Comprehensive Characterization of HIV-1 Molecular Epidemiology and Demographic History in the Brazilian Region Most Heavily Affected by AIDS.

UNLABELLED: The high incidence of AIDS cases and the dominance of HIV-1 subtype C infections are two features that distinguish the HIV-1 epidemic in the two southernmost Brazilian states (Rio Grande do Sul [RS] and Santa Catarina [SC]) from the epidemic in other parts of the country. Nevertheless, previous studies on HIV molecular epidemiology were conducted mainly in capital cities, and a more comprehensive understanding of factors driving this unique epidemic in Brazil is necessary. Blood samples were collected from individuals in 13 municipalities in the Brazilian southern region. HIV-1 env and pol genes were submitted to phylogenetic analyses for assignment of subtype, and viral population phylodynamics were reconstructed by applying Skygrid and logistic coalescent models in a Bayesian analysis. A high prevalence of subtype C was observed in all sampled locations; however, an increased frequency of recombinant strains was found in RS, with evidence for new circulating forms (CRFs). In the SC state, subtype B and C epidemics were associated with distinct exposure groups. Although logistic models estimated similar growth rates for HIV-1 subtype C (HIV-1C) and HIV-1B, a Skygrid plot reveals that the former epidemic has been expanding for a longer time. Our results highlight a consistent expansion of HIV-1C in south Brazil, and we also discuss how heterosexual and men who have sex with men (MSM) transmission chains might have impacted the current prevalence of HIV-1 subtypes in this region. IMPORTANCE: The AIDS epidemic in south Brazil is expanding rapidly, but the circumstances driving this condition are not well known. A high prevalence of HIV-1 subtype C was reported in the capital cities of this region, in contrast to the subtype B dominance in the rest of the country. This study sought to comparatively investigate the HIV-1 subtype B and C epidemics by sampling individuals from several cities in the two states with the highest AIDS incidences in Brazil. Our analyses showed distinct epidemic growth curves for the two epidemics, and we also found evidence suggesting that separate transmission chains may be impacting the viral phylodynamics and the emergence of new recombinant forms.

Contribution of Epidemiological Predictors in Unraveling the Phylogeographic History of HIV-1 Subtype C in Brazil.

UNLABELLED: The phylogeographic history of the Brazilian HIV-1 subtype C (HIV-1C) epidemic is still unclear. Previous studies have mainly focused on the capital cities of Brazilian federal states, and the fact that HIV-1C infections increase at a higher rate than subtype B infections in Brazil calls for a better understanding of the process of spatial spread. A comprehensive sequence data set sampled across 22 Brazilian locations was assembled and analyzed. A Bayesian phylogeographic generalized linear model approach was used to reconstruct the spatiotemporal history of HIV-1C in Brazil, considering several potential explanatory predictors of the viral diffusion process. Analyses were performed on several subsampled data sets in order to mitigate potential sample biases. We reveal a central role for the city of Porto Alegre, the capital of the southernmost state, in the Brazilian HIV-1C epidemic (HIV-1C_BR), and the northward expansion of HIV-1C_BR could be linked to source populations with higher HIV-1 burdens and larger proportions of HIV-1C infections. The results presented here bring new insights to the continuing discussion about the HIV-1C epidemic in Brazil and raise an alternative hypothesis for its spatiotemporal history. The current work also highlights how sampling bias can confound phylogeographic analyses and demonstrates the importance of incorporating external information to protect against this. IMPORTANCE: Subtype C is responsible for the largest HIV infection burden worldwide, but our understanding of its transmission dynamics remains incomplete. Brazil witnessed a relatively recent introduction of HIV-1C compared to HIV-1B, but it swiftly spread throughout the south, where it now circulates as the dominant variant. The northward spread has been comparatively slow, and HIV-1B still prevails in that region. While epidemiological data and viral genetic analyses have both independently shed light on the dynamics of spread in isolation, their combination has not yet been explored. Here, we complement publically available sequences and new genetic data from 13 cities with epidemiological data to reconstruct the history of HIV-1C spread in Brazil. The combined approach results in more robust reconstructions and can protect against sampling bias. We found evidence for an alternative view of the HIV-1C spatiotemporal history in Brazil that, contrary to previous explanations, integrates seamlessly with other observational data.

Association between cytokine gene polymorphisms and outcome of hepatitis B virus infection in southern Brazil.

A number of studies have demonstrated associations between cytokine gene polymorphisms and outcome of hepatitis B virus (HBV) infection. However, no general consensus has been reached, possibly due to differences between ethnic groups. In this study, 345 individuals living in southern Brazil, including 196 chronic HBV carriers and 149 subjects who had spontaneously recovered from acute infection, were enrolled to evaluate the influence of cytokine gene polymorphisms on the outcome of HBV infection. Most participants were of European descent. Genotyping of IL2-330 G/T, IL4-589C/T, IL6-174 G/C, IL10-592C/A, IL10-1082 A/G, IL17A-197 G/A, IL17A-692 T/C, TNF-α-238 G/A, and TNF-α-308 G/A single nucleotide polymorphisms was performed by using the minisequencing (single base extension) method. By multivariable analysis, a statistically significant association was found between genotypic profile AA + GA in TNF-α-308 and chronic HBV infection (OR, 1.82; 95%CI, 1.01-3.27; P = 0.046). In southern Brazil, the carriers of the -308A allele in the TNF-α gene promoter have a moderately higher risk of becoming chronic carriers in case of HBV infection. In addition, patients with chronic active hepatitis B (n = 60) exhibited a decreased frequency (3.3%) of the TNF-238A allele when compared to that (14.8%) found among asymptomatic HBV carriers (n = 136), suggesting that this could be a protective factor against liver injury (OR, 0.17; 95%CI, 0.04-0.076; P = 0.023). J. Med. Virol. 88:1759-1766, 2016. © 2016 Wiley Periodicals, Inc.

Co-circulation HIV-1 subtypes B, C, and CRF31_BC in a drug-naïve population from Southernmost Brazil: analysis of primary resistance mutations.

In Southernmost Brazil HIV-1 subtypes B, C, and CRF31_BC co-circulates and, since 1996 with the implementation of free access to highly active antiretroviral treatment (HAART), this epidemic is under a quite characteristic selective pressure. The profile of mutations and polymorphisms in the protease (PR) and reverse transcriptase (RT) genes of HIV-1 from untreated patients living in Porto Alegre, Southernmost Brazil were evaluated in order to identify the subtypes and circulating drug resistant genotypes. Blood samples from 99 HIV-1 positive drugs-naïve patients were collected from 2006 to 2007 in Porto Alegre, Brazil. HIV PR and RT genes were amplified, sequenced, and subtyped. The HIV-1 genotyping was performed by partial sequence analysis of the pol in the HIV Drug Resistance Database of Stanford University. Phylogenetic analyses allowed to classify the HIV samples according to their subtypes: B (26.2%), C (39.4%), F (1.1%), CRF31_CB (19.2%), and URF (14.1%). Eight (8.1%) samples showed primary resistance mutations according to the Calibrated Population Resistance tool based in the 2009 Surveillance Drug Resistance Mutation list. Two samples presented resistance mutations to PI, three NRTI and three NNRTI. There was no significant association between presence of resistant genotypes and subtypes, but resistance mutations seem to be less frequent in the subtype C. In addition, this study describes for the first time the mutational profile of CRF31_BC to PI, NRTI, and NNRTI. Genetic analyses of HIV-1 from naïve patients are a promising and important method for surveillance of HIV infection.

Increased IL-8 levels in HIV-infected individuals who initiated ART with CD4+ T cell counts <350 cells/mm3 - A potential hallmark of chronic inflammation.

The identification of inflammatory markers in HIV+ individuals on ART is fundamental since chronic ART-controlled HIV infection is linked to an increased inflammatory state. In this context, we assessed plasma levels of pro-inflammatory cytokines (IL-1ß, IL-8, and IL-12p70) of HIV+ individuals who initiated ART after immunosuppression (CD4+ T cell counts <350 cells/mm3). HIV+ individuals were stratified according to two extreme phenotypes: Slow Progressors (SPs; individuals with at least 8 years of infection before ART initiation) and Rapid Progressors (RPs; individuals who needed to initiate ART within 1-4 years after infection). A control group was composed of HIV-uninfected individuals. We found increased IL-8 levels (median: 5.13 pg/mL; SPs and RPs together) in HIV-infected individuals on ART as compared to controls (median: 3.2 pg/mL; p = 0.04), although no association with the progression profile (slow or rapid progressors) or CD4+ T cell counts at sampling was observed. This result indicates that IL-8 is a general marker of chronic inflammation in HIV+ individuals on ART, independently of CD4+ T cell counts at the beginning of the treatment or of the potential progression profile of the patient. In this sense, IL-8 may be considered a possible target for novel therapies focused on reducing inflammation in chronic HIV infection.

High CXCL10/IP-10 levels are a hallmark in the clinical evolution of the HIV infection.

The aim of this study was to investigate the modulation of plasma CXCL10, CCL20, CCL22, CCL2, CCL17 and CCL24 levels in HIV-positive patients grouped according to extreme phenotypes of progression to AIDS, and at different stages of HIV infection. HIV-positive individuals with extreme phenotypes of AIDS progression (n=58) at different clinical stages (chronic individuals, both pre-HAART and under-HAART) and HIV-negative controls (n=20) were evaluated. Additionally, HIV-positive individuals that initiated HAART with >350CD4+T-cells/mm3 were compared with those who initiated treatment with <350CD4+T-cells/mm3. Plasma levels of six chemokines were quantified by a Luminex assay. Higher CXCL10 levels were observed in individuals immediately before their CD4+T-cell levels were indicative for HAART (pre-HAART), independently of their progressor status, i.e. slow (SPs) or rapid progressors (RPs). SPs pre-HAART showed higher CXCL10 levels compared to elite controllers and RPs under HAART (pc=0.009 and pc=0.007, respectively). CXCL10 levels were higher in SPs HAART CD4<350 (initiated HAART with <350 CD4+T-cells) when compared with SPs HAART CD4>350 (initiated HAART with >350 CD4+T-cells) (1096 vs. 360.33pg/mL, p=0.0101). Normalisation of CXCL10 levels seems to depend on the CD4+T-cell nadir at HAART initiation. CCL20 levels were higher in chronic SPs, SPs pre-HAART, SPs HAART and RPs HAART compared with the HIV-negative controls, indicating persistent CCL20 expression. In conclusion, our results indicate that CXCL10 levels are a hallmark in the clinical evolution of HIV infection. However, our results must be verified in a study evaluating a larger number of AIDS progressors.

Endosomal toll-like receptor gene polymorphisms and susceptibility to HIV and HCV co-infection - Differential influence in individuals with distinct ethnic background.

The genetic background of human populations can influence the susceptibility and outcome of infection diseases. Toll-like receptors (TLRs) have been previously associated with susceptibility to human immunodeficiency virus (HIV) infection, disease progression and hepatitis C, virus (HCV) co-infection in different populations, although mostly in Europeans. In this study, we investigated the genetic role of endosomal TLRs on susceptibility to HIV infection and HCV co-infection through the analysis of TLR7 rs179008, TLR8 rs3764880, TLR9 rs5743836 and TLR9 rs352140 polymorphisms in 789 Brazilian individuals (374 HIV+ and 415 HIV-), taking into account their ethnic background. Amongst the 357 HIV+ individuals with available data concerning HCV infection, 98 were positive. In European descendants, the TLR9 rs5743836 C carriers displayed a higher susceptibility to HIV infection [dominant, Odds Ratio (OR)=1.53; 95% CI: 1.05-2.23; P=0.027]. In African descendants, TLR9 rs5743836 CT genotype was associated with protection to HIV infection (codominant, OR=0.51; 95% CI: 0.30-0.87; P=0.013). Also, the TLR9 rs352140 AA variant genotype was associated with susceptibility to HIV+/HCV+ co-infection in African descendants (recessive, OR=2.92; 95% CI: 1.22-6.98, P=0.016). These results are discussed in the context of the different ethnic background of the studied individuals highlighting the influence of this genetic/ethnic background on the susceptibility to HIV infection and HIV/HCV co-infection in Brazilian individuals.

Short-Term Dynamic and Local Epidemiological Trends in the South American HIV-1B Epidemic.

The human displacement and sexual behavior are the main factors driving the HIV-1 pandemic to the current profile. The intrinsic structure of the HIV transmission among different individuals has valuable importance for the understanding of the epidemic and for the public health response. The aim of this study was to characterize the HIV-1 subtype B (HIV-1B) epidemic in South America through the identification of transmission links and infer trends about geographical patterns and median time of transmission between individuals. Sequences of the protease and reverse transcriptase coding regions from 4,810 individuals were selected from GenBank. Maximum likelihood phylogenies were inferred and submitted to ClusterPicker to identify transmission links. Bayesian analyses were applied only for clusters including ≥5 dated samples in order to estimate the median maximum inter-transmission interval. This study analyzed sequences sampled from 12 South American countries, from individuals of different exposure categories, under different antiretroviral profiles, and from a wide period of time (1989-2013). Continentally, Brazil, Argentina and Venezuela were revealed important sites for the spread of HIV-1B among countries inside South America. Of note, from all the clusters identified about 70% of the HIV-1B infections are primarily occurring among individuals living in the same geographic region. In addition, these transmissions seem to occur early after the infection of an individual, taking in average 2.39 years (95% CI 1.48-3.30) to succeed. Homosexual/Bisexual individuals transmit the virus as quickly as almost half time of that estimated for the general population sampled here. Public health services can be broadly benefitted from this kind of information whether to focus on specific programs of response to the epidemic whether as guiding of prevention campaigns to specific risk groups.

Role of SEP15 Gene Polymorphisms in the Time of Progression to AIDS.

AIMS: HIV infection is a chronic disease that requires intensive treatment in its later phases, including dietary supplementation. Several studies have suggested clinical improvements in patients with high levels of selenium, linking these levels with a longer progression to AIDS. The objective of this study was to verify the association of two polymorphisms in the SEP15 gene, which encodes a selenoprotein that is responsible for the transport of selenium in cells, with the time of progression to AIDS in HIV-1-infected patients. METHODS: Blood samples were obtained from 139 HIV-1-positive individuals after they provided informed consent. DNA was isolated and genotyped using real-time polymerase chain reaction for the presence of SEP15 single nucleotide polymorphisms (rs5859 and rs561104). Questionnaires on sociodemographic features and behavior were answered, and the time of progression to AIDS was estimated based on a medical chart analysis. RESULTS: The allelic and genotypic frequencies did not differ between rapid and nonrapid progressors; however, the presence of the AA genotype of the rs5859 polymorphism was associated with a shorter time of progression to AIDS compared with GG homozygotes (hazard ratio = 3.62, 95% CI = 1.55-8.43, p = 0.003). CONCLUSION: These findings show the importance of genetic analysis of the SEP15 gene in individual patients with regard to predicting time of progression to AIDS.

New Insights about Treg and Th17 Cells in HIV Infection and Disease Progression.

Treg and Th17 cell subsets are characterized by the expression of specific transcriptional factors and chemokine receptor as well as by secretion of specific cytokine and chemokines. These subsets are important to the differentiation, expansion, homing capacity, and recruitment of several different immune cell populations to the site of infection. Whereas Treg cells maintain self-tolerance and control the activation and expansion of autoreactive CD4(+) T effector cells through an anti-inflammatory response, Th17 cells, in an exacerbated unregulated proinflammatory response, can promote autoimmunity. Despite such apparently opposite functions, Th17 and Treg cells share common characteristics, and their differentiation pathways are interconnected. Recent studies have revealed quite intricate relations between Treg and Th17 cells in HIV infection and progression to AIDS. Considering Treg cells, different subsets were already investigated in the context of HIV infection, indicating a fluctuation in the total number and frequency throughout the disease course. This review focuses on the recent findings regarding the role of regulatory T and Th17 cells in the context of HIV infection, highlighting the importance of the balance between these two subsets on disease progression.

Analysis of single-nucleotide polymorphisms in the APOBEC3H gene of domestic cats (Felis catus) and their association with the susceptibility to feline immunodeficiency virus and feline leukemia virus infections.

Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are widely distributed retroviruses that infect domestic cats (Felis catus). Restriction factors are proteins that have the ability to hamper retroviruses' replication and are part of the conserved mechanisms of anti-viral immunity of mammals. The APOBEC3 protein family is the most studied class of restriction factors; they are cytidine deaminases that generate hypermutations in provirus DNA during reverse transcription, thus causing hypermutations in the viral genome, hindering virus replication. One of the feline APOBEC3 genes, named APOBEC3H, encodes two proteins (APOBEC3H and APOBEC3CH). In other mammals, APOBEC3H single-nucleotide polymorphisms (SNPs) can alter the stability and cellular localization of the encoded protein, thus influencing its subcellular localization and reducing its anti-viral effect. In cats, the association of APOBEC3H SNPs with susceptibility to retroviral infections was not yet demonstrated. Therefore, this study aimed the investigation on the variability of APOBEC3H and the possible association with FIV/FeLV infections. DNA obtained from whole blood of fifty FIV- and/or FeLV-infected cats and fifty-nine FIV- and/or FeLV-uninfected cats were used as templates to amplify two different regions of the APOBEC3H, with subsequent sequencing and analysis. The first region was highly conserved among all samples, while in the second, six single-nucleotide variation points were identified. One of the SNPs, A65S (A65I), was significantly correlated with the susceptibility to FIV and/or FeLV infections. On the other hand, the haplotype analysis showed that the combination "GGGGCC" was positively correlated with the lack of FIV and/or FeLV infections. Our results indicate that, as previously shown in other mammals, variability of restriction factors may contribute to susceptibility of domestic cats to retroviral infections; however, these results should be confirmed by more extensive analysis and in vitro experiments.

Naturally occurring resistance mutations to HIV-1 entry inhibitors in subtypes B, C, and CRF31_BC.

BACKGROUND: Entry inhibitors are a class of antiretroviral (ARV) drugs that prevent HIV replication by blocking viral entry into the host cell. The investigation of naturally occurring mutations associated with entry inhibitors across subtypes is required because genetic differences between HIV-1 variants may influence the emergence of drug resistance. Despite the importance of subtype C, which predominates globally, the majority of studies include only subtype B strains. OBJECTIVES: To investigate the presence of natural resistance mutations to entry inhibitors in HIV-1 subtypes B, C, and CRF31_BC strains. STUDY DESIGN: Eighty samples were collected from antiretroviral-naïve patients. The gp41 gene from 67 patients and the gp120 gene from 65 patients were partially sequenced. Resistance mutations to entry inhibitors Enfuvirtide, Maraviroc, and Vicriviroc were screened. RESULTS: ENF resistance-associated mutations of HR1 and HR2 on gp41 were not associated with any subtype. However, the major polymorphisms detected in HR1: N42S, L54M, and A67T were most prevalent in subtype C (p<0.001). Mutations A316T and R315Q in gp120, which are related to MVC and VCV reduced susceptibility respectively, were predominant in subtype C (p<0.05). CONCLUSIONS: This study shows that many more resistance-associated mutations to entry inhibitors in ARV-naïve patients occur in subtype C compared with subtype B strains. However, further studies will be necessary to elucidate if the differential genetic background of HIV subtypes can affect the efficacy of treatment with entry inhibitors.

Reviewing the history of HIV-1: spread of subtype B in the Americas.

The dispersal of HIV-1 subtype B (HIV-1B) is a reflection of the movement of human populations in response to social, political, and geographical issues. The initial dissemination of HIV-1B outside Africa seems to have included the passive involvement of human populations from the Caribbean in spreading the virus to the United States. However, the exact pathways taken during the establishment of the pandemic in the Americas remain unclear. Here, we propose a geographical scenario for the dissemination of HIV-1B in the Americas, based on phylogenetic and genetic statistical analyses of 313 available sequences of the pol gene from 27 countries. Maximum likelihood and bayesian inference methods were used to explore the phylogenetic relationships between HIV-1B sequences, and molecular variance estimates were analyzed to infer the genetic structure of the viral population. We found that the initial dissemination and subsequent spread of subtype B in the Americas occurred via a single introduction event in the Caribbean around 1964 (1950-1967). Phylogenetic trees present evidence of several primary outbreaks in countries in South America, directly seeded by the Caribbean epidemic. Cuba is an exception insofar as its epidemic seems to have been introduced from South America. One clade comprising isolates from different countries emerged in the most-derived branches, reflecting the intense circulation of the virus throughout the American continents. Statistical analysis supports the genetic compartmentalization of the virus among the Americas, with a close relationship between the South American and Caribbean epidemics. These findings reflect the complex establishment of the HIV-1B pandemic and contribute to our understanding between the migration process of human populations and virus diffusion.