Antonio Charlys da Costa

Characterization of Partial and Near Full-Length Genomes of HIV-1 Strains Sampled from Recently Infected Individuals in São Paulo, Brazil

Background Genetic variability is a major feature of human immunodeficiency virus type 1 (HIV-1) and is considered the key factor frustrating efforts to halt the HIV epidemic. A proper understanding of HIV-1 genomic diversity is a fundamental prerequisite for proper epidemiology, genetic diagnosis, and successful drugs and vaccines design. Here, we report on the partial and near full-length genomic (NFLG) variability of HIV-1 isolates from a well-characterized cohort of recently infected patients in São Paul, Brazil. Methodology HIV-1 proviral DNA was extracted from the peripheral blood mononuclear cells of 113 participants. The NFLG and partial fragments were determined by overlapping nested PCR and direct sequencing. The data were phylogenetically analyzed. Results Of the 113 samples (90.3% male; median age 31 years; 79.6% homosexual men) studied, 77 (68.1%) NFLGs and 32 (29.3%) partial fragments were successfully subtyped. Of the successfully subtyped sequences, 88 (80.7%) were subtype B sequences, 12 (11%) BF1 recombinants, 3 (2.8%) subtype C sequences, 2 (1.8%) BC recombinants and subclade F1 each, 1 (0.9%) CRF02 AG, and 1 (0.9%) CRF31 BC. Primary drug resistance mutations were observed in 14/101 (13.9%) of samples, with 5.9% being resistant to protease inhibitors and nucleoside reverse transcriptase inhibitors (NRTI) and 4.9% resistant to non-NRTIs. Predictions of viral tropism were determined for 86 individuals. X4 or X4 dual or mixed-tropic viruses (X4/DM) were seen in 26 (30.2%) of subjects. The proportion of X4 viruses in homosexuals was detected in 19/69 (27.5%). Conclusions Our results confirm the existence of various HIV-1 subtypes circulating in São Paulo, and indicate that subtype B account for the majority of infections. Antiretroviral (ARV) drug resistance is relatively common among recently infected patients. The proportion of X4 viruses in homosexuals was significantly higher than the proportion seen in other study populations.

A diverse group of small circular ssDNA viral genomes in human and non-human primate stools.

Viral metagenomics sequencing of fecal samples from outbreaks of acute gastroenteritis from the US revealed the presence of small circular ssDNA viral genomes encoding a replication initiator protein (Rep). Viral genomes were ∼2.5 kb in length, with bi-directionally oriented Rep and capsid (Cap) encoding genes and a stem loop structure downstream of Rep. Several genomes showed evidence of recombination. By digital screening of an in-house virome database (1.04 billion reads) using BLAST, we identified closely related sequences from cases of unexplained diarrhea in France. Deep sequencing and PCR detected such genomes in 7 of 25 US (28 percent) and 14 of 21 French outbreaks (67 percent). One of eighty-five sporadic diarrhea cases in the Gambia was positive by PCR. Twenty-two complete genomes were characterized showing that viruses from patients in the same outbreaks were closely related suggesting common origins. Similar genomes were also characterized from the stools of captive chimpanzees, a gorilla, a black howler monkey, and a lemur that were more diverse than the human stool-associated genomes. The name smacovirus is proposed for this monophyletic viral clade. Possible tropism include mammalian enteric cells or ingested food components such as infected plants. No evidence of viral amplification was found in immunodeficient mice orally inoculated with smacovirus-positive stool supernatants. A role for smacoviruses in diarrhea, if any, remains to be demonstrated.

Spread of Chikungunya Virus East/Central/South African Genotype in Northeast Brazil

We investigated an outbreak of exanthematous illness in Maceió by using molecular surveillance; 76% of samples tested positive for chikungunya virus. Genetic analysis of 23 newly generated genomes identified the East/Central/South African genotype, suggesting that this lineage has persisted since mid-2014 in Brazil and may spread in the Americas and beyond.

Lack of evidence to support the association of a single IL28B genotype SNP rs12979860 with the HTLV-1 clinical outcomes and proviral load

BackgroundThe Interleukin 28B (IL28B) rs12979860 polymorphisms was recently reported to be associated with the human T-cell leukemia virus type 1 (HTLV-1) proviral load (PvL) and the development of the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP).MethodsIn an attempt to examine this hypothesis, we assessed the association of the rs12979860 genotypes with HTLV-1 PvL levels and clinical status in 112 unrelated Brazilian subjects (81 HTLV-1 asymptomatic carriers, 24 individuals with HAM/TSP and 7 with Adult T cell Leukemia/Lymphoma (ATLL)).ResultsAll 112 samples were successfully genotyped and their PvLs compared. Neither the homozygote TT nor the heterozygote CT mutations nor the combination genotypes (TT/CT) were associated with a greater PvL. We also observed no significant difference in allele distribution between asymptomatic carriers and patients with HTLV-1 associated HAM/TSP.ConclusionsOur study failed to support the previously reported positive association between the IL28B rs12979860 polymorphisms and an increased risk of developing HAM/TSP in the Brazilian population.

A new protoparvovirus in human fecal samples and cutaneous T cell lymphomas (mycosis fungoides)

We genetically characterized seven nearly complete genomes in the protoparvovirus genus from the feces of children with diarrhea. The viruses, provisionally named cutaviruses (CutaV), varied by 1-6% nucleotides and shared ~76% and ~82% amino acid identity with the NS1 and VP1 of human bufaviruses, their closest relatives. Using PCR, cutavirus DNA was found in 1.6% (4/245) and 1% (1/100) of diarrhea samples from Brazil and Botswana respectively. In silico analysis of pre-existing metagenomics datasets then revealed closely related parvovirus genomes in skin biopsies from patients with epidermotropic cutaneous T-cell lymphoma (CTCL or mycosis fungoides). PCR of skin biopsies yielded cutavirus DNA in 4/17 CTCL, 0/10 skin carcinoma, and 0/21 normal or noncancerous skin biopsies. In situ hybridization of CTCL skin biopsies detected viral genome within rare individual cells in regions of neoplastic infiltrations. The influence of cutavirus infection on human enteric functions and possible oncolytic role in CTCL progression remain to be determined.

Epidemic establishment and cryptic transmission of Zika virus in Brazil and the Americas

Zika virus (ZIKV) transmission in the Americas was first confirmed in May 2015 in Northeast Brazil1. Brazil has the highest number of reported ZIKV cases worldwide (>200,000 by 24 Dec 20162) as well as the greatest number of cases associated with microcephaly and other birth defects (2,366 confirmed cases by 31 Dec 20162). Following the initial detection of ZIKV in Brazil, 47 countries and territories in the Americas have reported local ZIKV transmission, with 22 of these reporting ZIKV-associated severe disease3. Yet the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of such information for interpreting past trends in reported microcephaly. To address this we generated 53 complete or partial ZIKV genomes, mostly from Brazil, including data generated by the ZiBRA project – a mobile genomics lab that travelled across Northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Joint analyses of viral genomes with ecological and epidemiological data estimate that the ZIKV epidemic first became established in NE Brazil by March 2014 and likely disseminated from there, both nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates of the international spread of ZIKV from Brazil coincide with periods of high vector suitability in recipient regions and indicate the duration of pre-detection cryptic transmission in those regions. NE Brazil’s role in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the virus’ basic reproduction number. One Sentence Summary Virus genomes reveal the establishment of Zika virus in Northeast Brazil and the Americas, and provide an appropriate timeframe for baseline (pre-Zika) microcephaly in different regions.

Genomic and epidemiological characterisation of a dengue virus outbreak among blood donors in Brazil.

Outbreaks caused by Dengue, Zika and Chikungunya viruses can spread rapidly in immunologically naïve populations. By analysing 92 newly generated viral genome sequences from blood donors and recipients, we assess the dynamics of dengue virus serotype 4 during the 2012 outbreak in Rio de Janeiro. Phylogenetic analysis indicates that the outbreak was caused by genotype II, although two isolates of genotype I were also detected for the first time in Rio de Janeiro. Evolutionary analysis and modelling estimates are congruent, indicating a reproduction number above 1 between January and June, and at least two thirds of infections being unnoticed. Modelling analysis suggests that viral transmission started in early January, which is consistent with multiple introductions, most likely from the northern states of Brazil, and with an increase in within-country air travel to Rio de Janeiro. The combination of genetic and epidemiological data from blood donor banks may be useful to anticipate epidemic spread of arboviruses.

Virome analysis of antiretroviral-treated HIV patients shows no correlation between T-cell activation and anelloviruses levels.

BACKGROUND Abnormally high levels of T-cell activation can persist in HIV-infected subjects despite effective anti-retroviral therapy (ART) and has been associated with negative health outcomes. The nature of the antigenic drivers or other causes of this residual T-cell activation remain uncertain. Anelloviruses are universally acquired soon after birth, resulting in persistent viremia, and considered part of the commensal human virome. Reduced immunocompetence results in increased anellovirus levels. OBJECTIVES To test whether increased levels of anelloviruses or other viruses in plasma are associated with higher levels of persistent T-cell activation during ART. STUDY DESIGN Two amplification methods combined with next generation sequencing were used to detect all viruses and estimate relative anellovirus levels in plasma from 19 adults on effective ART who exhibited a wide range of T-cell activation levels. RESULTS Nucleic acids from HBV and HCV were detected in one patient each while pegivirus A (GBV-C) was found in three patients. Anellovirus DNA was detected in all patients with some individuals carrying up to eight different genotypes. Specific anellovirus genotypes or higher level of co-infections were not detected in subjects with higher levels of T-cell activation. No association was detected between relative plasma anellovirus DNA levels and the percentage of activated CD4 or CD8 T cells. CONCLUSIONS Human anelloviruses were detected in all HIV suppressed subjects, exhibited a wide range of viremia levels, and were genetically highly diverse. The level of persistent T-cell activation was not correlated with the level of viremia or genotypes present indicating that anellovirus antigens are unlikely to be a dominant source of antigens driving chronic T-cell activation.

Genetic variability and high proportion of HIV-1 BF1 recombinant strains among vertically infected children in São Paulo, Brazil

The enormous genetic variability of human immunodeficiency virus type 1 (HIV-1) continues to present a major challenge for vaccine design and frustrate efforts to halt the epidemic. A proper understanding of this phenomenon is a prerequisite for proper epidemiology, genetic diagnosis, and successful drugs and vaccines. In this study, we undertook a detailed molecular epidemiological investigation on HIV-1 vertically-infected children born from 1993 to 2008 in the state of Sao Paulo, Brazil.

Wuhan large pig roundworm virus identified in human feces in Brazil

We report here the complete genome sequence of a bipartite virus, herein denoted WLPRV/human/BRA/TO-34/201, from a sample collected in 2015 from a two-year-old child in Brazil presenting acute gastroenteritis. The virus has 98–99% identity (segments 2 and 1, respectively) with the Wuhan large pig roundworm virus (unclassified RNA virus) that was recently discovered in the stomachs of pigs from China. This is the first report of a Wuhan large pig roundworm virus detected in human specimens, and the second genome described worldwide. However, the generation of more sequence data and further functional studies are required to fully understand the ecology, epidemiology, and evolution of this new unclassified virus.