result of an inability to assemble the ExeD secretin in the outer membrane. The localization and multimerization of overproduced ExeD in these mutants further suggests that the ExeAB complex plays either a direct or indirect role in the transport of ExeD into the outer membrane.