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.

Characterization of Phietavirus Henu 2 in the virome of individuals with acute gastroenteritis.

There is a growing interest in phages as potential biotechnological tools in human health owing to the antibacterial activity of these viruses. In this study, we characterized a new member (named PhiV_005_BRA/2016) of the recently identified phage species Phietavirus Henu 2. PhiV_005_BRA/2016 was detected through metagenomic analysis of stool samples of individuals with acute gastroenteritis. PhiV_005_BRA/2016 contains double-stranded linear DNA (dsDNA), it has a genome of 43,513 base pairs (bp), with a high identity score (99%) with phage of the genus Phietavirus, species of Phietavirus Henu 2. Life style prediction indicated that PhiV_005_BRA/2016 is a lysogenic phage whose the main host is methicillin-resistant Staphylococcus aureus (MRSA). Indeed, we found PhiV_005_BRA/2016 partially integrated in the genome of distinct MRSA strains. Our findings highlights the importance of large-scale screening of bacteriophages to better understand the emergence of multi-drug resistant bacterial.

Novel Chaphamaparvovirus in Insectivorous Molossus molossus Bats, from the Brazilian Amazon Region.

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.

Aedes aegypti Totivirus identified in mosquitoes in the Brazilian Amazon region.

The totiviridae family contains viruses with double-stranded RNA genomes of 4.6-7.0 kpb, which encode a capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), and they are approximately 40 nm in diameter with icosahedral symmetry. Totiviruses were first isolated from mosquitoes collected in Shaanxi Province (China). Here, we report a new Aedes aegypti Totivirus (AaTV) identified in mosquitoes from the Amazon rainforest. Mosquitoes (Diptera: Culicidae) were collected from a forest reserve belonging to the Amazon forest in the city of Macapá, Amapá state, Northern Brazil. A viral sequence with a 5748 nucleotide length that was nearly identical to Aedes aegypti Totivirus (AaTV), here named Aedes aegypti Totivirus BR59AP, was detected. A detailed molecular analysis was performed and shows that AaTV-BR59AP is highly related to the AaTV strain from the Caribbean region. We emphasize the importance of the characterization of new viruses in mosquitoes to deepen our understanding of viral diversity in insects and their potential role in disease.

Expanding known viral diversity in plants: virome of 161 species alongside an ancient canal.

BACKGROUND: Since viral metagenomic approach was applied to discover plant viruses for the first time in 2006, many plant viruses had been identified from cultivated and non-cultivated plants. These previous researches exposed that the viral communities (virome) of plants have still largely uncharacterized. Here, we investigated the virome in 161 species belonging to 38 plant orders found in a riverside ecosystem. RESULTS: We identified 245 distinct plant-associated virus genomes (88 DNA and 157 RNA viruses) belonging to 27 known viral families, orders, or unclassified virus groups. Some viral genomes were sufficiently divergent to comprise new species, genera, families, or even orders. Some groups of viruses were detected that currently are only known to infect organisms other than plants. It indicates a wider host range for members of these clades than previously recognized theoretically. We cannot rule out that some viruses could be from plant contaminating organisms, although some methods were taken to get rid of them as much as possible. The same viral species could be found in different plants and co-infections were common. CONCLUSIONS: Our data describe a complex viral community within a single plant ecosystem and expand our understanding of plant-associated viral diversity and their possible host ranges.

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.

Chikungunya Virus Asian Lineage Infection in the Amazon Region Is Maintained by Asiatic and Caribbean-Introduced Variants.

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.

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.

Virome in the cloaca of wild and breeding birds revealed a diversity of significant viruses.

BACKGROUND: Wild birds may harbor and transmit viruses that are potentially pathogenic to humans, domestic animals, and other wildlife. RESULTS: Using the viral metagenomic approach, we investigated the virome of cloacal swab specimens collected from 3182 birds (the majority of them wild species) consisting of > 87 different species in 10 different orders within the Aves classes. The virus diversity in wild birds was higher than that in breeding birds. We acquired 707 viral genomes from 18 defined families and 4 unclassified virus groups, with 265 virus genomes sharing < 60% protein sequence identities with their best matches in GenBank comprising new virus families, genera, or species. RNA viruses containing the conserved RdRp domain with no phylogenetic affinity to currently defined virus families existed in different bird species. Genomes of the astrovirus, picornavirus, coronavirus, calicivirus, parvovirus, circovirus, retrovirus, and adenovirus families which include known avian pathogens were fully characterized. Putative cross-species transmissions were observed with viruses in wild birds showing > 95% amino acid sequence identity to previously reported viruses in domestic poultry. Genomic recombination was observed for some genomes showing discordant phylogenies based on structural and non-structural regions. Mapping the next-generation sequencing (NGS) data respectively against the 707 genomes revealed that these viruses showed distribution pattern differences among birds with different habitats (breeding or wild), orders, and sampling sites but no significant differences between birds with different behavioral features (migratory and resident). CONCLUSIONS: The existence of a highly diverse virome highlights the challenges in elucidating the evolution, etiology, and ecology of viruses in wild birds. Video Abstract.

Human-stool-associated tusavirus (Parvoviridae) in domestic goats and sheep.

In this study, genetic counterparts of the human-stool-associated tusavirus (subfamily Parvovirinae, family Parvoviridae) with >97% and 95-100% amino acid sequence identity in the parvoviral NS1 and VP1 protein were identified in faecal specimens from domestic goats (Capra hircus) and sheep (Ovis aries) in Hungary. Eleven (17.8%) of the 62 faecal specimens from goats and 12 (25.5%) of the 47 from sheep both from less than 12 months old animals were positive for tusavirus DNA by PCR, while none of the specimens collected from cattle and swine were positive. Thus, it cannot be ruled out that tusavirus infection in humans is of zoonotic origin.

A novel parvovirus (family Parvoviridae) in a freshwater fish, zander (Sander lucioperca).

In this study, a novel parvovirus (zander/M5/2015/HUN, OK236393) was detected in faecal specimens from a fish - zander or pikeperch (Sander lucioperca) - and genetically characterized using viral metagenomics and PCR methods. The NS1 and VP1 proteins of zander/M5/2015/HUN share <30% aa sequence identity, respectively, with the corresponding proteins of known members of the family Parvoviridae. Out of 62 faecal specimens collected from 13 freshwater fish species, three (4.8%) samples were positive by PCR for the novel parvovirus - all from zander. This is the second parvovirus detected in fish - after the disease-causing tilapia parvovirus of the subfamily Hamaparvovirinae - and it potentially represents a novel genus in the subfamily Parvovirinae.

Enteric Viruses Nucleic Acids Distribution along the Digestive Tract of Rhesus Macaques with Idiopathic Chronic Diarrhea.

Idiopathic chronic diarrhea (ICD) is a little understood common clinical problem in captive rhesus macaques claiming 33% of medical culls unrelated to research. The eukaryotic virome in digestive tract tissues collected at necropsy from nine animals with ICD was characterized using viral metagenomics. We compared the distribution of viral reads in tissues and mucosal scrapings from the stomach, duodenum, jejunum, ileum, and the proximal, transverse, and distal colons. In situ hybridization (ISH) using viral probes were performed on fixed tissues. Deep sequencing revealed multiple viruses in the Parvoviridae and Picornaviridae family. Tissues and mucosal scraping from the same locations showed closely related viral reads contents while different gut tissues from the same animal varied widely. ISH showed punctuated staining for both RNA and DNA viruses in the distal colon. Parvovirus staining was also detected in the stomach/duodenum/jejunum in distinct oval-shaped structures. The location of enteric viral nucleic acid differed widely between different viral families and along the length of the digestive tract.

Composition of Eukaryotic Viruses and Bacteriophages in Individuals with Acute Gastroenteritis.

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.

Idiopathic Chronic Diarrhea in Rhesus Macaques Is Not Associated with Enteric Viral Infections.

While recent changes in treatment have reduced the lethality of idiopathic chronic diarrhea (ICD), this condition remains one of the most common causes of rhesus macaque deaths in non-human primate research centers. We compared the viromes in fecal swabs from 52 animals with late stage ICD and 41 healthy animals. Viral metagenomics targeting virus-like particles was used to identify viruses fecally shed by each animal. Five viruses belonging to the Picornaviridae, one to the Caliciviridae, one to the Parvoviridae, and one to the Adenoviridae families were identified. The fraction of reads matching each viral species was then used to estimate and compare viral loads in ICD cases versus healthy controls. None of the viruses detected in fecal swabs were strongly associated with ICD.

Investigation of Three Newly Identified Equine Parvoviruses in Blood and Nasal Fluid Samples of Clinically Healthy Horses and Horses with Acute Onset of Respiratory Disease.

Three newly identified equine parvoviruses (equine parvovirus hepatitis (EqPV-H), equine parvovirus CSF (EqPV-CSF) and equine copivirus (Eqcopivirus)) have recently been discovered in horses with respiratory signs. However, the clinical impact of these three equine parvoviruses has yet to be determined. Nasal fluid samples and blood from 667 equids with acute onset of fever and respiratory signs submitted to a diagnostic laboratory were analyzed for the presence of common equine respiratory pathogens (equine influenza virus, equine herpesvirus-1/-4, equine rhinitis A and B virus, S. equi subspecies equi) as well as EqPV-H, EqPV-CSF and Eqcopivirus by qPCR. An additional 87 clinically healthy horses served as controls. One hundred and seventeen sick horses tested qPCR-positive for at least one of the three parvoviruses. Co-infections with common respiratory pathogens and parvoviruses were seen in 39 sick equids. All 87 clinically healthy horses tested qPCR-negative for all tested common respiratory pathogens and 10 healthy horses tested qPCR-positive for one of the equine parvoviruses. When the frequency of detection for EqPV-H, EqPV-CSF and Eqcopivirus of equids with respiratory signs was compared to that of clinically healthy horses, the difference was not statistically significant (p > 0.05), suggesting that the three recently identified equine parvoviruses do not contribute to the clinical picture of equids with respiratory disease.

New Parvoviruses and Picornavirus in Tissues and Feces of Foals with Interstitial Pneumonia.

Six foals with interstitial pneumonia of undetermined etiology from Southern California were analyzed by viral metagenomics. Spleen, lung, and colon content samples obtained during necropsy from each animal were pooled, and nucleic acids from virus-like particles enriched for deep sequencing. The recently described equine copiparvovirus named eqcopivirus, as well as three previously uncharacterized viruses, were identified. The complete ORFs genomes of two closely related protoparvoviruses, and of a bocaparvovirus, plus the partial genome of a picornavirus were assembled. The parvoviruses were classified as members of new ungulate protoparvovirus and bocaparvovirus species in the Parvoviridae family. The picornavirus was classified as a new species in the Salivirus genus of the Picornaviridae family. Spleen, lung, and colon content samples from each foal were then tested for these viral genomes by nested PCR and RT-PCR. When present, parvoviruses were detected in both feces and spleen. The picornavirus, protoparvovirus, and eqcopivirus genomes were detected in the lungs of one animal each. Three foals were co-infected with the picornavirus and either a protoparvovirus, bocaparvovirus, or eqcopivirus. Two other foals were infected with a protoparvovirus only. No viral infection was detected in one animal. The complete ORFs of the first equine protoparvoviruses and bocaparvovirus, the partial ORF of the third equine picornavirus, and their detection in tissues of foals with interstitial pneumonia are described here. Testing the involvement of these viruses in fatal interstitial pneumonia or other equine diseases will require larger epidemiological and/or inoculation studies.

Human astrovirus types 1, 4 and 5 circulating among children with acute gastroenteritis in a rural Brazilian state, 2010-2016.

This study combined conventional epidemiology of human astroviruses. From 2010 to 2016, 232 stool samples from children under 5 years of age were screened using NGS and conventional RT-PCR followed by genetic analysis in order to investigate the genotypic diversity of classical human astrovirus (HAstV) circulating in Tocantins State, Brazil. HAstV was detected in 16 cases (6.9%). Seven specimens (43.7%; 7/16) were positive according RT-PCR and next-generation sequencing (NGS) to investigate the molecular to both NGS and RT-PCR. NGS and RT-PCR individually revealed six (37.5%; 6/16) and three (18.8%; 3/16) additional positive samples, respectively. Sequencing of the HAstV-positive samples revealed HAstV-1a (9/16), HAstV-4c (3/16), and HAstV-5c (4/16) lineages.

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.

Novel picornavirus (family Picornaviridae) from freshwater fishes (Perca fluviatilis, Sander lucioperca, and Ameiurus melas) in Hungary.

In this study, a novel picornavirus (perchPV/M9/2015/HUN, GenBank accession no. MW590713) was detected in eight (12.9%) out of 62 faecal samples collected from three (Perca fluviatilis, Sander lucioperca, and Ameiurus melas) out of 13 freshwater fish species tested and genetically characterized using viral metagenomics and RT-PCR methods. The complete genome of perchPV/M9/2015/HUN is 7,741 nt long, excluding the poly(A) tail, and has the genome organization 5'UTRIRES-?/P1(VP0-VP3-VP1)/P2(2A1NPG↓P-2A2H-box/NC-2B-2C)/P3(3A-3BVPg-3CPro-3DPol)/3'UTR-poly(A). The P1, 2C, and 3CD proteins had 41.4%, 38.1%, and 47.3% amino acid sequence identity to the corresponding proteins of Wenling lepidotrigla picornavirus (MG600079), eel picornavirus (NC_022332), and Wenling pleuronectiformes picornavirus (MG600098), respectively, as the closest relatives in the genus Potamipivirus. PerchPV/M9/2015/HUN represents a potential novel fish-origin species in an unassigned genus in the family Picornaviridae.

Circovirus in Blood of a Febrile Horse with Hepatitis.

Circoviruses infect vertebrates where they can result in a wide range of disease signs or in asymptomatic infections. Using viral metagenomics we analyzed a pool of five sera from four healthy and one sick horse. Sequences from parvovirus-H, equus anellovirus, and distantly related to mammalian circoviruses were recognized. PCR identified the circovirus reads as originating from a pregnant mare with fever and hepatitis. That horse's serum was also positive by real time PCR for equine parvovirus H and negative for the flavivirus equine hepacivirus. The complete circular genome of equine circovirus 1 strain Charaf (EqCV1-Charaf) was completed using PCR and Sanger sequencing. EqCV1 replicase showed 73-74% identity to those of their closest relatives, pig circoviruses 1/2, and elk circovirus. The closest capsid proteins were from the same ungulate circoviruses with 62-63% identity. The overall nucleotide identity of 72% to its closest relative indicates that EqCV1 is a new species in the Circovirus genus, the first reported in genus Equus. Whether EqCV1 alone or in co-infections can result in disease and its prevalence in different equine populations will require further studies now facilitated using EqCV1's genome sequence.