Environmental Changes and the Impact on the Human Infections by Dengue, Chikungunya and Zika Viruses in Northern Brazil, 2010-2019.

Environmental changes are among the main factors that contribute to the emergence or re-emergence of viruses of public health importance. Here, we show the impact of environmental modifications on cases of infections by the dengue, chikungunya and Zika viruses in humans in the state of Tocantins, Brazil, between the years 2010 and 2019. We conducted a descriptive and principal component analysis (PCA) to explore the main trends in environmental modifications and in the cases of human infections caused by these arboviruses in Tocantins. Our analysis demonstrated that the occurrence of El Niño, deforestation in the Cerrado and maximum temperatures had correlations with the cases of infections by the Zika virus between 2014 and 2016. El Niño, followed by La Niña, a gradual increase in precipitation and the maximum temperature observed between 2015 and 2017 were shown to have contributed to the infections by the chikungunya virus. La Niña and precipitation were associated with infections by the dengue virus between 2010 and 2012 and El Niño contributed to the 2019 outbreak observed within the state. By PCA, deforestation, temperatures and El Niño were the most important variables related to cases of dengue in humans. We conclude from this analysis that environmental changes (deforestation and climate change) presented a strong influence on the human infections caused by the dengue, chikungunya and Zika viruses in Tocantins from 2010 to 2019.

Detection of human feces pecovirus in newly diagnosed HIV patients in Brazil.

Circular single stranded DNA viruses (CRESS DNA) encoding a homologous replication-associated protein (REP) have been identified in most of eukaryotic groups. It is not clear yet the role in human diseases or details of the life cycle of these viruses. Recently, much interest has been raised in the evolutionary history of CRESS DNA owing to the increasing number of new sequences obtained by Next-Generation Sequencing (NGS) in distinct host species. In this study we describe two full-length CRESS DNA genomes obtained of two newly diagnosed HIV patients from São Paulo State, Brazil. The initial BLASTx search indicated that both sequences (named SP-FFB/2020 and SP-MJMS/2020) are highly similar (98%) to a previous CRESS DNA sequence detected in human fecal sample from Peru in 2016 and designated as pecovirus (Peruvian stool-associated circo-like virus). This study reported for the first time the Human feces pecovirus in the feces of two newly diagnosed HIV patients in Brazil. Our comparative analysis showed that although pecoviruses in South America share an identical genome structure they diverge and form distinct clades. Thus, we suggest the circulation of different species of pecoviruses in Latin America. Nevertheless, further studies must be done to examine the pathogenicity of this virus.

Metagenomics applied to the detection of diarrhea viruses in humans: Systematic Review.

To analyze the application of the metagenomics method in the identification of viral infectious agents that lead to diarrhea outbreaks. This study is a systematic review, which looked for publications on the following platforms: PubMed, Scientific Electronic Library Online (SciELO), LILACS and CAPES periodicals, conducted according to the PRISMA methodology, investigating in the literary composition studies related to metagenomics applied in the identification of viral infectious agents, which lead to diarrhea in humans. 1198 publications were identified. Of these, after analyzes and exclusions at different stages, 18 studies remained, which directly corresponded to the theme. Diarrhea was presented as a universal health concern. Despite the emergence of vaccines, cases of diarrhea remain persistent in poor populations. In this context, metagenomics emerges as a primary tool in detecting enteric viruses and identifying new viruses, revolutionizing health diagnoses, knowledge of viral diversity, and health surveillance, contributing to the correct etiology of infectious agents that would never be identified by conventional methods. The 18 articles studied point to advances in research in viral metagenomics of diarrheal samples, contributing to the discernment of diarrhea outbreaks, and properly associating with their etiological agents, they are presented in an innovative way for studies on the understanding of viral diversity.

Serological and molecular epidemiology of the Dengue, Zika and Chikungunya viruses in a risk area in Brazil.

BACKGROUND: The co-circulation of types of arbovirus in areas where they are endemic increased the risk of outbreaks and limited the diagnostic methods available. Here, we analyze the epidemiological profile of DENV, CHIKV and ZIKV at the serological and molecular level in patients with suspected infection with these arboviruses in the city of Juazeiro do Norte, Ceará, Brazil. METHODS: In 2016, the Central Public Health Laboratory (LACEN) of Juazeiro do Norte received 182 plasma samples from patients who visited health facilities with symptoms compatible with arbovirus infection. The LACEN performed serological tests for detection of IgM/IgG to DENV and CHIKV. They then sent these samples to the Retrovirology Laboratory of the Federal University of São Paulo and Faculty of Medical of the ABC where molecular analyses to confirm the infection by DENV, ZIKV and CHIKV were performed. The prevalence of IgM/IgG antibodies and of infections confirmed by RT-qPCR were presented with 95% confidence interval. RESULTS: In serologic analysis, 125 samples were positive for antibodies against CHIKV and all were positive for antibodies against DENV. A higher prevalence of IgG against CHIKV (63.20% with 95% CI: 45.76-70.56) than against DENV (95.05% with 95% CI: 78.09-98.12) was observed. When the samples were submitted to analysis by RT-qPCR, we observed the following prevalence: mono-infection by ZIKV of 19.23% (95% CI: 14.29-34.82) patients, mono-infection by CHIKV of 3.84% (95% CI: 2.01-5.44) and co-infection with ZIKV and CHIKV of 1.09% (95% CI: 0.89-4.56). CONCLUSION: The serologic and molecular tests performed in this study were effective in analyzing the epidemiological profile of DENV, CHIKV and ZIKV in patients with suspected infection by these arboviruses in the city of Juazeiro do Norte, Ceará/Brazil.

Diversity of enteric and non-enteric human adenovirus strains in Brazil, 2006-2011.

During 2006-2011, 5035 fecal samples were tested by PCR for human adenovirus (HAdV) and sequenced. HAdV was detected in 198 cases (3.9%), with the highest rate in children ≤ 5 years. Enteric HAdVs were the most prevalent genotypes (78%; 146/187): HAdV-F41 (63.6%; 119/187), HAdV-F40 (12.3%; 23/187), HAdV-A12 (1.6%; 3/187) and HAdV-A31 (0.5%; 1/187). Non-enteric HAdVs were detected in 22% (41/187): HAdV-C1 (8.0%; 15/187), HAdV-C2 (6.9%; 13/187), HAdV-C5 (4.3%; 8/187), HAdV-D8 (1.3%; 2/187), HAdV-B21 (0.5%; 1/187), HAdV-B3 (0.5%; 1/187) and HAdV-C6 (0.5%; 1/187). This 6-year retrospective study points out a high diversity of HAdV types circulating in Brazil and highlights the need to carry out molecular epidemiological studies of HAdV among patients with acute diarrheal infection on a regular basis.

Nicotinamide activates latent HIV-1 ex vivo in ART suppressed individuals, revealing higher potency than the association of two methyltransferase inhibitors, chaetocin and BIX01294

ABSTRACT Background: Latent HIV-1 is a major hurdle in obtaining HIV-1 sustained virological remission (SVR). Here we explored histone deacetylation inhibition property of nicotinamide (NAM; n = 17) for the first time in comparison to a combination of methyltransferase inhibitors (MTIs; Chaetocin and BIX01294; n = 25) to reactivate latent HIV ex vivo in CD8-depleted PBMCs from antiretroviral treated aviremic individuals. Results: NAM reactivated HIV-1 from 13/17 (76.4%) samples compared to 20/25 (80.0%) using MTIs with mean viral load (VLs) of 4.32 and 3.22 log10 RNA copies/mL, respectively (p = 0.004). Mean purging time after NAM and MTIs stimulation was 5.1 and 6.75 days, respectively (p = 0.73). Viral purging in autologous cultures exhibited blunted HIV recovery with fluctuating VLs followed by a complete viral extinction when expanded in allogenic system. Electron microscopy from five supernatants revealed anomalous viral particles, with lack of complete viral genomes when characterized by ultradeep sequencing through metagenomics approach (n = 4). Conclusion: NAM alone was more potent HIV-1 activator than combination of MTIs, with potential of clinical use.

Detection and characterization of Ilheus and Iguape virus genomes in historical mosquito samples from Southern Brazil.

In Brazil, flaviviruses have caused massive outbreaks. Surveillance programs designed to monitor virus activity in vectors provides a system for mapping disease distribution and for identifying specific vector species for targeted control. The present study aimed to describe the detection, whole genome characterization and phylogenetic analysis of Ilheus virus (ILHV) and Iguape virus (IGUV) strains obtained from historical mosquito's samples. Twelve isolates of pooled mosquito specimens (inoculated in neonate mouse brain) collected in the state of São Paulo, Brazil, in 1993, 1994 and 1997 were investigated. Viral RNA was extracted and analyzed by qRT-PCR using Flavivirus genus-specific primers. Positive samples were sequenced and underwent phylogenetic analyses. Flavivirus was detected in 50% of the specimens. Positive samples were successfully Sanger sequenced. Three Anopholes cruzii pools collected in 1994 were positive for IGUV. One Culex sp. pool, one Anopheles triannulatus pool, and one Coquillettidia juxtamansonia pool, collected in 1994, were positive for ILHV. Metagenomic sequencing successfully characterize one ILHV and four IGUV full genomes, and revealed a high degree of homology between the Brazilian ILHV and IGUV strains and isolates available in GenBank. Phylogenetic analysis of partial ILHV NS5 gene revealed three distinct lineages (clades), an indication of genetic heterogeneity in strains circulating in Brazil. Nucleotide insertions and a high-level of nucleotide diversity were observed in the NS1 protein and capsid region of IGUV strains, respectively. Detection of ILHV and IGUV in mosquitoes from Southeastern Brazil confirms the historical circulation of these viruses in this area. Furthermore, this first evidence of ILHV in Anopheles triannulatus suggests the potential importance of Anopheles mosquitoes in the IGUV transmission cycle. Genomic and phylogenetic analysis of these viruses provided insights into their diversity and evolution, which are important for the emergence patterns of flaviviruses and their evolutionary trends in Brazil, an endemic country for several arbovirus. in In-depth studies of ILHV and IGUV including vector competence and molecular studies are needed to shed light on their epidemiology and potential risk of future emergence.

Nicotinamide activates latent HIV-1 ex vivo in ART suppressed individuals, revealing higher potency than the association of two methyltransferase inhibitors, chaetocin and BIX01294.

BACKGROUND: Latent HIV-1 is a major hurdle in obtaining HIV-1 sustained virological remission (SVR). Here we explored histone deacetylation inhibition property of nicotinamide (NAM; n=17) for the first time in comparison to a combination of methyltransferase inhibitors (MTIs; Chaetocin and BIX01294; n=25) to reactivate latent HIV ex vivo in CD8-depleted PBMCs from antiretroviral treated aviremic individuals. RESULTS: NAM reactivated HIV-1 from 13/17 (76.4%) samples compared to 20/25 (80.0%) using MTIs with mean viral load (VLs) of 4.32 and 3.22 log10 RNA copies/mL, respectively (p=0.004). Mean purging time after NAM and MTIs stimulation was 5.1 and 6.75 days, respectively (p=0.73). Viral purging in autologous cultures exhibited blunted HIV recovery with fluctuating VLs followed by a complete viral extinction when expanded in allogenic system. Electron microscopy from five supernatants revealed anomalous viral particles, with lack of complete viral genomes when characterized by ultradeep sequencing through metagenomics approach (n=4). CONCLUSION: NAM alone was more potent HIV-1 activator than combination of MTIs, with potential of clinical use.

First identification of mammalian orthoreovirus type 3 by gut virome analysis in diarrheic child in Brazil.

Diarrhea remains one of the most common causes of deaths in children. Although many studies have investigated the prevalence of enteric pathogens around the globe some diarrheal episodes remain unexplained. It is possible that some yet-unidentified viral agents could be related to these cases of gastroenteritis. By using viral metagenomics techniques, we screened 251 fecal samples of children between 0.5 to 2.5-year-old with acute diarrhea not associated with common pathogens. These children live in rural areas and have different levels of contact with animals such as pigs, cows and bats. Here we report a complete genome of one mammalian orthoreovirus (MRV) type 3, denoted TO-151/BR, detected in a female child in the state of Tocantins (north of Brazil). Brazilian TO-151/BR strain was classified as MRV-3 based on S1 phylogeny and was closely related to porcine Asian strains. Phylogenetic analyses showed that other segments were more similar to MRV-3s of different geographic locations and hosts, including human and bats, highlighting genome reassortment and lack of host-specific barriers. This is the first report of MRV-3 in South America and a hypothesis of a silent long-term circulation of this virus in Brazil has been raised.

The rare enterovirus c99 and echovirus 29 strains in Brazil: potential risks associated to silent circulation.

Human enteroviruses (EVs) are associated with a wide spectrum of human diseases. Here we report the complete genome sequences of one EV-C99 strain and one E29 strain obtained from children suffering from acute gastroenteritis, without symptoms of enteroviral syndromes. This is the first report of EV-C99 in South America, and the second E29 genome described worldwide. Continuous surveillance on EVs is vital to provide further understanding of the circulation of new or rare EV serotypes in the country. The present study also highlights the capacity of EVs to remain in silent circulation in populations.

Recombinant Strains of Human Parechovirus in Rural Areas in the North of Brazil.

We characterized the 24 nearly full-length genomes of human parechoviruses (PeV) from children in the north of Brazil. The initial phylogenetic analysis indicated that 17 strains belonged to genotype 1, 5 to genotype 4, and 1 to genotype 17. A more detailed analysis revealed a high frequency of recombinant strains (58%): A total of 14 of our PeV-As were chimeric, with four distinct recombination patterns identified. Five strains were composed of genotypes 1 and 5 (Rec1/5); five strains shared a complex mosaic pattern formed by genotypes 4, 5, and 17 (Rec4/17/5); two strains were composed of genotypes 1 and 17 (Rec1/17); and two strains were composed of genotype 1 and an undetermined strain (Rec1/und). Coalescent analysis based on the Vp1 gene, which is free of recombination, indicated that the recombinant strains most likely arose in this region approximately 30 years ago. They are present in high frequencies and are circulating in different small and isolated cities in the state of Tocantins. Further studies will be needed to establish whether the detected recombinant strains have been replacing parental strains or if they are co-circulating in distinct frequencies in Tocantins.

Human sapovirus GI.2 and GI.3 from children with acute gastroenteritis in northern Brazil.

Human sapoviruses (HSaV) are considered important causative agents of acute gastroenteritis in humans worldwide. However, knowledge of the genetic characteristics of the whole genome of HSaV in Brazil is limited. Here we report the complete genome sequences of six HSaVs GI.2 and two GI.3 strains obtained from children with acute gastroenteritis in the Northern region of Brazil. Next generation sequencing was used to obtain the full genome and molecular characterization of the genome was performed. Phylogenetic analysis of the genome was also performed. Only one complete HSaV GI.2 genome characterization in the country precedes that of the present study. This is the first complete genome sequence of genotype GI.3 in Brazil. The data obtained in this investigation can contribute to the augmentation of the database on the molecular diversity of HSaVs strains circulating in Brazil, and to the improvement of current typing protocols.

Epizootics due to Yellow Fever Virus in São Paulo State, Brazil: viral dissemination to new areas (2016-2017).

Beginning in late 2016 Brazil faced the worst outbreak of Yellow Fever in recent decades, mainly located in southeastern rural regions of the country. In the present study we characterize the Yellow Fever Virus (YFV) associated with this outbreak in São Paulo State, Brazil. Blood or tissues collected from 430 dead monkeys and 1030 pools containing a total of 5,518 mosquitoes were tested for YFV by quantitative RT-PCR, immunohistochemistry (IHC) and indirect immunofluorescence. A total of 67 monkeys were YFV-positive and 3 pools yielded YFV following culture in a C6/36 cell line. Analysis of five nearly full length genomes of YFV from collected samples was consistent with evidence that the virus associated with the São Paulo outbreak originated in Minas Gerais. The phylogenetic analysis also showed that strains involved in the 2016-2017 outbreak in distinct Brazilian states (i.e., Minas Gerais, Rio de Janeiro, Espirito Santo) intermingled in maximum-likelihood and Bayesian trees. Conversely, the strains detected in São Paulo formed a monophyletic cluster, suggesting that they were local-adapted. The finding of YFV by RT-PCR in five Callithrix monkeys who were all YFV-negative by histopathology or immunohistochemistry suggests that this YFV lineage circulating in Sao Paulo is associated with different outcomes in Callithrix when compared to other monkeys.

Discovery of Cucumis melo endornavirus by deep sequencing of human stool samples in Brazil.

The nearly complete genome sequences of two Cucumis melo endornavirus (CmEV) strains were obtained using deep sequencing while investigating fecal samples for the presence of gastroenteritis viruses. The Brazilian CmEV BRA/TO-23 (aa positions 116-5027) and BRA/TO-74 (aa positions 26-5057) strains were nearly identical to the reference CmEV CL-01 (USA) and SJ1 (South Korea) strains, showing 97% and 98% of nucleotide and amino acid identity, respectively. Endornaviruses are not known to be associated with human disease and their presence may simply reflect recent dietary consumption. Metagenomic analyses offered an opportunity to identify for the first time in Brazil a newly described endornavirus species.

Detection of RNA-Dependent RNA Polymerase of Hubei Reo-Like Virus 7 by Next-Generation Sequencing in Aedes aegypti and Culex quinquefasciatus Mosquitoes from Brazil.

Advancements in next-generation sequencing and bioinformatics have expanded our knowledge of the diversity of viruses (pathogens and non-pathogens) harbored by mosquitoes. Hubei reo-like virus 7 (HRLV 7) was recently detected by the virome analysis of fecal samples from migratory birds in Australia. We now report the detection of RNA-dependent RNA polymerase sequences of HRLV 7 in pools of Aedes aegypti and Culex quinquefasciatus mosquitoes species from the Brazilian Amazon forest. Phylogenetic inferences indicated that all HRLV 7 strains fall within the same independent clade. In addition, HRLV 7 shared a close ancestral lineage with the Dinovernavirus genus of the Reoviridae family. Our findings indicate that HRLV 7 is present in two species of mosquitoes.

The rare enterovirus c99 and echovirus 29 strains in Brazil: potential risks associated to silent circulation

Human enteroviruses (EVs) are associated with a wide spectrum of human diseases. Here we report the complete genome sequences of one EV-C99 strain and one E29 strain obtained from children suffering from acute gastroenteritis, without symptoms of enteroviral syndromes. This is the first report of EV-C99 in South America, and the second E29 genome described worldwide. Continuous surveillance on EVs is vital to provide further understanding of the circulation of new or rare EV serotypes in the country. The present study also highlights the capacity of EVs to remain in silent circulation in populations.

Human sapovirus GI. 2 and GI. 3 from children with acute gastroenteritis in northern Brazil

Human sapoviruses (HSaV) are considered important causative agents of acute gastroenteritis in humans worldwide. However, knowledge of the genetic characteristics of the whole genome of HSaV in Brazil is limited. Here we report the complete genome sequences of six HSaVs GI.2 and two GI.3 strains obtained from children with acute gastroenteritis in the Northern region of Brazil. Next generation sequencing was used to obtain the full genome and molecular characterization of the genome was performed. Phylogenetic analysis of the genome was also performed. Only one complete HSaV GI.2 genome characterization in the country precedes that of the present study. This is the first complete genome sequence of genotype GI.3 in Brazil. The data obtained in this investigation can contribute to the augmentation of the database on the molecular diversity of HSaVs strains circulating in Brazil, and to the improvement of current typing protocols.

A Novel Highly Divergent Strain of Cell Fusing Agent Virus (CFAV) in Mosquitoes from the Brazilian Amazon Region.

Classical insect-specific flaviviruses (cISFs) have been widely detected in different countries in the last decades. Here, we characterize the near full-length genomes of two cISFs detected in mosquitoes collected in the city of Macapá, state of Amapá, Amazon region of Brazil. A total of 105 pools of female mosquitos were analyzed by next-generation sequencing (NGS). Comparative genomics and phylogenetic analysis identified three strains of cell fusing agent virus (CFAV) and two of Culex flavivirus (CxFV). All sequences were obtained from pools of Culex sp., except for one sequence of CFAV detected in a pool of Aedes aegypti. Both CxFV strains are phylogenetically related to a strain isolated in 2012 in the Southeast region of Brazil. The CFAV strains are the first of this species to be identified in Brazil and one of them is highly divergent from other strains of CFAV that have been detected worldwide. In conclusion, CFAV and CxFV, circulate in mosquitoes in Brazil. One strain of CFAV is highly divergent from others previously described, suggesting that a novel strain of CFAV is present in this region.

Recombination Located over 2A-2B Junction Ribosome Frameshifting Region of Saffold Cardiovirus.

Here we report the nearly full-length genome of a recombinant Saffold virus strain (SAFV-BR-193) isolated from a child with acute gastroenteritis. Evolutionary analysis performed using all available near-full length Saffold picornavirus genomes showed that the breakpoint found in the Brazilian strain (SAFV-BR-193) is indeed a recombination hotspot. Notably, this hotspot is located just one nucleotide after the ribosomal frameshift GGUUUUU motif in the SAFV genome. Empirical studies will be necessary to determine if this motif also affects the binding affinity of RNA-dependent RNA-polymerase (RdRp) and therefore increases the changes of RdRp swap between molecules during the synthesis of viral genomes.

Near full length genome of a recombinant (E/D) cosavirus strain from a rural area in the central region of Brazil.

In the present article we report the nearly full length genome of a Cosavirus strain (BRTO-83) isolated from a child with acute gastroenteritis, and who is an inhabitant of a rural area in the central region of Brazil. The sample was previously screened and negative for both: common enteric viruses (i.e. rotavirus and norovirus), bacteria, endoparasites and helminthes. Evolutionary analysis and phylogenetic inferences indicated that the Brazilian BRTO-83 Cosavirus strain was a recombinant virus highly related to the E/D recombinant NG385 strain (Genbank JN867757), which was isolated in Nigeria from an acute flaccid paralysis patient. This is the first report of a recombinant E/D Cosavirus strain detected in Brazil, and the second genome described worldwide. Further surveillance and molecular studies are required to fully understand the epidemiology, distribution and evolution of the Cosavirus.