Circulation of picobirnavirus in Neotropical free-ranging mammals.

Picobirnavirus (PBV) is a family of non-enveloped double-stranded RNA viruses with bisegmented genomes. Segment 1 encodes the capsid protein and segment 2 encodes RNA-dependent RNA polymerase. They exhibit high genomic heterogeneity and infect a wide range of vertebrate hosts, including humans. The objective of this study was to expand our knowledge of the circulation of PBV in free-living animals from two regions (Brazil and Argentina) of the Atlantic Forest. Fecal samples were analyzed from free-living animals: tapir, brocket deer, peccary, and different species of rodents and marsupials. A total of 133 samples were collected and analyzed by RT-PCR, of which 44 (33.08%) were PBV-positive. Nine amplicons were sequenced, five species from Argentina and four from Brazil, and phylogenetic analysis was performed. The nucleotide and amino acid identities of the PBV strains detected in animals from Argentina and Brazil were between 66.3% and 82.5% and between 55.3% and 74.2%, respectively. The analysed strains presented conserved nucleotide blocks without distinction of the host species. The phylogenetic tree showed that PBV strains from Atlantic Forest animals belonging to genogroup I were grouped into different clusters, without defining groups according to host species (human or animal) or the geographical area of detection. This is the first study on PBV in free-living animals in the Atlantic Forest. Our analysis suggested that PBV strains can infect different animal species, leading to PBV transmission between animals and humans. This reinforces the hypothesis of previous crossover points in the ecology and evolution of heterologous PBV strains.

Parallel evolution of picobirnaviruses from distinct ancestral origins.

IMPORTANCE: Picobirnaviruses (PBVs) are highly heterogeneous viruses encoding a capsid and RdRp. Detected in a wide variety of animals with and without disease, their association with gastrointestinal and respiratory infections, and consequently their public health importance, has rightly been questioned. Determining the "true" host of Picobirnavirus lies at the center of this debate, as evidence exists for them having both vertebrate and prokaryotic origins. Using integrated and time-stamped phylogenetic approaches, we show they are contemporaneous viruses descending from two different ancestors: avian Reovirus and fungal Partitivirus. The fungal PBV-R2 species emerged with a single segment (RdRp) until it acquired a capsid from vertebrate PBV-R1 and PBV-R3 species. Protein and RNA folding analyses revealed how the former came to resemble the latter over time. Thus, parallel evolution from disparate hosts has driven the adaptation and genetic diversification of the Picobirnaviridae family.

Abundant dsRNA picobirnaviruses show little geographic or host association in terrestrial systems.

Picobirnaviruses are double-stranded RNA viruses known from a wide range of host species and locations but with unknown pathogenicity and host relationships. Here, we examined the diversity of picobirnaviruses from cattle and gorillas within and around Bwindi Impenetrable Forest National Park (BIFNP), Uganda, where wild and domesticated animals and humans live in relatively close contact. We use metagenomic sequencing with bioinformatic analyses to examine genetic diversity. We compared our findings to global Picobirnavirus diversity using clustering-based analyses. Picobirnavirus diversity at Bwindi was high, with 14 near-complete RdRp and 15 capsid protein sequences, and 497 new partial viral sequences recovered from 44 gorilla samples and 664 from 16 cattle samples. Sequences were distributed throughout a phylogenetic tree of globally derived picobirnaviruses. The relationship with Picobirnavirus diversity and host taxonomy follows a similar pattern to the global dataset, generally lacking pattern with either host or geography.

Picobirnaviruses in animals: a review.

Picobirnaviruses (PBVs) are small non enveloped viruses with bi-segmented ds RNA. They have been observed in a wide variety of vertebrates, including mammals and birds with or without diarrhoea, as well as in sewage samples since its discovery (1988). The source of the viruses is uncertain. True hosts of PBVs and their role as primary pathogens or secondary opportunistic agents or innocuous viruses in the gut remains alien. The mechanisms by which they play a role in pathogenicity are still unclear based on the fact that they can be found in both symptomatic and asymptomatic cases. There is a need to determine their tropism since they have not only been associated with viral gastroenteritis but also been reported in the respiratory tracts of pigs. As zoonotic agents with diverse hosts, the importance of epidemiological and surveillance studies cannot be overstated. The segmented genome of PBV might pose a serious public health issue because of the possibility of continuous genetic reassortment. Aware of the growing attention being given to emerging RNA viruses, we reviewed the current knowledge on PBVs and described the current status of PBVs in animals.

The enigma of picobirnaviruses: viruses of animals, fungi, or bacteria?

Picobirnaviruses are small double-stranded RNA viruses first discovered in 1988 in stool samples from patients with diarrhea. It has generally been assumed that picobirnaviruses infect animal hosts and that they are potential agents of diarrhea, but there is still no direct evidence demonstrating that picobirnaviruses infect animals. In the metagenomic era, virome studies have broadened our understanding of picobirnavirus genetic diversity and genome organization, expanded the types of animals in which they have been detected, and identified novel associations with human disease. Most importantly, from the wealth of new sequencing data and comparative genomic analyses, a provocative new hypothesis has emerged that picobirnaviruses may not infect animals, but rather that they may infect evolutionarily simpler denizens of the gastrointestinal tract: bacteria and/or fungi. Depending on whether the true hosts of picobirnaviruses are animals, fungi, or bacteria, the mechanisms by which they impact animal biology will vary dramatically.

Molecular characterization of picobirnaviruses in small ruminants with diarrhea in Turkey.

Picobirnaviruses (PBVs), detected in a wide range of host species, are viruses of which limited information is available about their pathogenic potential, ecology, or evolutionary characteristics. In this study, a molecular analysis of segment 2 encoding the PBV RNA-dependent RNA-polymerase (RdRp) in small ruminants with diarrhea in Turkey was undertaken. A total of 66 fecal samples or gut contents from diarrheic small ruminants including 55 sheep and 11 goats were screened. Four samples (6.06%), obtained from sheep in different farms, yielded the expected amplicon size for the genogroup I RdRp gene fragment, whereas no positivity was detected for genogroup II PBVs. Phylogenetic analysis revealed high levels of genetic diversity among the genogroup I PBVs. Additionally, all PBV infected sheep were also positive for rotavirus A. This study, reporting the presence of the PBVs in sheep Turkey for the first time, contributes to the molecular characterization and epidemiology of PBVs.

Genetic diversity, frequency and concurrent infections of picobirnaviruses in diarrhoeic calves in Turkey.

Calf diarrhoea is one of the major problems in cattle farming with high morbidity and mortality in herds. Two enteric viruses, bovine rotavirus (BRV) and bovine coronavirus (BCoV), are the leading cause of gastroenteritis in young calves, whereas picobirnaviruses (PBVs) are often associated with diarrhoea. In the present study, the faecal specimens of 127 diarrhoeic bovines (less than 1-month-old) were employed to investigate the infection frequencies of these three pathogens. Results indicated that frequencies of BRV and BCoV in diarrhoeic calves were 38.58% and 29.92%, respectively. The 7.08% of bovine calf samples (9 out of 127) were found to be positive for PBV genogroup I. Sequence analysis further revealed the high genetic heterogeneity within representative PBV sequences. Additionally, both PBV-BCoV (n = 2) and BCoV-BRV-PBV (n = 1) co-infections were detected in bovine calves for the first time. Consequently, our findings pointed out the highly divergent nature of PBVs without regard to exact host or territory and the occasional co-existence with other enteric agents.

Detection and molecular characterization of picobirnaviruses in the wild birds: Identification of a novel picobirnavirus possessing yeast mitochondrial genetic code.

Picobirnaviruses (PBVs) are bi-segmented dsRNA viruses that have been detected in various animal species including vertebrates and invertebrates. In this study, 17 complete or incomplete PBV segment-2 and one unsegmented PBV-like virus sequence were identified in fecal samples from different bird species using viral metagenomic approach. The bird PBV and PBV-like virus retained the conservative motifs that are conserved in dsRNA2 of common PBVs. The RdRp of these 17 PBVs shared the highest Amino acid (aa) identity of 45.90%∼94.19% with previous animal and human PBVs, while the RdRp of the unsegment PBV-like virus shared the highest aa sequence identity of 31.93% with one chicken PBV (GenBank No. MW837829). The unsegmented PBV-like virus unexpectedly used the yeast mitochondrial genetic code (transl_table=3) for all ORFs translation. In addition, the prokaryotic RBS sequence was not only detected upstream to ORF2 at position 360AGGAGG365 of this unsegmented PBV-like virus, but also found upstream to ORF of bird PBV dsRNA2. The presence of the prokaryotic ribosomal binding site in the bird PBV genomes, and the finding of one novel unsegmented PBV-like virus using the yeast mitochondrial genetic code for translation supported recent speculations that PBVs may actually infect prokaryotic or fungal host cells. This study enhanced our understanding of PBVs and provided data support for exploring the real host of PBVs.

Emergence of a Distinct Picobirnavirus Genotype Circulating in Patients Hospitalized with Acute Respiratory Illness.

Picobirnaviruses (PBV) are found in a wide range of hosts and typically associated with gastrointestinal infections in immunocompromised individuals. Here, a divergent PBV genome was assembled from a patient hospitalized for acute respiratory illness (ARI) in Colombia. The RdRp protein branched with sequences previously reported in patients with ARI from Cambodia and China. Sputa from hospitalized individuals (n = 130) were screened by RT-qPCR which enabled detection and subsequent metagenomic characterization of 25 additional PBV infections circulating in Colombia and the US. Phylogenetic analysis of RdRp highlighted the emergence of two dominant lineages linked to the index case and Asian strains, which together clustered as a distinct genotype. Bayesian inference further established capsid and RdRp sequences as both significantly associated with ARI. Various respiratory-tropic pathogens were detected in PBV+ patients, yet no specific bacteria was common among them and four individuals lacked co-infections, suggesting PBV may not be a prokaryotic virus nor exclusively opportunistic, respectively. Competing models for the origin and transmission of this PBV genotype are presented that attempt to reconcile vectoring by a bacterial host with human pathogenicity. A high prevalence in patients with ARI, an ability to reassort, and demonstrated global spread indicate PBV warrant greater public health concern.

Ocurrence of rotavirus and picobirnavirus in wild and exotic avian from amazon forest.

The present study reports the occurrence of rotavirus A (RVA), rotavirus D (RVD), rotavirus F (RVF), rotavirus G (RVG), and picobirnavirus (PBV) in fecal specimens of wild (n = 22), and exotic birds (n = 1) from different cities of Pará state. These animals were hospitalized at Veterinary Hospital of the Federal University of Pará, Brazil, in a period from January 2018 to June 2019. The animals exhibited different clinical signs, such as diarrhea, malnutrition, dehydration, and fractures. The results showed 39.1% (9/23) of positivity for RVA by RT-qPCR. Among these, one sample (1/9) for the NSP3 gene of T2 genotype was characterized. About 88.9% (8/9) for the VP7 gene belonging to G1, G3 equine like and G6 genotypes, and 55.5% (5/9) for the VP4 gene of P[2] genotype were obtained. In the current study, approximately 4.5% of the samples (1/23) revealed coinfection for the RVA, RVD and RVF groups. Furthermore, picobirnavirus (PBV) was detected in one of the 23 samples tested, and was classified in the Genogroup I. The findings represent the first report of RVA, RVD, RVF, RVG, and PBV genotypes in wild birds in Brazil, and due to wide distribution it can implies potential impacts of RVs, and PBVs on avian health, and other animals contributing to construction of new knowledge, and care perspectives.

Understanding the Genetic Diversity of Picobirnavirus: A Classification Update Based on Phylogenetic and Pairwise Sequence Comparison Approaches.

Picobirnaviruses (PBVs) are small, double stranded RNA viruses with an ability to infect a myriad of hosts and possessing a high degree of genetic diversity. PBVs are currently classified into two genogroups based upon classification of a 200 nt sequence of RdRp. We demonstrate here that this phylogenetic marker is saturated, affected by homoplasy, and has high phylogenetic noise, resulting in 34% unsolved topologies. By contrast, full-length RdRp sequences provide reliable topologies that allow ancestralism of members to be correctly inferred. MAFFT alignment and maximum likelihood trees were established as the optimal methods to determine phylogenetic relationships, providing complete resolution of PBV RdRp and capsid taxa, each into three monophyletic groupings. Pairwise distance calculations revealed these lineages represent three species. For RdRp, the application of cutoffs determined by theoretical taxonomic distributions indicates that there are five genotypes in species 1, eight genotypes in species 2, and three genotypes in species 3. Capsids were also divided into three species, but sequences did not segregate into statistically supported subdivisions, indicating that diversity is lower than RdRp. We thus propose the adoption of a new nomenclature to indicate the species of each segment (e.g., PBV-C1R2).

Molecular Epidemiology and Characterization of Picobirnavirus in Wild Deer and Cattle from Australia: Evidence of Genogroup I and II in the Upper Respiratory Tract.

Picobirnaviruses (PBVs) have been detected in several species of animals worldwide; however, data pertaining to their presence in Australian wild and domestic animals are limited. Although PBVs are mostly found in faecal samples, their detection in blood and respiratory tract samples raises questions concerning their tropism and pathogenicity. We report here PBV detection in wild deer and cattle from southeastern Australia. Through metagenomics, the presence of PBV genogroups I (GI) and II (GII) were detected in deer serum and plasma. Molecular epidemiology studies targeting the partial RNA-dependent RNA polymerase gene were performed in a wide range of specimens (serum, faeces, spleen, lung, nasal swabs, and trachea) collected from wild deer and cattle, with PCR amplification obtained in all specimen types except lung and spleen. Our results reveal the predominance of GI and concomitant detection of both genogroups in wild deer and cattle. In concordance with other studies, the detected GI sequences displayed high genetic diversity, however in contrast, GII sequences clustered into three distinct clades. Detection of both genogroups in the upper respiratory tract (trachea and nasal swab) of deer in the present study gives more evidence about the respiratory tract tropism of PBV. Although much remains unknown about the epidemiology and tropism of PBVs, our study suggests a wide distribution of these viruses in southeastern Australia.

High Prevalence of Genogroup I and Genogroup II Picobirnaviruses in Dromedary Camels.

Picobirnaviruses (PBVs) are small non-enveloped bisegmented double-stranded RNA viruses found in humans, mammals, and birds. Increasing molecular epidemiology studies suggest a high sequence diversity of PBVs in numerous hosts and the environment. In this study, using 229 fecal samples from dromedary camels in Dubai, 52.8% were positive for PBVs, of which 77.7% and 41.3% were positive for genogroup I and II, respectively, and 19.0% were positive for both genotypes. Phylogenetic analysis showed high diversity among the sequences of genogroup I and II dromedary PBVs. Marked nucleotide polymorphisms were observed in 75.5% and 46.0% of genogroup I and II RNA-dependent RNA polymerase (RdRp) sequences, respectively, suggesting the co-existence of multiple strains in the same specimen. Both high genetic diversity and prevalence of genogroup I and II PBV in dromedaries were observed. In fact, the prevalence of genogroup II PBV in dromedaries is the highest among all animals to date. The complete/near-complete core genomes of five genogroup I and one genogroup II dromedary PBVs and partial segment 1 and 2 of both genotypes were also sequenced. The dromedary PBV genome organizations were similar to those of other animals. Genetic reassortment and mutation are both important in the ecology and evolution of PBVs.

Detection and partial molecular characterization of Picobirnavirus in swine from the state of Minas Gerais, Brazil.

Picobirnavirus (PBV) is a small two-segmented double-stranded RNA (dsRNA) virus that has been identified in diarrheic feces of a large range of animal hosts, including humans. For this reason, PBV has been recognized as an opportunistic agent of gastrointestinal disease. Even under these circumstances, there is a lack of studies regarding this pathogen. Not outstanding, in Brazil, the single description of the PBV occurrence in pigs was provided in the 1980s. Hence, this study aimed to verify the PBV occurrence in Brazilian swine farms and to perform molecular characterization of the identified strains. High genetic variability was found in the analyzed sequences. Further studies comprehending the infection of swine by PBV in Brazilian herds should be performed to provide more accurate information on its epidemiology and to discuss the role of the virus in gastrointestinal diseases.

Cryo-electron Microscopy Structure, Assembly, and Mechanics Show Morphogenesis and Evolution of Human Picobirnavirus.

Despite their diversity, most double-stranded-RNA (dsRNA) viruses share a specialized T=1 capsid built from dimers of a single protein that provides a platform for genome transcription and replication. This ubiquitous capsid remains structurally undisturbed throughout the viral cycle, isolating the genome to avoid triggering host defense mechanisms. Human picobirnavirus (hPBV) is a dsRNA virus frequently associated with gastroenteritis, although its pathogenicity is yet undefined. Here, we report the cryo-electron microscopy (cryo-EM) structure of hPBV at 2.6-Å resolution. The capsid protein (CP) is arranged in a single-shelled, ∼380-Å-diameter T=1 capsid with a rough outer surface similar to that of dsRNA mycoviruses. The hPBV capsid is built of 60 quasisymmetric CP dimers (A and B) stabilized by domain swapping, and only the CP-A N-terminal basic region interacts with the packaged nucleic acids. hPBV CP has an α-helical domain with a fold similar to that of fungal partitivirus CP, with many domain insertions in its C-terminal half. In contrast to dsRNA mycoviruses, hPBV has an extracellular life cycle phase like complex reoviruses, which indicates that its own CP probably participates in cell entry. Using an in vitro reversible assembly/disassembly system of hPBV, we isolated tetramers as possible assembly intermediates. We used atomic force microscopy to characterize the biophysical properties of hPBV capsids with different cargos (host nucleic acids or proteins) and found that the CP N-terminal segment not only is involved in nucleic acid interaction/packaging but also modulates the mechanical behavior of the capsid in conjunction with the cargo.IMPORTANCE Despite intensive study, human virus sampling is still sparse, especially for viruses that cause mild or asymptomatic disease. Human picobirnavirus (hPBV) is a double-stranded-RNA virus, broadly dispersed in the human population, but its pathogenicity is uncertain. Here, we report the hPBV structure derived from cryo-electron microscopy (cryo-EM) and reconstruction methods using three capsid protein variants (of different lengths and N-terminal amino acid compositions) that assemble as virus-like particles with distinct properties. The hPBV near-atomic structure reveals a quasisymmetric dimer as the structural subunit and tetramers as possible assembly intermediates that coassemble with nucleic acids. Our structural studies and atomic force microscopy analyses indicate that hPBV capsids are potentially excellent nanocages for gene therapy and targeted drug delivery in humans.

High detection rates and genetic diversity of picobirnaviruses (PBVs) in pigs on St. Kitts Island: Identification of a porcine PBV strain closely related to simian and human PBVs.

We report here high rates (75.38%, 49/65) of detection of genogroup I (GI) PBVs in diarrheic pigs on the Caribbean island of St. Kitts. High quality gene segment-2 sequences encoding a significant region (~350 amino acid (aa) residues) of the putative RNA-dependent RNA polymerase (RdRp) were obtained for 23 PBV strains. The porcine PBV strains from St. Kitts exhibited high genetic diversity among themselves (deduced aa identities of 56-100%) and with other PBVs (maximum deduced aa identities of 64-97%), and retained the three domains that are conserved in putative RdRps of PBVs. The nearly complete gene segment-2 sequence (full-length minus partial 3'- untranslated region) of a porcine PBV strain (strain PO36 from St. Kitts) that is closely related (deduced aa identities of 96-97%) to simian and human GI PBVs was determined using a combination of the non-specific primer-based amplification method and conventional RT-PCR. The complete putative RdRp sequence of strain PO36 preserved the various features that are maintained in PBVs from various species. For the first time, several co-circulating PBV strains from pigs were characterized for a significant region (~350 aa) of the putative RdRp, providing important insights into the genetic diversity of PBVs in a porcine population. Taken together, these observations corroborated growing evidence that PBVs can be highly prevalent and show limited correlation globally with host species or geography. This is the first report on detection of PBVs in pigs from the Caribbean region.

Molecular Detection and Characterization of Picobirnavirus in Environmental Water in Thailand.

BACKGROUND: Enteric viruses are responsible for waterborne and foodborne infections affecting a large number of people around the world. Picobirnavirus (PBV) is a highly versatile virus, detected in a wide range of hosts and has been reported to be associated with gastroenteritis in humans and animals. METHODS: Molecular screening of environmental water samples for PBV was performed over a period of two years from November 2016 to July 2018. The virus was detected by RT multiplex-PCR, nucleotide sequencing, and phylogenetic analysis. RESULTS: Out of 125 water samples, 1.6% (2 samples) tested positive for PBV. Nucleotide sequence analysis showed that both PBV strains detected in this study belonged to PBV genotype II and most closely related to the human PBV genotype II reference strains previously detected in China, the Netherlands, and the USA. CONCLUSIONS: This study reports the first detection of PBV genotype II in environmental water in Thailand. Our result highlights the need for better sanitation and disposal of waste water within this area.

Virome diversity analysis reveals novel enteroviruses and a human picobirnavirus in stool samples from African green monkeys with diarrhea.

It is important to identify viruses in animals because most infectious diseases in humans are caused by viruses of zoonotic origin. African green monkey is a widely used non-human primate model in biomedical investigations. In this study, total RNAs were extracted from stool samples of 10 African green monkeys with diarrhea. High-throughput sequencing was used to characterize viromes. PCR and Sanger sequencing were used to determine the full genome sequences. Great viral diversity was observed. The dominant viruses were enteroviruses and picobirnaviruses. Six enterovirus genomes and a picobirnavirus RNA-dependent RNA polymerase sequence were characterized. Five enteroviruses belonged to two putative new genotypes of species Enterovirus J. One enterovirus belonged to EV-A92. The picobirnavirus RNA-dependent RNA polymerase sequence had the highest nucleotide similarity (93.48%) with human picobirnavirus isolate GPBV6C2. The present study helped to identify the potential zoonotic viruses in African green monkeys. Further investigations are required to elucidate their pathogenic roles in animals and humans.

Detection and Molecular Characterization of Picobirnaviruses (PBVs) in the Mongoose: Identification of a Novel PBV Using an Alternative Genetic Code.

We report high rates of detection (35.36%, 29/82) of genogroup-I (GI) picobirnaviruses (PBVs) in non-diarrheic fecal samples from the small Indian mongoose (Urva auropunctata). In addition, we identified a novel PBV-like RNA-dependent RNA polymerase (RdRp) gene sequence that uses an alternative mitochondrial genetic code (that of mold or invertebrate) for translation. The complete/nearly complete gene segment-2/RdRp gene sequences of seven mongoose PBV GI strains and the novel PBV-like strain were obtained by combining a modified non-specific primer-based amplification method with conventional RT-PCRs, facilitated by the inclusion of a new primer targeting the 3'-untranslated region (UTR) of PBV gene segment-2. The mongoose PBV and PBV-like strains retained the various features that are conserved in gene segment-2/RdRps of other PBVs. However, high genetic diversity was observed among the mongoose PBVs within and between host species. This is the first report on detection of PBVs in the mongoose. Molecular characterization of the PBV and PBV-like strains from a new animal species provided important insights into the various features and complex diversity of PBV gene segment-2/putative RdRps. The presence of the prokaryotic ribosomal binding site in the mongoose PBV genomes, and analysis of the novel PBV-like RdRp gene sequence that uses an alternative mitochondrial genetic code (especially that of mold) for translation corroborated recent speculations that PBVs may actually infect prokaryotic or fungal host cells.

Novel Picobirnaviruses in Respiratory and Alimentary Tracts of Cattle and Monkeys with Large Intra- and Inter-Host Diversity.

Picobirnaviruses (PBVs) are mostly found in animal alimentary samples. In this study, among 576 respiratory specimens from 476 mammals and 100 chickens, genogroup I PBVs were detected in three cattle and three monkeys, and a genogroup II PBV-positive sample was collected from one cattle specimen. More than one PBV sequence type was observed in two and one genogroup I PBV-positive samples from cattle and monkeys, respectively. Twenty-four complete/near-complete segments 2 (nine from respiratory and 15 from alimentary samples) from the cattle and monkey genogroup I PBVs and one complete segment 2 from the cattle genogroup II PBV were sequenced. Similar to other studies, the cattle PBVs also showed a high diversity. In contrast, the monkey PBVs observed in this study were clustered into three distinct clades. Within each clade, all the sequences showed >99% amino acid identities. This unique phenomenon is probably due to the fact that monkeys in our locality reside in separated troops with minimal inter-troop contact.