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The HIV-1 epidemic in Brazil is predominantly characterized by subtype B, except in the southern states, where subtype C (HIV-1C) is more prevalent. Continuous monitoring of this profile is essential to maintain an accurate understanding of the molecular landscape of the HIV epidemic in Brazil. In this study, we isolated and sequenced seven new HIV-1C strains from the state of Bahia, located in the Northeast region of Brazil. To reconstruct the phylogenetic history of HIV-1C in the Northeast and investigate its connections with other regions of the country and globally, we first compiled a dataset of 3631 HIV-1C sequences from Brazil, Africa, and Europe. As expected, most of the new HIV-1C sequences from Bahia (n = 6) clustered within the well-known Brazilian clade. However, one sequence from Bahia clustered within the African clade, suggesting a possible new introduction of HIV-1C into Brazil. Furthermore, our findings indicate that the HIV-1C cases in Bahia likely originated from southern states, particularly Santa Catarina. This study provides valuable insights into the molecular profile of the HIV epidemic in Brazil, expanding our understanding of HIV-1C beyond the Southern region.

Doravirine (DOR) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) with efficacy against some NNRTI-resistant mutants. Although DOR resistance mutations are established for HIV-1 subtype B, it is less clear for non-B subtypes. This study investigated prevalent NNRTI resistance mutations on DOR susceptibility in HIV-1 subtype C. Prevalent drug resistance mutations were identified from a South African genotypic drug resistance testing database. Mutations, single or in combination, were introduced into replication-defective pseudoviruses and assessed for DOR susceptibility in vitro. The single V106M and Y188L mutations caused high-level resistance while others did not significantly impact DOR susceptibility. We observed an agreement between our in vitro and the Stanford HIVdb predicted susceptibilities. However, the F227L mutation was predicted to cause high-level DOR resistance but was susceptible in vitro. Combinations of mutations containing K103N, V106M or Y188L caused high-level resistance, in agreement with the predictions. These mutations are frequently observed in patients failing efavirenz- or nevirapine-based first-line regimens. However, they are also observed in those failing a protease inhibitor-based second-line regimen, as we have observed in our database. Genotypic drug resistance testing is therefore vital prior to the initiation of DOR-based treatment for those previously exposed to efavirenz or nevirapine.

HIV-1 subtype C is the most abundant strain of HIV-1 infections worldwide and was found in the first known patients diagnosed with HIV/AIDS in Bulgaria in 1986. However, there is limited information on the molecular-epidemiological characteristics of this strain in the epidemic of the country. In this study, we analyze the evolutionary history of the introduction and dissemination of HIV-1 subtype C in Bulgaria using global phylogenetic analysis, Bayesian coalescent-based approach, and molecular clock methods. All available samples with HIV-1 subtype C from individuals diagnosed with HIV/AIDS between 1986 and 2017 were analyzed. Men and women were equally represented, and 24.3% of patients reported being infected abroad. The global phylogenetic analysis indicated multiple introductions of HIV-1 subtype C from various countries of the world. The reconstruction of a Bayesian time-scaled phylogenies showed that several Bulgarian strains segregated together in clusters, while others were intermixed in larger clades containing strains isolated from both European and non-European countries. The time-scale of HIV-1 subtype C introductions in Bulgaria demonstrates the early introduction of these viruses in the country. Our in-depth phylogenetic and phylogeographic analyses are compatible with a scenario of multiple early introductions in the country followed by limited local distribution in the subsequent years. HIV-1 subtype C was introduced in the early years of the epidemic, originating from different countries of the world. Due to the comprehensive measures for prevention and control in the early years of the epidemic in Bulgaria, HIV-1 subtype C was not widely disseminated among the general population of the country.

Rhesus macaques can be readily infected with chimeric simian-human immunodeficiency viruses (SHIV) as a suitable virus challenge system for testing the efficacy of HIV vaccines. Three Chinese-origin rhesus macaques (ChRM) were inoculated intravenously (IV) with SHIVC109P4 in a rapid serial in vivo passage. SHIV recovered from the peripheral blood of the final ChRM was used to generate a ChRM-adapted virus challenge stock. This stock was titrated for the intrarectal route (IR) in 8 ChRMs using undiluted, 1:10 or 1:100 dilutions, to determine a suitable dose for use in future vaccine efficacy testing via repeated low-dose IR challenges. All 11 ChRMs were successfully infected, reaching similar median peak viraemias at 1–2 weeks post inoculation but undetectable levels by 8 weeks post inoculation. T-cell responses were detected in all animals and Tier 1 neutralizing antibodies (Nab) developed in 10 of 11 infected ChRMs. All ChRMs remained healthy and maintained normal CD4+ T cell counts. Sequence analyses showed >98% amino acid identity between the original inoculum and virus recovered at peak viraemia indicating only minimal changes in the env gene. Thus, while replication is limited over time, our adapted SHIV can be used to test for protection of virus acquisition in ChRMs.

Maturation inhibitors represent a new underdeveloped class of antiretroviral agents that block virus maturation by binding to the target of protease (PR)-Gag precursor (Pr55Gag). Development of a maturation inhibitor is based on a number of small molecules that are capable of blocking the cleavage event between p24-CA and spacer peptide 1. The bulk of the literature on HIV antiretroviral therapy and drug resistance has primarily been derived from HIV-1B, and relatively less is known about the context of HIV-1C that is responsible for more than 95% of HIV infections of India and half of these infections globally. We and others have shown that the presence of maturation inhibitor resistance mutations would make HIV-1B particles either less fit or dependent on these drugs to replicate. By contrast, it is unclear how HIV-1C would naturally acquire these mutations yet remain replication competent in the absence of the selective pressure of maturation inhibitors. Bevirimat, the first-in-class MI, was found to be inactive against HIV-C due to polymorphisms in the SP1 region. We have identified novel second generation of HIV maturation inhibitors with high potency against HIV clade C (IC50 values in the low nM range). The mutations identified during selection experiments revealed the putative binding pocket of these compounds on HIV-1 subtype C Gag. While working on the role of CA-C terminal domain (CA-CTD) domain in HIV-1 Gag assembly and release, we have found that core glycine-rich residues of the β-turn motif are crucial in Gag-membrane binding, multimerization, and assembly. Furthermore, we have identified a novel mutation in CA-CTD that is dependent on both classes of MIs. In conclusion, our studies provide insights into the mechanistic action of MIs on HIV-1 Gag processing and stabilization.

The HIV-1 subtype C has been substituting the subtype B population in southern Brazil. This phenomenon has been previously described in other countries, suggesting that subtype C may possess greater fitness than other subtypes. The HIV-1 long-terminal repeat (LTR) is an important regulatory region critical for the viral life cycle. Sequence insertions immediately upstream of the viral enhancer are known as the most frequent naturally occurring length polimorphisms (MFNLP). Previous reports demonstrated that the MFNLP could lead to the duplication of transcription factor binding sites (TFBS) enhancing the activity of the HIV-1 subtype C LTR. Here, we amplified and sequenced the LTR obtained from proviral DNA samples collected from patients infected with subtype C from the Southern Region of Brazil (naïve or treatment failure) and Mozambique (only naïve). We confirm the presence of different types of insertions in the LTR sequences of both the countries leading to the creation of additional TFBS. In the Brazilian clinical samples, the frequency of the sequence insertion was significantly higher in subjects experiencing treatment failure than in antiretroviral naïve patients.

The Human Immunodeficiency Virus type 1 (HIV-1) subtype C is currently the predominant subtype worldwide. Cell culture studies of Sub-Saharan African subtype C proviral plasmids are hampered by the low replication capacity of the resulting viruses, although viral loads in subtype C infected patients are as high as those from patients with subtype B. Here, we describe the sequencing and construction of a new HIV-1 subtype C proviral clone (pZAC), replicating more than one order of magnitude better than the previous subtype C plasmids. We identify the env-region for being the determinant for the higher viral titers and the pZAC Env to be M-tropic. This higher replication capacity does not lead to a higher cytotoxicity compared to previously described subtype C viruses. In addition, the pZAC Vpu is also shown to be able to down-regulate CD4, but fails to fully counteract CD317.