Detection of haemagglutinin D222 polymorphisms in influenza A(H1N1)pdm09-infected patients by ultra-deep pyrosequencing.

This study was aimed at establishing the genetic heterogeneity of influenza virus haemagglutinin (HA) gene quasi-species and the polymorphisms at codon 222, by application of ultra-deep pyrosequencing (UDPS) to respiratory samples from patients hospitalized for influenza A(H1N1)pdm09 infection, presenting with severe or moderate-mild disease. HA diversity was significantly higher in samples collected from patients with severe manifestations than in those from patients with moderate-mild manifestations (p 0.02). D222 polymorphism was detected in 40.7% of patients by UDPS, and in only 7.1% by Sanger sequencing. D222E, D222G, D222N and D222A were observed in 37.0%, 11.1%, 7.4% and 3.7% of patients, respectively; 10.7% of samples harboured more than two variants. The relative frequency of each single variant showed a wide range of intrapatient variation. D222G/N/A were detected, as either minor or predominant variants, only in severe cases, whereas D222E was equally represented in severe and moderate-mild infections. Other amino acid variants were observed at different positions within the analysed HA fragment. Consistent with higher heterogeneity, non-D222 variants were more frequently detected in severe cases than in moderate-mild cases. In addition, seven non-D222 mutations carried by minority variants, not previously described, were observed.

Analysis of HIV drug-resistant quasispecies in plasma, peripheral blood mononuclear cells and viral isolates from treatment-naive and HAART patients.

The pattern of HIV-1 reverse transcriptase and protease mutations conferring resistance to antiretroviral drugs was studied in five treatment-naive patients and five HIV-infected patients receiving HAART [two reverse transcriptase inhibitors + one protease inhibitor] for > or = 1 year. Direct sequencing was performed on plasma HIV RNA, HIV DNA from peripheral blood mononuclear cells (PBMCs), and RNA from viral isolates. In addition, reverse transcriptase and protease PCR products from PBMCs HIV DNA, plasma HIV RNA, and viral isolate RNA were cloned in a plasmid to study the quasispecies distribution of drug-resistance associated mutations. Direct sequencing of HIV DNA from PBMCs and HIV RNA from plasma and viral isolates did not show the presence of drug resistance associated mutations in both reverse transcriptase and protease of HIV from all five treatment-naive patients. On the contrary, mutation analysis obtained by cloning plasma HIV RNA and PBMCs DNA showed the presence of drug-resistance related mutations at a low frequency in both HIV enzymes of four out of five treatment-naive patients. On the other hand, direct sequencing of plasma HIV RNA showed the presence of several reverse transcriptase and protease mutations in all five treated patients. Mutation analysis performed by cloning PBMCs HIV DNA, and HIV RNA from plasma and viral isolates, revealed additional reverse transcriptase and protease changes compared to direct sequencing of the relevant biological samples. All the additional changes were observed in a minority of clones. In conclusion, the data suggest that less frequent drug-resistant viral variants not detected by direct sequencing of PBMCs, plasma samples, or viral isolates are present in both treatment-naive and treatment-experienced HIV patients. These findings may have important implications in the understanding of the selection process of drug-resistant variants under drug pressure.