Gonzalo Bello

Zika virus in the Americas: Early epidemiological and genetic findings

Zika virus genomes from Brazil The Zika virus outbreak is a major cause for concern in Brazil, where it has been linked with increased reports of otherwise rare birth defects and neuropathology. In a phylogenetic analysis, Faria et al. infer a single introduction of Zika to the Americas and estimated the introduction date to be about May to December 2013—some 12 months earlier than the virus was reported. This timing correlates with major events in the Brazilian cultural calendar associated with increased traveler numbers from areas where Zika virus has been circulating. A correlation was also observed between incidences of microcephaly and week 17 of pregnancy. Science, this issue p. 345 Virus sequencing indicates that Zika arrived in Brazil during the middle of 2013, coincident with a surge in air travelers. Brazil has experienced an unprecedented epidemic of Zika virus (ZIKV), with ~30,000 cases reported to date. ZIKV was first detected in Brazil in May 2015, and cases of microcephaly potentially associated with ZIKV infection were identified in November 2015. We performed next-generation sequencing to generate seven Brazilian ZIKV genomes sampled from four self-limited cases, one blood donor, one fatal adult case, and one newborn with microcephaly and congenital malformations. Results of phylogenetic and molecular clock analyses show a single introduction of ZIKV into the Americas, which we estimated to have occurred between May and December 2013, more than 12 months before the detection of ZIKV in Brazil. The estimated date of origin coincides with an increase in air passengers to Brazil from ZIKV-endemic areas, as well as with reported outbreaks in the Pacific Islands. ZIKV genomes from Brazil are phylogenetically interspersed with those from other South American and Caribbean countries. Mapping mutations onto existing structural models revealed the context of viral amino acid changes present in the outbreak lineage; however, no shared amino acid changes were found among the three currently available virus genomes from microcephaly cases. Municipality-level incidence data indicate that reports of suspected microcephaly in Brazil best correlate with ZIKV incidence around week 17 of pregnancy, although this correlation does not demonstrate causation. Our genetic description and analysis of ZIKV isolates in Brazil provide a baseline for future studies of the evolution and molecular epidemiology of this emerging virus in the Americas.

Origin and evolutionary history of Hiv-1 subtype C in Brazil

Objective:To investigate the origin and to reconstruct the onset date of the HIV-1 subtype C epidemic in Brazil. Design:Three independent datasets of subtype C sequences isolated from HIV-1-positive patients from southern Brazil over a period of 15 years (1991–2006) were analyzed: 82 env V3 sequences (213nt), 40 env C2–C5 sequences (559nt), and 72 pol sequences (960nt). Methods:Brazilian sequences were compared with other subtype C reference strains from the database using basic local alignment search tool, phylogenetic analyses, and searching of specific amino acid signature patterns. Evolutionary parameters were estimated using a Bayesian coalescent-based method under either strict or relaxed molecular clock models. Results:HIV-1 subtype C sequences from Brazil and Burundi formed a monophyletic cluster at both env and pol regions and shared specific amino acid signatures in the protease region when compared with other viruses of the same subtype from around the world. All Brazilian strains arose as a monophyletic subcluster within the Burundi-Brazilian lineage, whereas isolates from Burundi appeared at the origin of the clade. Evolutionary analyses of both env and pol genomic regions indicate that the age of the most recent common ancestor of the Brazilian subtype C clade dates back to the early 1980s. Conclusion:The subtype C epidemic in the southern Brazilian region was initiated by the introduction of a single founder strain closely related to subtype C strains from Burundi. Our results suggest that this founder event probably took place around the early 1980s, roughly a decade before the previous estimates.

Phylogeography and evolutionary history of dengue virus type 3

In this study, we revisited the phylogeography of the three of major DENV-3 genotypes and estimated its rate of evolution, based on the analysis of the envelope (E) gene of 200 strains isolated from 31 different countries around the world over a time period of 50 years (1956-2006). Our phylogenetic analysis revealed a geographical subdivision of DENV-3 population in several country-specific clades. Migration patterns of the main DENV-3 genotypes showed that genotype I was mainly circumspect to the maritime portion of Southeast-Asia and South Pacific, genotype II stayed within continental areas in South-East Asia, while genotype III spread across Asia, East Africa and into the Americas. No evidence for rampant co-circulation of distinct genotypes in a single locality was found, suggesting that some factors, other than geographic proximity, may limit the continual dispersion and reintroduction of new DENV-3 variants. Estimates of the evolutionary rate revealed no significant differences among major DENV-3 genotypes. The mean evolutionary rate of DENV-3 in areas with long-term endemic transmissions (i.e., Indonesia and Thailand) was similar to that observed in the Americas, which have been experiencing a more recent dengue spread. We estimated the origin of DENV-3 virus around 1890, and the emergence of current diversity of main DENV-3 genotypes between the middle 1960s and the middle 1970s, coinciding with human population growth, urbanization, and massive human movement, and with the description of the first cases of DENV-3 hemorrhagic fever in Asia.

Evolutionary history of HIV-1 subtype B and F infections in Brazil

Objective:To reconstruct the onset date of the HIV-1 B and F epidemics in Brazil based on virus diversification over time. Design:We studied HIV-1 env V3 sequences (210nt) with a known sampling year isolated from HIV-1 positive patients from Brazil between 1989 and 1997: 101 subtype B sequences and 41 subtype F sequences. Methods:HIV-1 V3 env sequences were grouped by year of collection and the relationship between the sampling years of HIV-1 sequences and their genetic distance to the reconstructed common ancestor (intra-population divergence) or to other sequences from the same year (intra-population diversity) was examined by using linear regression analysis. Results:Regression analysis of nucleotide distances, revealed a highly significant positive correlation between sampling years of subtype B and F V3 sequences and their intra-population divergence (P < 0.001) or diversity (P < 0.0001). In both subtype populations, the divergence and diversity increased at a rate of 0.5 and 0.9% per year, respectively. Considering these evolutionary rates, we estimate the onset of the subtype B and F HIV-1 epidemics in Brazil during early 1970s and early 1980s, respectively. Conclusions:The consistent correlation between divergence and diversity of the V3 sequences with their sampling years indicates that the molecular clock is operational in the evolution of the HIV-1 in Brazils epidemic, and show that subtypes B and F are evolving at a similar rate over time. The dating results suggest a discontinuous introduction of these subtypes in the Brazilian population.

HIV-1 genetic diversity and drug resistance among treatment naïve patients from Southern Brazil: An association of HIV-1 subtypes with exposure categories

BACKGROUND The AIDS epidemic in Southern Brazil has unique features, showing co-circulation of HIV-1 subtypes C, B and recombinant forms. Florianópolis has the second highest AIDS incidence among Brazilian capitals, but limited information is available about HIV molecular epidemiology and prevalence of primary drug resistance. OBJECTIVES To investigate the molecular epidemiology of HIV-1 in Florianópolis and to describe the prevalence of primary HIV-1 drug resistance mutations (DMRs). STUDY DESIGN Epidemiological and clinical data from 82 untreated patients from Florianópolis (2008-2009) were analyzed. The HIV-1 subtype at envelope, protease, reverse transcriptase and integrase regions were determined by phylogenetic and bootscaning analyses and the drug resistance profile were analyzed at the Stanford HIV Drug Resistance Database. RESULTS The most frequent HIV-1 genetic form was subtype C (65.8%) followed by mosaics BC (18.3%), subtype B (13.4%), subtype F1 (1.2%) and BCF1 recombinant (1.2%). HIV-1 subtype C and BC recombinants were much more frequent in the heterosexual exposure category, whereas subtype B was more common in the MSM exposure category. DRMs were seen in 11% of the sequences, 2.4% of them were related to PI, 5% to NRTI, 3.6% to NNRTI and 1.2% was related to INTI. CONCLUSIONS The present study confirms the high prevalence of subtype C and BC recombinants in Santa Catarina State and revealed a significant difference in the subtype distribution among distinct virus exposure categories. This study also shows a relative high prevalence of protease/reverse transcriptase primary drug resistance mutations and corroborates the usefulness of the integrase inhibitors in southern Brazil.

Immune activation and antibody responses in non-progressing elite controller individuals infected with HIV-1

An extremely rare subset of patients infected with HIV‐1 designated as “non‐progressing elite controllers” appears to be able to maintain stable CD4+ T‐cell counts and a median plasma viremia below the detection limit of current ultrasensitive assays (<50–80 copies/ml of plasma) for >10 years in the absence of antiretroviral therapy. Lymphocyte subsets (CD4+, CD8+), immune activation markers (HLA‐DR+, CD38+, Beta‐2‐microglobulin), and HIV‐specific antibody responses were longitudinally examined in four non‐progressing elite controllers over more than 5 years. Two control groups of seronegative healthy individuals and untreated patients infected with HIV‐1 presenting detectable viremia were also included. None of the non‐progressing elite controllers displayed the high T‐cell activation levels generally seen in the seropositive individuals, keeping them within the normal range. Three non‐progressing elite controllers showed no significant immune system abnormalities when compared to seronegative individuals, displaying a low proportion of HIV‐1‐specific binding antibodies and low avidity index, similar to those observed for individuals infected recently with HIV‐1. One non‐progressing elite controller exhibited CD8+ T‐cell counts and β2‐M levels above normal ranges and developed a low but “mature” (high‐avidity) HIV‐1‐specific antibody response. Thus, the non‐progressing elite controllers are able to maintain normal T‐cell activation levels, which may contribute to prevent, or greatly reduce, the damage of the immune system typically induced by the HIV‐1 over time. They are, however, immunologically heterogeneous and very low levels of antigen exposure seem to occur in these patients, sufficient for sustaining a low, but detectable, HIV‐1‐specific immunity. J. Med. Virol. 81:1681–1690, 2009.

Temporal dynamics of norovirus GII.4 variants in Brazil between 2004 and 2012.

Noroviruses (NoVs) are the major cause of acute gastroenteritis outbreaks, and, despite a wide genetic diversity, genotype II.4 is the most prevalent strain worldwide. Mutations and homologous recombination have been proposed as mechanisms driving the epochal evolution of the GII.4, with the emergence of new variants in 1–3-year intervals causing global epidemics. There are no data reporting the dynamics of GII.4 variants along a specific period in Brazil. Therefore, to improve the understanding of the comportment of these variants in the country, the aim of this study was to evaluate the circulation of NoV GII.4 variants during a 9-year period in 3 out of 5 Brazilian regions. A total of 147 samples were sequenced, and a phylogenetic analysis of subdomain P2 demonstrated the circulation of six GII.4 variants, Asia_2003, Hunter_2004, Den Haag_2006b, Yerseke_2006a, New Orleans_2009, and Sydney_2012, during this period. The most prevalent variant was Den Haag_2006b, circulating in different Brazilian regions from 2006 to 2011. A Bayesian coalescent analysis was used to calculate the mean evolutionary rate of subdomain P2 as 7.3×10−3 (5.85×10−3–8.82×10−3) subst./site/year. These analyses also demonstrated that clade Den Haag_2006b experienced a rapid expansion in 2005 and another in 2008 after a period of decay. The evaluation of the temporal dynamics of NoV GII.4 in Brazil revealed a similar pattern, with few exceptions, to the worldwide observation. These data highlight the importance of surveillance for monitoring the emergence of new strains of NoV GII.4 and its impact on cases of acute gastroenteritis.

Epidemic history of Hepatitis C virus in Brazil

Hepatitis C virus (HCV) subtypes 1a, 1b and 3a are the most prevalent strains in Brazil, but very little is known about the epidemic history of these subtypes in the country. A total of 231 HCV NS5B gene sequences (subtype 1a=89, subtype 1b=56, and subtype 3a=86) isolated in Brazil between 1995 and 2007 were analyzed in the present study. Sequences (328-pb) were subjected to phylogenetic analyses and statistical tests of phylogenetic mixing by sampling location and risk group. Our results revealed important variations in the pattern of HCV transmission among subtypes. Transmission of subtype 1a was characterized by dissemination of one major Brazilian lineage with a random virus exchange between different geographical regions but not between IDU and non-IDU populations. Transmission of subtype 1b was characterized by concurrent dissemination of multiple HCV lineages with a restricted virus exchange between country regions and risk groups. Transmission of subtype 3a was characterized by simultaneous spreading of multiple HCV lineages and random phylogenetic mixing by risk group and sampling location. Epidemic histories of major subtypes 1a, 1b and 3a Brazilian clades were estimated using a Bayesian coalescent approach. Our results indicate that all major HCV Brazilian clades probably start to circulate in the country during the second half of the 20th century and displayed roughly similar epidemic histories characterized by an initial phase of exponential expansion and by reduction of growth rates since 1980-1995. This suggests that the expansion of HCV may have been effectively controlled in Brazil.

Short Communication: Evidences of Recent Decline in the Expansion Rate of the HIV Type 1 Subtype C and CRF31_BC Epidemics in Southern Brazil

The HIV-1 epidemic in southern Brazil is characterized by the high prevalence of subtype C and CRF31_BC infections but little is known about the population dynamics of these strains over time. We used a total of 82 env and 72 pol HIV-1 subtype C sequences collected from 1991 to 2006 and 47 pol CRF31_BC sequences collected from 1998 to 2006 from Brazilian patients to reconstruct the demographic history of these HIV-1 strains. Estimations of demographic history were performed using a Bayesian Markov Chain Monte Carlo coalescent-based approach as implemented in the BEAST program. Our analyses indicate that subtype C and CRF31_BC epidemics experienced an initial period of fast exponential spread in the southern Brazilian population during the 1980s and early 1990s, but the spreading rate of these epidemics seems to have slowed down since the middle 1990s. The initial mean exponential growth rate of the subtype C epidemic was estimated to be around 0.70-0.90/year, whereas the estimated population growth rate of CRF31_BC was 1.3/year, more than two times higher than that previously described for this CRF. These results suggest for the first time that the growth rate of subtype C and CRF31_BC epidemics has been changing over time in southern Brazil with evidence for a deceleration in recent years. During the expansion phase, the CRF31_BC seems to have spread at a rate much higher than Brazilian parental subtypes B and C.

Phylodynamics of HIV-1 Subtype C Epidemic in East Africa

The HIV-1 subtype C accounts for an important fraction of HIV infections in east Africa, but little is known about the genetic characteristics and evolutionary history of this epidemic. Here we reconstruct the origin and spatiotemporal dynamics of the major HIV-1 subtype C clades circulating in east Africa. A large number (n = 1,981) of subtype C pol sequences were retrieved from public databases to explore relationships between strains from the east, southern and central African regions. Maximum-likelihood phylogenetic analysis of those sequences revealed that most (>70%) strains from east Africa segregated in a single regional-specific monophyletic group, here called CEA. A second major Ethiopian subtype C lineage and a large collection of minor Kenyan and Tanzanian subtype C clades of southern African origin were also detected. A Bayesian coalescent-based method was then used to reconstruct evolutionary parameters and migration pathways of the CEA African lineage. This analysis indicates that the CEA clade most probably originated in Burundi around the early 1960s, and later spread to Ethiopia, Kenya, Tanzania and Uganda, giving rise to major country-specific monophyletic sub-clusters between the early 1970s and early 1980s. The results presented here demonstrate that a substantial proportion of subtype C infections in east Africa resulted from dissemination of a single HIV local variant, probably originated in Burundi during the 1960s. Burundi was the most important hub of dissemination of that subtype C clade in east Africa, fueling the origin of new local epidemics in Ethiopia, Kenya, Tanzania and Uganda. Subtype C lineages of southern African origin have also been introduced in east Africa, but seem to have had a much more restricted spread.