Protease inhibitor resistance mutations in untreated Brazilian patients infected with HCV: novel insights about targeted genotyping approaches.

Several new direct-acting antiviral (DAA) drugs are being developed or are already approved for the treatment of chronic hepatitis C virus (HCV) infection. HCV variants presenting drug-resistant phenotypes were observed both in vitro and during clinical trials. The aim of this study was to characterize amino acid changes at positions previously associated with resistance in the NS3 protease in untreated Brazilian patients infected with HCV genotypes 1a and 1b. Plasma samples from 171 untreated Brazilian patients infected with HCV were obtained from the Department of Gastroenterology of Clinics Hospital (HCFMUSP) in São Paulo, Brazil. Nested PCR and Sanger sequencing were used to obtain genetic information on the NS3 protein. Bioinformatics was used to confirm subtype information and analyze frequencies of resistance mutations. The results from the genotype analysis using non-NS3 targeted methods were at variance with those obtained from the NS3 protease phylogenetic analyses. It was found that 7.4% of patients infected with HCV genotype 1a showed the resistance-associated mutations V36L, T54S, Q80K, and R155K, while 5.1% of patients infected with HCV genotype 1b had the resistance-associated mutations V36L, Q41R, T54S, and D168S. Notably, codons at positions 80 and 155 differed between samples from Brazilian patient used in this study and global isolates. The present study demonstrates that genotyping methods targeting the NS3 protein showed a difference of results when compared to mainstream methodologies (INNO-LiPA and polymerase sequencing). The resistance mutations present in untreated patients infected with HCV and codon composition bias by geographical location warrant closer examination.

Occurrence and phylogenetic characterization of a baculovirus isolated from Culex quinquefasciatus in São Paulo State, Brazil.

The aim of this study was to assess the occurrence of baculovirus infections in mosquitoes and characterize them by using molecular tools. Fortnightly collections were made of mosquito larvae in the city of Caraguatatuba. Six larvae of Culex quinquefasciatus were isolated that had white cysts (nodules) in epithelia cells of the posterior midgut, indicative of infection by a baculovirus. These larvae were subjected to DNA extraction. DNA was amplified, producing a fragment of around 600 nt of the lef-8 gene and 400 nt of Pif-2 gene. The sequences were aligned, using ClustalX 2.0, with partial sequences of lef-8 genes of baculoviruses isolated from members of other insect orders taken from the GenBank database and edited, and phylogenetic analysis was performed. Phylogenetic analysis performed with the lef-8 and pif-2 genes demonstrated that the baculovirus identified in Culex quinquefasciatus in the Caraguatatuba region is most closely related to the deltabaculovirus Culex nigripalpus nucleopolyhedrovirus.