SARS-CoV-2 shedding, infectivity, and evolution in an immunocompromised adult patient.

This study aimed to provide further insight into the evolutionary dynamics of SARS-CoV-2 by analyzing the case of a 40-year-old man who had previously undergone autologous hematopoietic stem cell transplantation due to a diffuse large B-cell lymphoma. He developed a persistent SARS-CoV-2 infection lasting at least 218 days and did not manifest a humoral immune response to the virus during this follow-up period. Whole-genome sequencing and viral cultures confirmed a persistent infection with a replication-positive virus that had undergone genetic variation for at least 196 days after symptom onset.

Metagenomic of Liver Tissue Identified at Least Two Genera of Totivirus-like Viruses in Molossus molossus Bats.

The Totiviridae family of viruses has a unique genome consisting of double-stranded RNA with two open reading frames that encode the capsid protein (Cap) and the RNA-dependent RNA polymerase (RdRpol). Most virions in this family are isometric in shape, approximately 40 nm in diameter, and lack an envelope. There are five genera within this family, including Totivirus, Victorivirus, Giardiavirus, Leishmaniavirus, and Trichomonasvirus. While Totivirus and Victorivirus primarily infect fungi, Giardiavirus, Leishmaniavirus, and Trichomonasvirus infect diverse hosts, including protists, insects, and vertebrates. Recently, new totivirus-like species have been discovered in fish and plant hosts, and through metagenomic analysis, a novel totivirus-like virus (named Tianjin totivirus) has been isolated from bat guano. Interestingly, Tianjin totivirus causes cytopathic effects in insect cells but cannot grow in mammalian cells, suggesting that it infects insects consumed by insectivorous bats. In this study, we used next-generation sequencing and identified totivirus-like viruses in liver tissue from Molossus molossus bats in the Amazon region of Brazil. Comparative phylogenetic analysis based on the RNA-dependent RNA polymerase region revealed that the viruses identified in Molossus bats belong to two distinct phylogenetic clades, possibly comprising different genera within the Totiviridae family. Notably, the mean similarity between the Tianjin totivirus and the totiviruses identified in Molossus bats is less than 18%. These findings suggest that the diversity of totiviruses in bats is more extensive than previously recognized and highlight the potential for bats to serve as reservoirs for novel toti-like viruses.

Genetic characterization of the rare Bruconha virus (Bunyavirales: Orthobunyavirus) isolated in Vale do Ribeira (Atlantic Forest biome), Southeastern Brazil.

Brazil is a great source of arbovirus diversity, mainly in the Amazon region. However, other biomes, especially the Atlantic Forest, may also be a hotspot for emerging viruses, including Bunyaviruses (Negarnaviricota: Bunyavirales). For instance, Vale do Ribeira, located in the Southeastern region, has been widely studied for virus surveillance, where Flavivirus, Alphavirus and Bunyaviruses were isolated during the last decades, including Bruconha virus (BRCV), a member of Orthobunyavirus genus Group C, in 1976. Recently, a new isolate of BRCV named Span321532 was obtained from an adult sentinel mouse placed in Iguape city in 2011, and a full-length genome was generated with nucleotide differences ranging between 1.5%, 5.3% and 5% (L, M and S segments, respectively) from the prototype isolated 35 years earlier. In addition, each segment placed BRCV into different clusters, showing the high variety within Bunyavirales. Although no evidence for reassortants was detected, this finding reiterates the need for new surveillance and genomic studies in the area considering the high mutation rates of arbovirus, and also to identify the hosts capable of supporting the continuous circulation of Orthobunyavirus.

Genetic characterization of the rare Bruconha virus (Bunyavirales: Orthobunyavirus) isolated in Vale do Ribeira (Atlantic Forest biome), Southeastern Brazil

ABSTRACT Brazil is a great source of arbovirus diversity, mainly in the Amazon region. However, other biomes, especially the Atlantic Forest, may also be a hotspot for emerging viruses, including Bunyaviruses (Negarnaviricota: Bunyavirales). For instance, Vale do Ribeira, located in the Southeastern region, has been widely studied for virus surveillance, where Flavivirus, Alphavirus and Bunyaviruses were isolated during the last decades, including Bruconha virus (BRCV), a member of Orthobunyavirus genus Group C, in 1976. Recently, a new isolate of BRCV named Span321532 was obtained from an adult sentinel mouse placed in Iguape city in 2011, and a full-length genome was generated with nucleotide differences ranging between 1.5%, 5.3% and 5% (L, M and S segments, respectively) from the prototype isolated 35 years earlier. In addition, each segment placed BRCV into different clusters, showing the high variety within Bunyavirales. Although no evidence for reassortants was detected, this finding reiterates the need for new surveillance and genomic studies in the area considering the high mutation rates of arbovirus, and also to identify the hosts capable of supporting the continuous circulation of Orthobunyavirus.

Characterization of insect-specific Culex flavivirus (Flaviviridae) nucleotide sequences in mosquitoes from urban parks in São Paulo, Brazil.

BACKGROUND: Despite their worldwide occurrence, the distribution and role of insect-specific flaviviruses (ISFs) remain unclear. METHODS: We evaluated the presence of ISFs in mosquitoes collected in São Paulo, Brazil, using reverse transcription and semi-nested polymerase chain reaction (PCR). Some of the positive samples were subjected to nanopore sequencing. RESULTS: Twelve mosquito pools (2.8%) tested positive for flavivirus infection. Nanopore sequencing was successfully performed on six samples. Phylogenetic analysis grouped these sequences into genotype 2 of Culex flavivirus (CxFV). CONCLUSIONS: The identification of CxFV genotype 2 at new locations in São Paulo highlights the importance of understanding the role of ISFs in mosquito vector competence.

A New Circular Single-Stranded DNA Virus Related with Howler Monkey Associated Porprismacovirus 1 Detected in Children with Acute Gastroenteritis.

Putative replication-associated protein (REP) and capsid-like (CAP) proteins are encoded by circular single-stranded DNA viruses (CRESS DNA), which have been found in samples from most eukaryotic groups. However, the details of these viruses' life cycles and their significance in diseases have yet to be established. We presented and analyzed two full-length CRESS DNA genomes acquired from two children diagnosed with acute gastroenteritis (GI) in the northeast state of Tocantins, Brazil, using next-generation sequencing and a virus-like filtration approach. Both sequences (named SmaCV3BR08 and SmaCV3BR291) are closely similar to a prior CRESS DNA sequence discovered in the feces of a new world monkey (Alouatta caraya) from the United States in 2009 and termed Howler monkey-associated porprismacovirus 1 (Genbank ID: NC 026317). According to our comparative study, these porprismacovirus genomes deviate by 10% at the nucleotide level. For comparative reasons, the divergence between our sequences (SmaCV3BR08 and SmaCV3BR291) and a porprismacovirus recently identified in a human fecal sample from Peru is 37%. These data suggest that there is a great diversity of porprismacoviruses in South America, perhaps more than two species. In addition, the finding of closely related sequences of porprismacoviruses in humans and native monkeys highlights the zoonotic potential of these viruses.

Coxsackievirus A6 strains causing an outbreak of hand-foot-and-mouth disease in Northeastern Brazil in 2018.

Hand-foot-and-mouth disease (HFMD) is a highly contagious viral disease commonly associated to Enteroviruses (EV). During 2018, Brazil faced massive HFMD outbreaks spread across the country. This study aimed to characterize the EV responsible for the HFMD outbreak that occurred in Paraiba State, Brazilian Northeastern region, in 2018, followed by a phylogenetic analysis to detail information on its genetic diversity. A total of 49 serum samples (one from each patient) collected from children ≤ 15 years old, clinically diagnosed with HFMD were tested for EV using conventional RT-PCR and RT-qPCR. EV infection was confirmed in 71.4% (35/49) of samples. The mean and median ages were 1.83 years and one year old, respectively. Twenty-two EV-positive samples were successfully sequenced and classified as EV-A species; 13 samples were also identified with the CV-A6 genotype. The phylogenetic analysis (VP1 region) of three samples revealed that the detected CV-A6 strains belonged to sub-lineage D3. The CV-A6 strains detected here clustered with strains from South America, Europe and West Asia strains that were also involved in HFMD cases during the 2017-2018 seasons, in addition to the previously detected Brazilian CV-A6 strains from 2012 to 2017, suggesting a global co-circulation of a set of different CV-A6 strains introduced in the country at different times. The growing circulation of the emerging CV-A6 associated with HFMD, together with the detection of more severe cases worldwide, suggests the need for a more intense surveillance system of HFMD in Brazil. In addition, this investigation was performed exclusively on serum samples, and the analysis of whole blood samples should be considered and could have shown advantages when employed in the diagnosis of enteroviral HFMD outbreaks.

Characterization of insect-specific Culex flavivirus (Flaviviridae) nucleotide sequences in mosquitoes from urban parks in São Paulo, Brazil

ABSTRACT Background: Despite their worldwide occurrence, the distribution and role of insect-specific flaviviruses (ISFs) remain unclear. Methods: We evaluated the presence of ISFs in mosquitoes collected in São Paulo, Brazil, using reverse transcription and semi-nested polymerase chain reaction (PCR). Some of the positive samples were subjected to nanopore sequencing. Results: Twelve mosquito pools (2.8%) tested positive for flavivirus infection. Nanopore sequencing was successfully performed on six samples. Phylogenetic analysis grouped these sequences into genotype 2 of Culex flavivirus (CxFV). Conclusions: The identification of CxFV genotype 2 at new locations in São Paulo highlights the importance of understanding the role of ISFs in mosquito vector competence.

Coxsackievirus A6 strains causing an outbreak of hand-foot-and-mouth disease in Northeastern Brazil in 2018

ABSTRACT Hand-foot-and-mouth disease (HFMD) is a highly contagious viral disease commonly associated to Enteroviruses (EV). During 2018, Brazil faced massive HFMD outbreaks spread across the country. This study aimed to characterize the EV responsible for the HFMD outbreak that occurred in Paraiba State, Brazilian Northeastern region, in 2018, followed by a phylogenetic analysis to detail information on its genetic diversity. A total of 49 serum samples (one from each patient) collected from children ≤ 15 years old, clinically diagnosed with HFMD were tested for EV using conventional RT-PCR and RT-qPCR. EV infection was confirmed in 71.4% (35/49) of samples. The mean and median ages were 1.83 years and one year old, respectively. Twenty-two EV-positive samples were successfully sequenced and classified as EV-A species; 13 samples were also identified with the CV-A6 genotype. The phylogenetic analysis (VP1 region) of three samples revealed that the detected CV-A6 strains belonged to sub-lineage D3. The CV-A6 strains detected here clustered with strains from South America, Europe and West Asia strains that were also involved in HFMD cases during the 2017-2018 seasons, in addition to the previously detected Brazilian CV-A6 strains from 2012 to 2017, suggesting a global co-circulation of a set of different CV-A6 strains introduced in the country at different times. The growing circulation of the emerging CV-A6 associated with HFMD, together with the detection of more severe cases worldwide, suggests the need for a more intense surveillance system of HFMD in Brazil. In addition, this investigation was performed exclusively on serum samples, and the analysis of whole blood samples should be considered and could have shown advantages when employed in the diagnosis of enteroviral HFMD outbreaks.

New Variants of Squash Mosaic Viruses Detected in Human Fecal Samples.

Squash mosaic virus (SqMV) is a phytovirus that infects great diversity of plants worldwide. In Brazil, the SqMV has been identified in the states of Ceará, Maranhão, Piauí, Rio Grande do Norte, and Tocantins. The presence of non-pathogenic viruses in animals, such as phytoviruses, may not be completely risk-free. Similarities in gene repertories between these viruses and viruses that affect animal species have been reported. The present study describes the fully sequenced genomes of SqMV found in human feces, collected in Tocantins, and analyzes the viral profile by metagenomics in the context of diarrhea symptomatology. The complete SqMV genome was obtained in 39 of 253 analyzed samples (15.5%); 97.4% of them belonged to children under 5 years old. There was no evidence that the observed symptoms were related to the presence of SqMV. Of the different virus species detected in these fecal samples, at least 4 (rotavirus, sapovirus, norovirus, parechovirus) are widely known to cause gastrointestinal symptoms. The presence of SqMV nucleic acid in fecal samples is likely due to recent dietary consumption and it is not evidence of viral replication in the human intestinal cells. Identifying the presence of SqMV in human feces and characterization of its genome is a relevant precursor to determining whether and how plant viruses interact with host cells or microorganisms in the human gastrointestinal tract.

Genomic constellation of human Rotavirus A strains identified in Northern Brazil: a 6-year follow-up (2010-2016).

Surveillance of Rotavirus A (RVA) throughout the national territory is important to establish a more complete epidemiological-molecular scenario of this virus circulation in Brazil. The aim of the present study was to investigate the genetic diversity of RVA strains circulating in Tocantins State (Northern Brazil) during six years of post-vaccination follow-up (2010-2016). A total of 248 stool samples were screened by next generation sequencing and 107 (43.1%) nearly full length RVA genome sequences were obtained; one sample was co-infected with two RVA strains (G2/G8P[4]). Six G and P genotypes combinations were detected: G12P[8] strains (78.6%), as well as the G3P[8] (9.3%) and G1P[8] (0.9%) were associated with a Wa-like genogroup backbone. All G2P[4] (5.6%) and G8P[4] (2.8%) strains, including the mixed G2/G8P[4] infection (0.9%) showed the DS-1-like genetic background. The two G12P[4] strains (1.9%) were associated with distinct genetic backbones: Wa-like and DS-1-like. The phylogenetic analysis revealed the circulation of lineages G1-I, G2-IV, G3-III, G8-I and G12-III, and P[4]-V and P[8]-III of the VP7 and VP4 genes, respectively. Conserved clustering pattern and low genetic diversity were observed regarding VP1-VP3 and VP6, as well as NSP1-5 segments. We identified the same RVA circulation pattern reported in other Brazilian regions in the period of 2010-2016, suggesting that rural and low-income areas may not have a different RVA genotypic distribution compared to other parts of the country. The unique presentation of whole-genome data of RVA strains detected in the Tocantins State provides a baseline for monitoring variations in the genetic composition of RVA in this area.

Genomic Analyses of Potential Novel Recombinant Human Adenovirus C in Brazil.

Human Adenovirus species C (HAdV-C) is the most common etiologic agent of respiratory disease. In the present study, we characterized the nearly full-length genome of one potential new HAdV-C recombinant strain constituted by Penton and Fiber proteins belonging to type 89 and a chimeric Hexon protein of types 1 and 89. By using viral metagenomics techniques, we screened out, in the states of Tocantins and Pará, Northern and North regions of Brazil, from 2010 to 2016, 251 fecal samples of children between 0.5 to 2.5 years old. These children were presenting acute diarrhea not associated with common pathogens (i.e., rotavirus, norovirus). We identified two HAdV-C strains in two distinct patients. Phylogenetic analysis performed using all complete genomes available at GenBank database indicated that one strain (HAdV-C BR-245) belonged to type 1. The phylogenetic analysis also indicated that the second strain (HAdV-C BR-211) was located at the base of the clade formed by the newly HAdV-C strains type 89. Recombination analysis revealed that strain HAdV-C BR-211 is a chimera in which the variable regions of Hexon gene combined HAdV-C1 and HAdV-C89 sequences. Therefore, HAdV-C BR-211 strain possesses a genomic backbone of type HAdV-C89 and a unique insertion of HAdV-C1 in the Hexon sequence. Recombination may play an important driving force in HAdV-C diversity and evolution. Studies employing complete genomic sequencing on circulating HAdV-C strains in Brazil are needed to understand the clinical significance of the presented data.

Prevalence of dengue, Zika and chikungunya viruses in Aedes (Stegomyia) aegypti (Diptera: Culicidae) in a medium-sized city, Amazon, Brazil.

Aedes aegypti is associated with epidemic diseases in Brazil, such as urban yellow fever, dengue, and more recently, chikungunya and Zika viruses infections. More information about Ae. aegypti infestation is fundamental to virological surveillance in order to ensure the effectiveness of control measures in use. Thus, the present study aims to identify and compare infestation and infectivity of Ae. aegypti females in Macapa city, Amapa State (Amazon region), Brazil, between the epidemiological weeks 2017/02 and 2018/20. A total number of 303 Ae. aegypti females were collected at 21 fixed collection points, 171 at the 10 collection points in the Marabaixo neighborhood and 132 at the 11 collection points in the Central neighborhood. Among the collected samples, only two were positive for dengue virus, with a 2.08% (2/96 pools) infectivity rate for Marabaixo. The difference between the medians of Ae. aegypti females captured in Central and Marabaixo sites was not statistically significant. The findings indicate similar mosquito infestation levels between the neighborhoods, and a low-level of mosquito infectivity, although dengue virus was found only in Marabaixo. Virological surveillance of Ae. aegypti was important to identify sites of infection and determine possible routes of transmission to enable health surveillance teams to adopt preventive strategies where infected mosquitoes are present and act faster.

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.

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.

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.