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Metagenomic studies of mosquito viromes demonstrated a more diverse composition than just an exclusive composition of pathogenic arboviruses transmitted to humans. In our study, the virome of 866 female mosquitoes collected throughout 2020 at the São Paulo Zoo, located in the city of São Paulo/SP—Brazil, was obtained. Specifically, in this paper, we describe a new virus found by viral RNA extraction and next-generation MiSeq sequencing of a group of 23 specimens of Anopheles (Nys.) strodei. The complete genome with a length of 9709 nucleotides was characterized by a positive orientation and a single strand, with a single large ORF, which encodes a polyprotein of 2987 amino acids. The phylogenetic analysis showed an association with the viral family Iflaviridae and the Riboviria realm. We carried out comparisons with translated sequences of the capsid regions of other iflavirus, and the identities in relation to our sequence were below the minimum limit of 90%, indicating that possibly it is a new species of iflavirus. Our findings contribute to expanding knowledge of virome composition among mosquito species in Brazil and globally. Moreover, we provide a viral genome reference specific to this geographic region and Culicidae family of mosquitoes. This resource facilitates future in silico recognition and assembly of viral genomes within metagenomic datasets.

CRESS-DNA encompasses a broad spectrum of viruses documented across diverse organisms such as animals, plants, diatoms, fungi, and marine invertebrates. Despite this prevalence, the full extent of these viruses’ impact on the environment and their respective hosts remains incompletely understood. Furthermore, an increasing number of viruses within this category lack detailed characterization. This investigation focuses on unveiling and characterizing viruses affiliated with the Genomoviridae family identified in liver samples from the bat Molossus molossus. Leveraging viral metagenomics, we identified seven sequences (MmGmV-PA) featuring a circular DNA genome housing two ORFs encoding replication-associated protein (Rep) and capsid protein (Cap). Predictions based on conserved domains typical of the Genomoviridae family were established. Phylogenetic analysis revealed the segregation of these sequences into two clades aligning with the genera Gemycirculavirus (MmGmV-06-PA and MmGmV-07-PA) and Gemykibivirus (MmGmV-01-PA, MmGmV-02-PA, MmGmV-03-PA, MmGmV-05-PA, and MmGmV-09-PA). At the species level, pairwise comparisons based on complete nucleotide sequences indicated the potential existence of three novel species. In summary, our study significantly contributes to an enhanced understanding of the diversity of Genomoviridae within bat samples, shedding light on previously undiscovered viral entities and their potential ecological implications.

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.

Abstract Classical insect-flaviviruses (cISFVs) and dual host-related insect-specific flavivirus (dISFV) are within the major group of insect-specific flavivirus. Remarkably dISFV are evolutionarily related to some of the pathogenic flavivirus, such as Zika and dengue viruses. The Evolutionary relatedness of dISFV to flavivirus allowed us to investigate the evolutionary principle of host adaptation. Additionally, dISFV can be used for the development of flavivirus vaccines and to explore underlying principles of mammalian pathogenicity. Here we describe the genetic characterization of a novel putative dISFV, termed Guapiaçu virus (GUAPV). Distinct strains of GUAPV were isolated from pools of Aedes terrens and Aedes scapularis mosquitoes. Additionally, we also detected viral GUAPV RNA in a plasma sample of an individual febrile from the Amazon region (North of Brazil). Although GUAPV did not replicate in tested mammalian cells, 3′UTR secondary structures duplication and codon usage index were similar to pathogenic flavivirus.

Chaphamaparvovirus (CHPV) is a recently characterized genus of the Parvoviridae family whose members can infect different hosts, including bats, which constitute the second most diverse order of mammals and are described worldwide as important transmitters of zoonotic diseases. In this study, we identified a new CHPV in bat samples from the municipality of Santarém (Pará state, North Brazil). A total of 18 Molossus molossus bats were analyzed using viral metagenomics. In five animals, we identified CHPVs. These CHPV sequences presented the genome with a size ranging from 3797 to 4284 bp. Phylogenetic analysis-based nucleotide and amino acid sequences of the VP1 and NS1 regions showed that all CHPV sequences are monophyletic. They are also closely related to CHPV sequences previously identified in bats in southern and southeast Brazil. According to the International Committee on Taxonomy of Viruses (ICTV) classification criteria for this species (the CHPV NS1 gene region must have 85% identity to be classified in the same species), our sequences are likely a new specie within the genus Chaphamaparvovirus, since they have less than 80% identity with other CHPV described earlier in bats. We also make some phylogenetic considerations about the interaction between CHPV and their host. We suggest a high level of specificity of CPHV and its hosts. Thus, the findings contribute to improving information about the viral diversity of parvoviruses and show the importance of better investigating bats, considering that they harbor a variety of viruses that may favor zoonotic events.

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.

The simultaneous transmission of two lineages of the chikungunya virus (CHIKV) was discovered after the pathogen’s initial arrival in Brazil. In Oiapoque (Amapá state, north Brazil), the Asian lineage (CHIKV-Asian) was discovered, while in Bahia state, the East-Central-South-African lineage (CHIKV-ECSA) was discovered (northeast Brazil). Since then, the CHIKV-Asian lineage has been restricted to the Amazon region (mostly in the state of Amapá), whereas the ECSA lineage has expanded across the country. Despite the fact that the Asian lineage was already present in the Amazon region, the ECSA lineage brought from the northeast caused a large outbreak in the Amazonian state of Roraima (north Brazil) in 2017. Here, CHIKV spread in the Amazon region was studied by a Zika–Dengue–Chikungunya PCR assay in 824 serum samples collected between 2013 and 2016 from individuals with symptoms of viral infection in the Amapá state. We found 11 samples positive for CHIKV-Asian, and, from these samples, we were able to retrieve 10 full-length viral genomes. A comprehensive phylogenetic study revealed that nine CHIKV sequences came from a local transmission cluster related to Caribbean strains, whereas one sequence was related to sequences from the Philippines. These findings imply that CHIKV spread in different ways in Roraima and Amapá, despite the fact that both states had similar climatic circumstances and mosquito vector frequencies.

Husavirus (HuV) is an unclassified virus of the order Picornavirales that has already been identified worldwide in various locations. The genetic, epidemiological, and pathogenic characteristics are, however, little understood. In children with acute gastroenteritis, this study used next-generation sequencing to recognize unknown sources of viruses. In particular, 251 fecal samples obtained from individuals were sequenced in southern, northeastern, and northern Brazil. all samples were also analyzed using culture methods and parasitological tests to classify other enteric pathogens such as bacteria, parasites, and viruses. 1.9% of the samples tested positive for HuV, for a total of 5 positive children, with a mean age of 2 year, with three males and two females. Detailed molecular characterization of full genomes showed that Brazilian HuVs' nucleotide divergence is less than 11%. The genetic gap between Brazilian sequences and the closest HuV reported previously, on the other hand, is 18%. The study showed that Brazilian sequences are closely related to the HuV defined in Viet Nam in 2013, further characterization based on phylogenetics. At least two divergent clades of HuV in South America were also seen in the phylogenetic study.

Protoparvoviruses, widespread among cats and wild animals, are responsible for leukopenia. Feline panleukopenia virus (FPLV) in domestic cats is genetically diverse and some strains may differ from those used for vaccination. The presence of FPLV in two domestic cats from Hebei Province in China was identified by polymerase chain reaction. Samples from these animals were used to isolate FPLV strains in CRFK cells for genome sequencing. Phylogenetic analysis was performed to compare our isolates with available sequences of FPLV, mink parvovirus (MEV) and canine parvovirus (CPV). The isolated strains were closely related to strains of FPLV/MEV isolated in the 1960s. Our analysis also revealed that the evolutionary history of FPLV and MEV is characterized by local adaptations in the Vp2 gene. Thus, it is likely that new FPLV strains are emerging to evade the anti-FPLV immune response.

To evaluate the impact of hypermutation on the HIV-1 dissemination at the population level we studied 7072 sequences HIV-1 gene vif retrieved from the public databank. From this dataset 854 sequences were selected because they had associated values of CD4+ T lymphocytes counts and viral loads and they were used to assess the correlation between clinical parameters and hypermutation. We found that the frequency of stop codons at sites 5, 11 and 79 ranged from 2.8x10-4 to 4.2x10-4. On the other hand, at codons 21, 38, 70, 89 and 174 the frequency of stop codons ranged from 1.4x10-3 to 2.5x10-3. We also found a correlation between clinical parameters and hypermutation where patients harboring proviruses with one or more stop codons at the tryptophan sites of the gene vif had higher CD4+ T lymphocytes counts and lower viral loads compared to the population. Our findings indicate that A3 activity potentially restrains HIV-1 replication because individuals with hypermutated proviruses tend to have lower numbers of RNA copies. However, owing to the low frequency of hypermutated sequences observed in the databank (44 out of 7072), it is unlikely that A3 has a significant impact to curb HIV-1 dissemination at the population level.

Metagenomics based on the next-generation sequencing (NGS) technique is a target-independent assay that enables the simultaneous detection and genomic characterization of all viruses present in a sample. There is a limited amount of data about the virome of individuals with gastroenteritis (GI). In this study, the enteric virome of 250 individuals (92% were children under 5 years old) with GI living in the northeastern and northern regions of Brazil was characterized. Fecal samples were subjected to NGS, and the metagenomic analysis of virus-like particles (VLPs) identified 11 viral DNA families and 12 viral RNA families. As expected, the highest percentage of viral sequences detected were those commonly associated with GI, including rotavirus, adenovirus, norovirus (94.8%, 82% and 71.2%, respectively). The most common co-occurrences, in a single individual, were the combinations of rotavirus-adenovirus, rotavirus-norovirus, and norovirus-adenovirus (78%, 69%, and 62%, respectively). In the same way, common fecal-emerging human viruses were also detected, such as parechovirus, bocaporvirus, cosavirus, picobirnavirus, cardiovirus, salivirus, and Aichivirus. In addition, viruses that infect plants, nematodes, fungi, protists, animals, and arthropods could be identified. A large number of unclassified viral contigs were also identified. We show that the metagenomics approach is a powerful and promising tool for the detection and characterization of different viruses in clinical GI 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.

Dengue virus (DENV) is a mosquito-borne viral pathogen that plagues many tropical-climate nations around the world, including Brazil. Molecular epidemiology is a growing and increasingly invaluable tool for understanding the dispersal, persistence, and diversity of this impactful virus. In this study, plasma samples (n = 824) from individuals with symptoms consistent with an arboviral febrile illness were analyzed to identity the molecular epidemiological dynamics of DENV circulating in the Brazilian state of Amapá. Twelve DENV type 1 (DENV-1) genomes were identified, which were phylogenetically related to the BR4 lineage of genotype V. Phylodynamics analysis suggested that DENV-1 BR-4 was introduced into Amapá around early 2010, possibly from other states in northern Brazil. We also found unique amino acids substitutions in the DENV-1 envelope and NS5 protein sequences in the Amapá isolates. Characterization of the DENV-1 BR-4 sequences highlights the potential of this new lineage to drive outbreaks of dengue in the Amazon region.

Echoviruses (E) are a diverse group of viruses responsible for various pathological conditions in humans including aseptic meningitis, myocarditis, and acute flaccid paralysis. The detection and identification of echovirus genotypes in clinical samples is challenging due to its high genetic diversity. Here, we report the complete genome sequences of nine echoviruses, obtained by next-generation sequencing of 238 fecal samples from individuals with gastroenteritis in regions of Brazil. Detected viruses were classified into six genotypes: Three E1 sequences (BRA/TO-028, BRA/TO-069 and BRA/TO-236), one E3 (BRA/TO-018), one E11 (BRA/TO-086), one E20 (BRA/TO-016), two E29 (BRA/TO-030 and BRA/TO-193), and one E30 sequence (BRA/TO-032). Phylogenetic analysis indicated that the echoviruses E1 and E29 circulating in Brazil are divergent from strains circulating worldwide. The genotype diversity identified in our study may under-represent the total echovirus diversity in Brazil because of the small sample size and the restricted geographical distribution covered by the survey.

Viruses are the most diverse and abundant microorganisms on earth, highly adaptive to a wide range of hosts. Viral diversity within invertebrate hosts has gained notoriety in recent years in public health as several such viruses have been of medical importance. Aedes aegypti serves as a vector for several viruses that have caused epidemics within the last year throughout Brazil; including Dengue, Zika and Chikungunya. This study aimed to identify new viral agents within Aedes aegypti mosquito in a city of the Amazonian region, where it is highly endemic. Metagenomic investigation was performed on 60 mosquito pools and viral RNA sequences present in their microbiota were characterized using genomic and phylogenetic tools. In total, we identified five putative novel virus species related to the Sobemovirus genus, Iflavirus genus and Permutatetraviridae family. These findings indicate a diverse taxonomy of viruses present in the mosquito microbiota of the Amazon, the region with the greatest invertebrate diversity in the world.

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.

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important known viral respiratory pathogens of both young and adult cattle. It is also named “heat stress in transport”, causing morbidity and mass death. New variants of BPIV3 have been detected or isolated in China since 2008. Here, we isolate one BPIV3 strain (named BPIV3 BJ) in Madin-Darby bovine kidney (MDBK) cells from nasal samples collected in China. Phylogenetic analysis showed that our isolate is related to BPIV3 of the genotype A. The comparison of BPIV3-BJ and the reference Chinese isolate NM09 showed that these strains are highly divergent. We found many differences in the amino acid composition in the nucleocapsid (NP) protein among these genotype A strains. Since the NP protein has been implicated in immunization studies, our BPIV3 isolate will be useful for the development of immune assays and vaccine studies. The diversity of BPIV3 lineages that we found in China indicated ongoing evolution for immune escape. Our study highlights the importance of genetic surveillance for determining the effect of BPIV3 variability on pathogen evolution and population-scale immunity.

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.

Enterovirus B73 is a new member of the Enterovirus B species. First detected in the USA, it has been subsequently identified in China, India, Oman, and the Netherlands. In this study, we characterize the first B73 strain (named TO-127) to be detected in South America. TO-127 was obtained from a child with acute gastroenteritis living in a rural area in Northern Brazil. The subject was not infected with any known enteric pathogens such as norovirus, rotavirus, helminths, or enteric bacteria. Analysis of the nearly full-length TO-127 genome (6993 nt) indicated a 74–75% nucleotide similarity with EV-B73 strains from other countries. Evolutionary analysis suggests that B73 is endemic and widespread.