title: A new and rapid genotypic assay for the detection of neuraminidase inhibitor resistant influenza A viruses of subtype H1N1, H3N2, and H5N1.
abstract: The neuraminidase of influenza viruses is the target of the inhibitors oseltamivir and zanamivir. Recent reports on influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAI) are a cause for concern. Several amino acid substitutions, each as a consequence of one single nucleotide mutation, are known to confer resistance to NAI. An increase of NAI-resistant viruses appears to be likely as a result of a wider application of NAI for treatment and prophylaxis of seasonal influenza infections. Monitoring the occurrence and spread of resistant viruses is an important task. Therefore, RT-PCR assays were developed with subsequent pyrosequencing analysis (PSQ-PCR). These assays allow a rapid, high-throughput and cost-effective screening of subtype A/H1N1, A/H3N2, and A/H5N1 viruses. Various specimens such as respiratory swabs, allantoic fluid, or cell-propagated viruses can be used and results are available within hours. Several A/H1N1, A/H3N2, and A/H5N1 viruses isolated from human and avian specimens were tested to evaluate the method. Positive controls encoding resistance-associated mutations were created using site-directed mutagenesis. The results obtained with these controls showed that the assay can discriminate clearly the wild-type virus from a mutant virus. The detection limit of minor virus variants within the viral quasispecies amounts to 10%.
authors: National Reference Centre for Influenza, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany.
authors: Susanne
authors: S
authors: Duwe
authors: Brunhilde
authors: B
authors: Schweiger
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D053122:Influenza A Virus, H3N2 Subtype / Q000145:classification / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D013997:Time Factors
chemical_list: D000998:Antiviral Agents; D009439:Neuraminidase