Angelica N. Martins

Phylogenetic and Genetic Analysis of Feline Immunodeficiency Virus gag, pol, and env Genes from Domestic Cats Undergoing Nucleoside Reverse Transcriptase Inhibitor Treatment or Treatment-Naïve Cats in Rio de Janeiro, Brazil

ABSTRACT Feline immunodeficiency virus (FIV) is the Lentivirus responsible for an immunodeficiency-like disease in domestic cats (Felis catus). FIV is divided into five phylogenetic subtypes (A, B, C, D, and E), based on genetic diversity. Knowledge of the geographical distribution of subtypes is relevant for understanding different disease progressions and for vaccine development. In this study, viral sequences of 26 infected cats from Rio de Janeiro, 8 undergoing treatment with zidovudine (AZT) for at least 5 years, were successfully amplified from blood specimens. gag capsid (CA), pol reverse transcriptase (RT), and env gp120 (V3-V4) regions were analyzed to determine subtypes and to evaluate potential mutations related to antiretroviral drug resistance among treated cats. Subtyping based on phylogenetic analysis was performed by the neighbor-joining and maximum likelihood methods. All of the sequences clustered with subtype B in the three regions, exhibiting low genetic variability. Additionally, we found evidence that the same virus is circulating in animals in close contact. The analysis of FIV RT sequences identified two new putative mutations related to drug resistance located in the RT “finger” domain, which has 60% identity to human immunodeficiency virus (HIV) sequence. Amino acid change K→R at codons 64 and 69 was found in 25% and 37.5% of the treated animals, respectively. These signatures were comparable to K65R and K70R thymidine-associated mutations found in the HIV-1 HXB2 counterpart. This finding strongly suggests a position correlation between the mutations found in FIV and the K65R and K70R substitutions from drug-resistant HIV-1 strains.

Genotypic and phenotypic characterization of human immunodeficiency virus type 1 isolates circulating in pregnant women from Mozambique.

This work evaluated HIV-1 subtypes from different geographic regions and phenotypic data from drug-naïve HIV-positive pregnant women from Mozambique. We analyzed 75 pol sequences from patients and the distribution of the subtypes in three regions of Mozambique and found that the majority of samples analyzed clustered with subtype C. In the northern region, multiple variants were found 5 (~18%) subtype A, 3 (~11%) subtype D and 2 (~7.1%) mosaics (A/C/D and C/D), whereas 18 (64.3%) isolates were subtype C, from a total of 28 samples. Already in the southern region, only one (5%) isolate of 20 samples was subtype D, and the other 19 (95%) isolates were subtype C. All 27 (100%) isolates from the central region grouped within clade C. No primary resistance mutations to IP, NNRTI or NRTI were found. There was no evidence of phenotypic resistance in any of the isolates tested, suggesting that neither the polymorphism in the protease, nor the one found at codon 215 of the RT gene caused an increase in phenotypic resistance. This finding suggests that HAART regimens indicated by WHO will probably be successful in Mozambique.

Enfuvirtide (T-20) Resistance-Related Mutations in HIV Type 1 Subtypes B, C, and F Isolates from Brazilian Patients Failing HAART

The synthetic peptide T-20 (enfuvirtide, EFV) represents the first compound approved by the FDA known as entry inhibitors (EIs). The resistance mutations associated with this new class of antiretroviral drug are located in the first heptad repeat (HR1) region of gp41. Amino acid changes in codons G36D/S, I37V, V38A/M/E, Q39H/R, Q40H, N42T, and N43D can confer resistance to EFV. In this work we investigated the presence of resistance mutations that occur in patients never treated with EFV and failing HAART with protease inhibitors (PIs), nucleoside reverse transcriptase (RT) inhibitors (NRTIs), and nonnucleoside RT inhibitors (NNRTIs). This knowledge can reveal whether this salvage therapy can be effective in patients failing HAART. For this, we amplified 65 samples from plasma isolates and than sequenced a fragment of 416 nt encompassing the HR1 and HR2 regions (amino acids 33-170 of gp41). The subtype distribution among the 65 isolates was 45 (69.23%) subtype B, 9 (13.85%) subtype C, 7 (10.77%) subtype F1, and 4 (6.15%) mosaics B/F1, B/C, F1/C, and C/F1/B. We found a high prevalence (7.6%) of EFV-associated mutation G36D in this cohort of patients failing HAART therapy, five isolates from subtype B (11.11% within this group). In contrast, when 1079 sequences from drug-naive patients were analyzed, only one showed the G36D substitution. This finding indicates a strong association between the selected position G36D and HAART therapy (p < 0.0001). The isolates that possess these mutations can develop resistance to EFV more rapidly. Nevertheless, more information about the impact of these mutations in salvage therapy with EFV in patients failing HAART must still be obtained.

ELUCIDATION OF THE MOLECULAR MECHANISM DRIVING DUPLICATION OF THE HIV-1 PTAP LATE DOMAIN

ABSTRACT HIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6Gag), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6Gag significantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6Gag confers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy. IMPORTANCE Resistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease inhibitor (PI) therapy. However, the reason that these duplications arise and their consequences for virus replication remain to be established. In this study, we examined the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients.

Association of hepatitis C virus NS5B variants with resistance to new antiviral drugs among untreated patients

Mutations located in the 109-amino acid fragment of NS5B are typically associated with resistance to interferon (IFN) and ribavirin (RIB) and to new antiviral drugs. The prevalence of these mutations was examined in 69 drug-naïve individuals with hepatitis C virus (HCV) infections in Rio de Janeiro, Brazil. Mutations related to non-response to IFN/RIB were observed in all subtypes studied (1a, 1b, 2b, 3a and 4). The most common mutation was Q309R, present in all subtypes, except subtype 2b with frequency above 20%. D244N was detected only in subtype 3a and A333E was detected only in subtype 2b. We did not detect the S282T, S326G or T329I mutations in any of the samples analysed. Of note, the C316N mutation, previously related to a new non-nucleoside compound (HCV796 and AG-021541), was observed in only eight of 33 (24%) samples from subtype 1b. Site 316 was under positive selection in this HCV variant. Our data highlight the presence of previously described resistance mutations in HCV genotypes from drug-naïve patients.

Natural transmission of feline immunodeficiency virus from infected queen to kitten

BackgroundFeline immunodeficiency virus (FIV) is a naturally occurring lentivirus that infects cats. The primary mode of transmission occurs through bite wounds, and other routes are difficult to observe in nature.FindingsThe purpose of this study was to evaluate FIV transmission from queen to kitten in a colony of naturally infected stray cats. With this aim, a queen was monitored over a period of three years. A blood sample was taken to amplify and sequence gag, pol and env regions of the virus from the queen, two kittens and other cats from the colony.ConclusionPhylogenetic analysis showed evidence of queen to kitten transmission.

Genotypic Analysis of the gp41 HR1 Region From HIV-1 isolates From Enfuvirtide-Treated and Untreated Patients

Objective:To evaluate the polymorphisms and resistance mutations in gp41 HR1 region of HIV-1. Methods:The study included 28 HIV-positive patients undergoing enfuvirtide (ENF) treatment or not from Porto Alegre, Rio Grande do Sul state, and Rio de Janeiro, Rio de Janeiro state, between 2006 and 2009. Resistance mutations and polymorphisms of the gp41 HR1 region were detected using the genomic DNA of 12 ENF-untreated patients and 16 patients in ENF treatment, encompassing subtypes B, C, and F1. Sample subtypes were determined by neighbor-joining phylogenetic analysis with a Kimuras two-parameter correction. Results:A high prevalence of polymorphisms unrelated to resistance was observed. Among ENF-untreated patients, 16% showed mutations related with resistance. Among patients in ENF treatment, 50% had resistance-related mutations. Overall, 17% of all isolates showed the N42S polymorphism related to ENF hypersusceptibility. The presence of ENF resistance mutations in the group of treated patients reduced viral load. The V38A substitution was the most frequent among treatment-experienced patients followed by the G36D/E, N42D, and V38M substitutions. Conclusions:The V38A substitution in the gp41 HR region was the most common resistance mutation among ENF-treated patients and was associated with increased viral load.