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).

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.

Re-emergence of porcine epidemic diarrhea virus in a piglet-producing farm in northwestern Germany in 2019.

BACKGROUND: Porcine epidemic diarrhea (PED) is a viral enteric disease of pigs. It affects all age classes of animals but lethality is mainly seen in suckling piglets. After its first appearance in England in 1971, Porcine epidemic diarrhea virus (PEDV) has spread worldwide. While sporadic outbreaks prevailed in Europe, the disease had high impact in Asia. Following particularly severe outbreaks in 2011, high impact cases were also reported in the United States and neighboring countries in 2013. Subsequently, outbreaks were also reported in several European countries including Germany. These outbreaks were less severe. This case report describes a recent case of PED re-emergence in Germany and the sequence analyses of the causative PEDV. CASE PRESENTATION: In spring 2019 5 years after re-introduction of PED into Central Europe, a piglet-producer in northwestern Germany experienced an outbreak that affected sows, their suckling piglets, and weaners. After initial confirmation of PEDV by real-time RT-PCR, fecal material and small intestine samples from affected pigs were subjected to metagenomic analyses employing next-generation sequencing. Phylogenetic analyses showed high identities among the PEDV sequences obtained from samples of different animals and a close relation to recent strains from Hungary and France. Compared to the PEDV strains analyzed in 2014, genetic drift could be confirmed. Changes were mainly observed in the spike protein encoding S gene segment. In addition, metagenomic analyses showed multiple Picobirnavirus reads in all investigated samples. CONCLUSION: This case report shows that PEDV is still circulating in Europe. The causative strains are moderately virulent and are still closely related to the so-called INDEL strains reported previously in Europe, including Germany. However, a genetic drift has taken place that can be seen in a novel cluster comprising strains from Germany, Hungary and France in 2019. Relevance and impact of the detected Picobirna sequences need further investigations.

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.

ICTV virus taxonomy profile: Picobirnaviridae.

Picobirnaviridae is a family of viruses with bi-segmented (rarely unsegmented) dsRNA genomes comprising about 4.4 kbp in total, with small, non-enveloped spherical virions. The family includes one genus (Picobirnavirus) grouping three genetic clusters with high sequence variability, two defined by viruses infecting vertebrates and a third with viruses found in invertebrates. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Picobirnaviridae, which is available at www.ictv.global/report/picobirnaviridae.

Molecular characterization of a novel picobirnavirus in a chicken.

Picobirnaviruses (PBVs) are bisegmented viruses with a wide geographical and host species distribution. The number of novel PBV sequences has been increasing with the help of the viral metagenomics. A novel picobirnavirus strain, pbv/CHK/M3841/HUN/2011, was identified by viral metagenomics; the complete segment 1 (MH327933) and 2 (MH327934) sequences were obtained by RT-PCR from a cloacal sample of a diseased broiler breeder pullet in Hungary. Although the conserved nucleotide (e.g., ribosome binding site) and amino acid motifs (e.g., ExxRxNxxxE, S-domain of the viral capsid and motifs in the RNA-dependent RNA polymerase) were identifiable in the chicken picobirnavirus genome, the putative segment 1 showed low (< 30%) amino acid sequence identity to the corresponding proteins of marmot and dromedary PBVs, while segment 2 showed higher (< 70%) amino acid sequence identity to a wolf PBV protein sequence. This is the first full-genome picobirnavirus sequence from a broiler breeder chicken, but the pathogenicity of this virus is still questionable.

Multiple divergent picobirnaviruses with functional prokaryotic Shine-Dalgarno ribosome binding sites present in cloacal sample of a diarrheic chicken.

Picobirnaviruses (PBVs) of family Picobirnaviridae have bisegmented (S1 and S2 segments), double-stranded RNA genomes. In this study a total of N = 12 complete chicken PBVs (ChPBV) segments (N = 5 of S1 and N = 7 of S2, Acc. Nos.: MH425579-90) were determined using viral metagenomic and RT-PCR techniques from a single cloacal sample of a diarrheic chicken. The identified ChPBV segments are unrelated to each other and distant from all of the currently known PBVs. In silico sequence analyses revealed the presence of conserved prokaryotic Shine-Dalgarno-like (SD-like) sequences upstream of the three presumed open reading frames (ORFs) of the S1 and a single presumed ORF of the S2 segments. According to the results of expression analyses in E. coli using 6xHis-tagged recombinant ChPBV segment 1 construct and Western blot these SD-like sequences are functional in vivo suggesting that S1 of study PBVs can contain three ORFs and supporting the bacteriophage-nature of PBVs.

High detection rate and genetic diversity of picobirnavirus in a sheep flock in Brazil.

This study reports the detection by RT-PCR and molecular characterization of partial RdRp gene of picobirnavirus (PBV) dsRNA in fecal samples (n = 100) from a meat sheep flock in southern Brazil. The analysis of the results allowed the identification of two important characteristics of PBV infection. The first was the high frequency of infection in the sheep flock evaluated where 62% of the analyzed fecal samples were PBV-positive. The second was the high genetic variability found in field strains of ovine PBV genogroup I circulating in animals of the same sheep flock.

Marmota himalayana in the Qinghai-Tibetan plateau as a special host for bi-segmented and unsegmented picobirnaviruses.

Wildlife has been considered the main source of novel viruses causing emerging infectious diseases. Marmota himalayana is endemic to the Qinghai-Tibetan Plateau, China. Here, based on a high-throughput method using Illumina RNA sequencing, we studied the RNA virome of M. himalayana and discovered multiple novel viruses, especially picobirnaviruses (PBVs), which have a bi-segmented genome and belong to the family Picobirnaviridae. A total of 63% of the viral contigs corresponded to PBVs, comprising 274 segment 1 and 56 segment 2 sequences. Unexpectedly, four unsegmented PBV genomes were also detected and confirmed by PCR and resequencing. According to the phylogenetic analysis, the following nine PBV assortment types are proposed: C1:GI, C2:GIV, C4:GI, C4:GV, C5:GI, C7:GI, C8:GIV, C8:GV and C8:GII. We hypothesize a model of segmentation for the PBV genome, mediated by a 6-bp direct repeat sequence, GAAAGG. The model is supported by detection of the segmentation-associated sequence GAAAGG not only in the 5' untranslated regions of segment 1 (221 in 289) and segment 2 (57 in 80) of bi-segmented PBVs but also in the 5' untranslated regions and junction sequences between the capsid and RdRp genes of unsegmented PBVs. Therefore, with RNA sequencing, we found an unexpected biodiversity of PBVs in M. himalayana, indicating that M. himalayana is a special host for PBVs. We also proposed a putative model of how bi-segmented PBVs could be converted into unsegmented PBVs, which sheds new light on the processes of RNA virus genome evolution.

Prevalence and molecular characterization of porcine Picobirnavirus in piglets of North East Region of India.

Picobirnaviruses (PBVs) have been recognized as one of the important causal viral agents of gastroenteritis in several animal species especially in young immunocompromised hosts. In this study, we report the prevalence and molecular epidemiology of porcine PBVs from North Eastern Hilly region of India. A total of 457 fecal samples from piglets were collected from local (n = 130) and cross (n = 327) breed piglets in different seasons for 2 years. All the samples were subjected to RNA-PAGE and RT-PCR analysis for detection of PBVs. A total of 4.59 and 11.15% samples were recorded as positive for PBVs by RNA-PAGE and RT-PCR, respectively. Rate of detection was higher from diarrhoeic animals (13.56%) compared to non-diarrhoeic (4.23%) animals. Higher prevalence rate was observed from unorganized farms (14.22%) compared to organized farms (8.0%) with slightly higher detection from cross breed (11.62%) compared to local breed (10.0%). Maximum cases of piglet diarrhea associated with PBVs were detected during summer (16.4%) and winter (14.39%) seasons compared to autumn (4.80%) and spring (6.45%). All the samples were positive for PBV genogroup I only. Based upon the sequence analysis, the isolates were unique and placed in separate clad and were not closely associated with any other Indian isolates of PBVs so far. Two isolates were closely related with one Chinese isolate recovered from sewage water. This is the first systematic study of prevalence of PBVs associated with piglet diarrhea.

Molecular detection and characterization of picobirnaviruses in piglets with diarrhea in Thailand.

Picobirnavirus (PBV) is a small, bi-segmented, double-stranded RNA virus. Taxonomically, the genus Picobirnavirus belongs to the Picobirnaviridae family. PBV infects a wide range of hosts and causes opportunistic infections, but its role in diarrheal disease remains unclear. To determine the prevalence and genetic diversity of porcine PBVs in Northern Thailand, 380 fecal samples collected from diarrheic and non-diarrheic piglets, raised in 22 pig farms, were tested for the presence of PBV. Reverse-transcription PCR (RT-PCR) was performed using primer sets specific to the RNA-dependent RNA polymerase (RdRp) gene. PBV was detected in 86 of 265 (32.5%) diarrheic piglets and in 26 of 115 (22.6%) non-diarrheic piglets. All the PBV strains detected in this study belonged to genogroup I and a high proportion of PBV-positive piglets were co-infected with group A rotavirus (RVA) and bocavirus (BoV). Phylogenetic analysis of representative genogroup I strains revealed remarkably high similarity between strains; these formed a monophyletic cluster with 97-100% sequence identity in the RdRp gene. The strains were also closely related to genogroup I PBV Chinese porcine strain. The findings indicate that PBV infection is common in piglets with and without diarrhea in Northern Thailand.

Reassortment among picobirnaviruses found in wolves.

We conducted a viral metagenomics study in diarrheic free-ranging wolves in Portugal, revealing for the first time the presence of reassortant picobirnaviruses. These viruses shared identical capsid segments together with diverse RNA-dependent RNA polymerase segments. Even though causality between these picobirnaviruses and diarrhea could not be established, the study nonetheless confirms for the first time that wolves are a potential reservoir for picobirnaviruses, which might play a role as enteric pathogens.

Faecal virome of red foxes from peri-urban areas.

Red foxes (Vulpes vulpes) are the most abundant carnivore species in the Northern Hemisphere. Since their populations are well established in peri-urban and urban areas, they represent a potential reservoir of viruses that transmit from wildlife to humans or domestic animals. In this study, we evaluated the faecal virome of juvenile and adult foxes from peri-urban areas in central Croatia. The dominating mammalian viruses were fox picobirnavirus and parvovirus. The highest number of viral reads (N=1412) was attributed to a new fox circovirus and complete viral genome was de novo assembled from the high-throughput sequencing data. Fox circovirus is highly similar to dog circoviruses identified in diseased dogs in USA and Italy, and to a recently discovered circovirus of foxes with neurologic disease from the United Kingdom. Our fox picobirnavirus was more closely related to the porcine and human picobirnaviruses than to known fox picobirnaviruses.

Viral metagenomics analysis of picobirnavirus-positive feces from children with sporadic diarrhea in China.

Picobirnaviruses (PBVs) infect humans and a wide range of animals and may cause diarrhea. The aim of this study was to investigate PBV infection and its association with diarrhea. Here, seven PBV RT-PCR-positive fecal samples from diarrheic children and four fecal samples from healthy children as controls were analyzed by viral metagenomics. The results indicated that all the seven diarrheic fecal samples contain high titers of PBV sequences, while three of the controls were negative, and one had low titers of PBV. Three of the diarrheic fecal samples were also positive for other viruses, including anellovirus, human gyrovirus, human parechovirus, and porcine stool-associated circular virus. PBV sequences from the seven patients were assembled, generating seven large contigs with the complete ORF of RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis of the amino acid sequences of RdRp indicated that the seven PBVs in the present study belonged to three different genogroups. Our data suggest that PBV might have been the cause of diarrhea in these seven children.