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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. [ABSTRACT FROM AUTHOR]

Simple Summary: One of the factors contributing to the emergence and resurgence of diseases is the persistent disruption and inadequate management of natural ecosystems. This poses a significant threat to many wildlife species, which are considered potential reservoirs of human diseases, as well as to the biodiversity of the Brazilian Amazon. Understanding the potential of enteric viruses is of great importance due to the substantial impact on both animal and human populations. Picobirnaviruses serve as examples of pathogens that infect various animal species, including mammals and non-mammals, either symptomatically or asymptomatically. They function as emerging opportunistic agents and are regarded as potential zoonotic pathogens. This study successfully detected picobirnavirus in fecal specimens collected from companion animals (mostly cats), livestock (mostly pigs), and wild animals inhabiting areas affected by anthropogenic changes. The data underscore the importance of monitoring viruses in animals in order to identify potential points of spillover to humans. This study aimed to detect picobirnavirus (PBV) in the fecal samples of wild and domestic animals from 2014 to 2016 in the Amazon biome. Fecal samples from different animals, including birds (n = 41) and mammals (n = 217), were used. The PAGE test showed negativity for PBV. However, 32 samples (12.4%, 32/258) showed positive results in RT-PCR analyses. Among the positive samples, pigs and cats, both with 28.12% (9/32), registered the highest frequencies. In a phylogenetic analysis, eight sequences from positive samples were grouped in the Genogroup 1 of PBV (PBV GI). PBV occurrence was significantly related to cats and pigs but not other mammals or birds, independently of their geographical origin. A nucleotide analysis demonstrated similarity among the feline group but the absence of a defined structure between the clades. PBVs are highly widespread viruses that can affect the most diverse types of hosts in the Amazon biome, including humans. [ABSTRACT FROM AUTHOR]

This study aimed to detect picobirnavirus (PBV) in the fecal samples of wild and domestic animals from 2014 to 2016 in the Amazon biome. Fecal samples from different animals, including birds (n = 41) and mammals (n = 217), were used. The PAGE test showed negativity for PBV. However, 32 samples (12.4%, 32/258) showed positive results in RT-PCR analyses. Among the positive samples, pigs and cats, both with 28.12% (9/32), registered the highest frequencies. In a phylogenetic analysis, eight sequences from positive samples were grouped in the Genogroup 1 of PBV (PBV GI). PBV occurrence was significantly related to cats and pigs but not other mammals or birds, independently of their geographical origin. A nucleotide analysis demonstrated similarity among the feline group but the absence of a defined structure between the clades. PBVs are highly widespread viruses that can affect the most diverse types of hosts in the Amazon biome, including humans.

The picobirnaviruses (Picobirnaviridae, Picobirnavirus, PBVs) are currently thought to be animal viruses, as they are usually found in animal stool samples. However, no animal model or cell culture for their propagation has yet been found. In 2018, a hypothetical assumption about PBVs belonging to prokaryotic viruses was put forward and experimentally substantiated. This hypothesis is based on the presence of Shine–Dalgarno sequences in the genome of all PBVs before three reading frames (ORF) at the ribosomal binding site, with which the prokaryotic genome is saturated, while in the eukaryotic genome such regions occur with low frequency. The genome saturation with the Shine–Dalgarno sequences, as well as the preservation of this saturation in the progeny, according to scientists, allows us to attribute PBVs to prokaryotic viruses. On the other hand, there is a possibility that PBVs belong to viruses of eukaryotic hosts – fungi or invertebrates, since PBV­like sequences similar to the genome of fungal viruses from the families of mitoviruses and partitiviruses have been identified. In this regard, the idea arose that, in terms of reproduction mode, PBVs resemble fungal viruses. The divergence of views on the true PBV host(s) has sparked discussions among scientists and required further research to elucidate their nature. The review highlights the results of the search for a PBV host. The reasons for the occurrence of atypical sequences among the PBV genome sequences that use an alter­ native mitochondrial code of lower eukaryotes (fungi and invertebrates) for the translation of viral RNA­dependent RNA polymerase (RdRp) instead of the standard genetic code are analyzed. The purpose of the review was to collect arguments in support of the hypothesis about the phage nature of PBVs and to find the most realistic explanation of the reasons for identifying non­standard genomic sequences for PBVs. Based on the hypothesis about the genealogical relationship of PBVs with RNA viruses from other families with similar segmented genomes, such as Reoviridae, Cystoviridae, Totiviridae and Partitiviridae, virologists support the assumption of a decisive role in the origin of atypical PBV­like reassortment strains between PBVs and viruses of the listed families. The collected arguments given in this review indicate a high probability of a phage nature of PBVs. The data presented in the review show that the belonging of PBV­like progeny to prokaryotic or eukaryotic viruses is determined not only by its genome saturation level with a prokaryotic motif, standard or mitochondrial genetic code. The primary structure of the gene encoding the viral capsid protein responsible for the presence or absence of specific proteolytic properties of the virus that determine its ability for independent horizontal transmission into new cells may also be a decisive factor.

ABSTRACT Picobirnaviruses (PBVs) are small, bi-segmented, double-stranded RNA viruses frequently associated with gastrointestinal and recently linked to respiratory infections. Detected in hosts from distant biological kingdoms, debate swirls as to their age and origins and whether they are prokaryotic or eukaryotic viruses. Our evolutionary analysis revealed a contemporaneous emergence for PBV, as PBV-R1&3 species arose ~350 years ago with both segments, whereas the more ancient species, PBV-R2, initially lacked capsid. Integrated phylogenetic reconstruction defined two origins for PBV, determining PBV-R1&3 species descended from Reovirus and PBV-R2 branched with Partitivirus ancestors. These results, coupled with the heterogeneity of Shine-Dalgarno motifs, argue against a prokaryotic origin. Epistatic interactions identified in the RdRp of PBV-R1&3 evidenced the constraints imposed by vertebrate host immunity, whereas its absence in PBV-R2 concurs with its fungal origin. After acquisition of capsid, PBV-R2 increased its adaptive and functional divergences in RdRp domains and the compactness of its RNA structure to enable encapsidation. While their pathogenicity remains an open question, picobirnaviruses likely originated from both fungal and avian hosts: parallel evolution mechanisms have driven the genetic similarities shared among present-day PBV species. 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.

Picobirnaviruses (PBVs) are double-stranded RNA viruses frequently detected in human and animal enteric viromes. Associations of PBVs with enteric graft-versus-host disease and type I diabetes during pregnancy have been established. Since their discovery in 1988, PBVs have been generally assumed to be animal-infecting viruses despite the lack of culture system, animal model, or detection in animal cells or tissues. Recent studies have proposed that bacteria or fungi could be the hosts of PBVs based on genomic analysis. Here, we functionally demonstrate that multiple PBVs of different genome organizations encode bacterial lysins that lyse Escherichia coli. Such genes are typically encoded only by bacteriophages supporting the model that PBVs infect bacterial hosts. Recognition of PBVs as RNA phages in the human gut would completely shift models of how PBVs could impact human health. In addition, expanding the RNA phage world beyond the two recognized clades to three clades has implications for our understanding of the evolution of RNA viruses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This article presents a general overview of the prevalence, genetic diversity and detection methods of picobirnaviruses (PBVs), which are small, non-enveloped icosahedral viruses with a segmented double-stranded RNA genome consisting of two segments taxonomically related to the genus Picobirnavirus of the family Picobirnaviridae. This review of scientific papers published in 1988–2019 provides data on the PBV distribution in the nature and a broad host range. PBV infection is characterized as opportunistic, the lack of understanding of the etiological role of PBVs in diarrhea is emphasized, since these viruses are detected both in symptomatic and asymptomatic cases. The concept of PBV infection as a chronic disease caused by a long-lasting persistence of the virus in the host is considered. Such factors as stress syndrome, physiological conditions, immune status and host age at the time of primary PBV infection influence the virus detection rate in humans and animals. The possible zoonotic nature of human PBV infection is noted due to the capacity for interspecies PBV transmission acquired during evolution as a result of the reassortment of the genome segments of different viruses infecting the same host. Data providing evidence that PBVs belong to eukaryotes and a challenging hypothesis stating that PBVs are bacterial viruses are presented. The need to intensify work on PBV detection because of their wide distribution, despite the complexity due to the lack of the cultivation system, is emphasized. Two strategies of RT-PCR as main PBV detection methods are considered. The genomes of individual representatives of the genus isolated from different hosts are characterized. Emphasis is placed on the feasibility of developing primers with broader specificity for expanding the range of identifiable representatives of the genus PBV due to a huge variety of their genotypes. The importance of effective monitoring of PBV prevalence for studying the zoonotic and anthroponotic potential using metagenomics analysis is highlighted, and so is the possibility of using PBV as a marker for environmental monitoring.

We describe the metagenomics-derived feline enteric virome in the faeces of 25 cats from a single shelter in California. More than 90 % of the recognizable viral reads were related to mammalian viruses and the rest to bacterial viruses. Eight viral families were detected: Astroviridae, Coronaviridae, Parvoviridae, Circoviridae, Herpesviridae, Anelloviridae, Caliciviridae and Picobirnaviridae. Six previously known viruses were also identified: feline coronavirus type 1, felid herpes 1, feline calicivirus, feline norovirus, feline panleukopenia virus and picobirnavirus. Novel species of astroviruses and bocaviruses, and the first genome of a cyclovirus in a feline were characterized. The RNA-dependent RNA polymerase region from four highly divergent partial viral genomes in the order Picornavirales were sequenced. The detection of such a diverse collection of viruses shed within a single shelter suggested that such animals experience robust viral exposures. This study increases our understanding of the viral diversity in cats, facilitating future evaluation of their pathogenic and zoonotic potentials.

Aim: This study aimed to study the prevalence of the coinfection of enteric bacterial and viral pathogens, namely Escherichia coli, Salmonella, Rotavirus, and Picobirnavirus from fecal samples of pre-weaned piglets in Northeast region of India. Materials and Methods: A total of 457 fresh fecal samples were collected from piglets under 9 weeks old during 2013-2015 from organized (n=225) and unorganized (n=232) farms of Manipur, Meghalaya, Mizoram, and Nagaland. Samples were collected from diarrheic (n =339) and non-diarrheic (n=118) piglets including local indigenous (n=130) and crossbreed (n=327) piglets in different seasons during the study period. The samples were processed for the isolation of E. coli and Salmonella and detection of their putative virulence genes by polymerase chain reaction (PCR). Samples were also processed for the detection of Rotavirus and Picobirnavirus by RNA-polyacrylamide agarose gel electrophoresis and reverse transcriptase-PCR (RT-PCR). Results: A total of 11 (2.40%) samples were found positive for two or more coinfecting enteric bacterial and viral pathogens. All the 11 positive fecal samples were recovered from diarrheic piglets. Salmonella Typhimurium (enterotoxin, stn gene) and Picobirnavirus genogroup 1 were found to be more frequent as coinfecting agents. Coinfection was recorded higher in unorganized (3.87%) compared to organized farm (0.88%). Again, higher detection was recorded in crossbreed (2.75%) than local indigenous piglets (1.53%). The occurrence of coinfection was found to be more common during summer (4.68%) followed by winter (2.27%) season. Conclusion: The present study highlighted the significance of E. coli, Salmonella, Rotavirus, and Picobirnavirus as important diarrheagenic pathogens causing coinfection in piglets in Northeast region of India. Probably, this is the first systematic study of the coinfection of four important diarrheagenic bacterial and viral agents associated with piglet diarrhea in India.

Picobirnaviruses (PBVs) are bisegmented double-stranded RNA viruses that have been detected in a wide variety of animal species including invertebrates and in environmental samples. Since PBVs are ubiquitous in feces/gut contents of humans and other animals with or without diarrhea, they were considered as opportunistic enteric pathogens of mammals and avian species. However, the virus remains to be propagated in animal cell cultures, or in gnotobiotic animals. Recently, the classically defined prokaryotic motif, the ribosomal binding site sequence, has been identified upstream of putative open reading frame/s in PBV and PBV-like sequences from humans, various animals, and environmental samples, suggesting that PBVs might be prokaryotic viruses. On the other hand, based on the detection of some novel PBV-like RNA-dependent RNA polymerase sequences that use the alternative mitochondrial genetic code (that of mold or invertebrates) for translation, and principal component analysis of codon usage bias for these sequences, it has been proposed that PBVs might be fungal viruses with a lifestyle reminiscent of mitoviruses. These contradicting observations warrant further studies to ascertain the true host/s of PBVs, which still remains controversial. In this minireview, we have focused on the various findings that have raised a debate on the true host/s of PBVs.

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. [ABSTRACT FROM AUTHOR]

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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.

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. [ABSTRACT FROM AUTHOR]

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).

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.

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., Competing Interests: L.J.P., M.G.B., and G.A.C. are all employees and shareholders of Abbott.

Picobirnaviruses (PBVs) have been characterized as opportunistic enteric pathogens detected in various domestic, zoo and wild animals, suggesting a wide host range of these viruses. It is thus important to monitor wild animals for the presence of various human and animal pathogens in order to identify a potential reservoir of infectious diseases. In this study, the first phylogenetic analysis of PBV from roe deer (Capreolus capreolus) was performed with a total of 70 investigated samples of feces from roe deer collected in 2014 and 2015 during a survey throughout Slovenia. A high detection rate of PBVs was observed with newly designed specific primers, 42 samples out of 70 (60%) being positive. Phylogenetic analysis of the partial RdRp gene showed that roe deer PBV sequences were distributed over the whole phylogenetic tree and were distributed between 7 highly supported groups and 12 separate branches within the PBV genogroup I. The animal PBV strain most closely related to roe deer PBV strains was the Rhesus macaque PBV/BGD/PbV-55 strain, with 89.1% nucleotide identity to that of PBV SLO/D80-14. Overall nucleotide sequence identity between PBV strains obtained from roe deer ranged from 60.4 to 100%, confirming the high genetic diversity with no subtypes related to host species or geographic location in general. This first phylogenetic survey of roe deer PBVs provides further knowledge concerning the putative host range and confirms the high genetic diversity of these PBVs. • High detection rate (60%) of roe deer Genogroup I PBVs is presented. • High genetic diversity of roe deer Genogroup I PBVs is presented. • The first phylogenetic analysis (partial RdRp) of roe deer PBVs was performed. • Roe deer PBV strains were distributed between 7 groups and 12 separate branches. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to study the prevalence of the coinfection of enteric bacterial and viral pathogens, namely Escherichia coli, Salmonella, Rotavirus, and Picobirnavirus from fecal samples of pre-weaned piglets in Northeast region of India. Materials and Methods: A total of 457 fresh fecal samples were collected from piglets under 9 weeks old during 2013-2015 from organized (n=225) and unorganized (n=232) farms of Manipur, Meghalaya, Mizoram, and Nagaland. Samples were collected from diarrheic (n =339) and non-diarrheic (n=118) piglets including local indigenous (n=130) and crossbreed (n=327) piglets in different seasons during the study period. The samples were processed for the isolation of E. coli and Salmonella and detection of their putative virulence genes by polymerase chain reaction (PCR). Samples were also processed for the detection of Rotavirus and Picobirnavirus by RNA-polyacrylamide agarose gel electrophoresis and reverse transcriptase-PCR (RT-PCR). Results: A total of 11 (2.40%) samples were found positive for two or more coinfecting enteric bacterial and viral pathogens. All the 11 positive fecal samples were recovered from diarrheic piglets. Salmonella Typhimurium (enterotoxin, stn gene) and Picobirnavirus genogroup 1 were found to be more frequent as coinfecting agents. Coinfection was recorded higher in unorganized (3.87%) compared to organized farm (0.88%). Again, higher detection was recorded in crossbreed (2.75%) than local indigenous piglets (1.53%). The occurrence of coinfection was found to be more common during summer (4.68%) followed by winter (2.27%) season. Conclusion: The present study highlighted the significance of E. coli, Salmonella, Rotavirus, and Picobirnavirus as important diarrheagenic pathogens causing coinfection in piglets in Northeast region of India. Probably, this is the first systematic study of the coinfection of four important diarrheagenic bacterial and viral agents associated with piglet diarrhea in India. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Twelve novel picobirnavirus segments were identified from a chicken cloacal sample. • Full-length sequences of five segment 1 and seven segment 2 were determined. • Prokaryotic Shine-Dalgarno-like (SD-like) sequences were present upstream of all ORFs. • In vivo expression analysis indicates the SD-like sequences are functional in E. coli. • The presence of functional SD-like sequences further support the phage nature of PBVs. [ABSTRACT FROM AUTHOR]

Sewage-associated viruses can cause several human and animal diseases, such as gastroenteritis, hepatitis, and respiratory infections. Therefore, their detection in wastewater can reflect current infections within the source population. To date, no viral study has been performed using the sewage of any large South American city. In this study, we used viral metagenomics to obtain a single sample snapshot of the RNA virosphere in the wastewater from Santiago de Chile, the seventh largest city in the Americas. Despite the overrepresentation of dsRNA viruses, our results show that Santiago’s sewage RNA virosphere was composed mostly of unknown sequences (88%), while known viral sequences were dominated by viruses that infect bacteria (60%), invertebrates (37%) and humans (2.4%). Interestingly, we discovered three novel genogroups within the Picobirnaviridae family that can fill major gaps in this taxa’s evolutionary history. We also demonstrated the dominance of emerging Rotavirus genotypes, such as G8 and G6, that have displaced other classical genotypes, which is consistent with recent clinical reports. This study supports the usefulness of sewage viral metagenomics for public health surveillance. Moreover, it demonstrates the need to monitor the viral component during the wastewater treatment and recycling process, where this virome can constitute a reservoir of human pathogens.

Picobirnaviruses (PBVs), family Picobirnaviridae, are bi-segmented, double-stranded RNA viruses. PBVs are considered opportunistic enteric pathogens. Gene segment-1 of PBV encodes the capsid protein, whilst gene segment-2 codes for RNA-dependent RNA polymerase (RdRp). Based on differences in gene segment-2, PBVs are classified into genogroup-I (GI) and GII. Although PBVs have been detected in a wide variety of host species, there are no reports on PBVs from mongoose so far. We report here high rates of detection (35.36%, 29/82) of GI PBVs in fecal samples from the small Indian mongoose (Herpestes auropunctatus) on the Caribbean island of St. Kitts. Applying a combination of a non-specific primer-based amplification method and conventional RT-PCR using a newly designed primer targeting the 3′-untranslated region (UTR), we could amplify and sequence the complete/nearly complete gene segment-2 of eight mongoose PBV strains. Except for a single strain, the gene segment-2 of the remaining mongoose PBV strains contained the putative open reading frame encoding the RdRp. The gene segment-2/putative RdRps of the mongoose PBV strains retained various features that are conserved in other PBVs (5′- and 3′-terminal nucleotide sequences, bacterial ribosomal binding site sequence in 5′-UTR, and the three domains in putative RdRps). On the other hand, phylogenetic analysis and sequence identities of the putative RdRps revealed high genetic diversity among the mongoose PBV strains and with those of PBVs from other host species. To our knowledge, this is the first report on detection and genetic diversity of PBVs from the mongoose, expanding the host range of PBVs and providing vital insights into the various features and evolution of putative RdRps of PBVs in a new host species.

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.

Here we investigate the cargo retention of individual human picobirnavirus (hPBV) virus-like particles (VLPs) which differ in the N-terminal of their capsid protein (CP): (i) hPBV CP contains the full-length CP sequence; (ii) hPBV Δ45-CP lacks the first 45 N-terminal residues; and (iii) hPBV Ht-CP is the full-length CP with a N-terminal 36-residue tag that includes a 6-His segment. Consequently, each VLP variant holds a different interaction with the ssRNA cargo. We used atomic force microscopy (AFM) to induce and monitor the mechanical disassembly of individual hPBV particles. First, while Δ45-CP particles that lack ssRNA allowed a fast tip indentation after breakage, CP and Ht-CP particles that pack heterologous ssRNA showed a slower tip penetration after being fractured. Second, mechanical fatigue experiments revealed that the increased length in 8% of the N-terminal (Ht-CP) makes the virus particles to crumble ∼10 times slower than the wild type N-terminal CP, indicating enhanced RNA cargo retention. Our results show that the three differentiated N-terminal topologies of the capsid result in distinct cargo release dynamics during mechanical disassembly experiments because of the different interaction with RNA.

Picobirnaviruses (PBVs) are double-stranded RNA viruses frequently detected in human and animal enteric viromes. Associations of PBVs with enteric graft-versus-host disease and type I diabetes during pregnancy have been established. Since their discovery in 1988, PBVs have been generally assumed to be animal-infecting viruses despite the lack of culture system, animal model, or detection in animal cells or tissues. Recent studies have proposed that bacteria or fungi could be the hosts of PBVs based on genomic analysis. Here, we functionally demonstrate that multiple PBVs of different genome organizations encode bacterial lysins that lyse Escherichia coli . Such genes are typically encoded only by bacteriophages supporting the model that PBVs infect bacterial hosts. Recognition of PBVs as RNA phages in the human gut would completely shift models of how PBVs could impact human health. In addition, expanding the RNA phage world beyond the two recognized clades to three clades has implications for our understanding of the evolution of RNA viruses.

Abstract We report here high rates of detection (54%, 21/39) of picobirnaviruses (PBVs) in feces/intestinal contents of free roaming, apparently healthy rats (Rattus spp.) on the Caribbean island of St. Kitts. One of the PBV strains, strain PBV/Rat/KNA/Rat9/2017, was molecularly characterized for complete gene segment-2. To determine the nucleotide (nt) sequence of full-length gene segment-2, the 5′- and 3′- portions of gene segment-2 of strain Rat9 containing an overlapping region were amplified using a non-specific primer-based amplification method with modifications. The complete gene segment-2 of PBV strain Rat9 was 1730 bp in length, encoding a putative RNA-dependent RNA polymerase (RdRp) of 535 amino acid (aa). By nt and deduced aa sequence identities and phylogenetic analysis, the complete gene segment-2 of strain Rat9 exhibited high genetic diversity with those of PBVs from other host species. On the other hand, 5′- and 3′- end nt sequences of gene segment-2, and the three domains of putative RdRp that are conserved in PBVs were retained in strain Rat9. To our knowledge, this is the first report on molecular characterization of complete gene segment-2 of a PBV strain from Rattus spp. , providing important insights into the putative RdRp, and genetic diversity and evolution of PBV in rats. The high detection rates of PBV in free roaming rats on St. Kitts emphasizes the importance of further studies on epidemiology and genetic makeup of PBVs in Rattus spp. Highlights • High detection rates of picobirnaviruses (PBV) in free roaming rats (Rattus spp.) • First report on molecular characterization of complete gene segment-2 of PBV in a rat • Complete gene segment-2 of rat PBV strain was genetically distinct from other PBVs. [ABSTRACT FROM AUTHOR]

Picobirnavirus (PBV) is recognized as a putative cause of diarrhea and respiratory illnesses. Although PBV has been reported in several mammalian (including humans) and avian host species, data pertaining to its presence in small ruminants are limited. We report, here, PBV infection in small ruminants (ovine and caprine), in India. From January 2015 to December 2017, 400 samples were tested for the presence of PBV, using an RT-PCR assay specific for the genome segment-2. The overall rate of PBV infection was 35.75% (143/400), being higher in caprines (42.35%, 83/196) than in ovines (29.42%, 60/204). Viral genogrouping showed the predominance of PBV genogroup I (GG-I; 53.15%, 76/173), the detection of genogroup II (GG-II; 3.49%, 5/143), a concomitant infection with GG-I and GG-II (38.47%, 55/143), and un-typeable strains (4.9%, 7/143). Of note, these PBV strains exhibit low sequence identity (11.2% to 70.7%) to other reported PBV isolates from humans and other animals. By phylogenetic analysis, camel PBV isolates from the United Arab Emirates (UAE) and the reference human GG-I strain (1-CHN-97) from China were found to be the nearest neighbors of PBV strains. Furthermore, sequence analysis revealed the possible appearance of a new genogroup/genetic cluster and the existence of high genetic heterogeneity in the circulating PBV strains. Although much remains to be understood about the epidemiology and impact of PBV, the present study demonstrates the high prevalence of GG-I, the detection of GG-II, and the possible emergence of new genogroup/genetic cluster in small ruminant populations in India. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Currently, the Leviviridae and Cystoviridae are the only two recognized families of prokaryotic RNA viruses. Picobirnaviruses, which are bisegmented double-stranded RNA viruses commonly found in animal stool samples, are currently thought to be animal viruses, but have not been propagated in cell culture or in an animal model. We hypothesize that picobirnaviruses are prokaryotic RNA viruses. We identified and analyzed the genomes of 38 novel picobirnaviruses and determined that a classical bacterial sequence motif, the ribosomal binding site (RBS), is present in the 5′ untranslated regions (5′ UTRs) of all of the novel as well as all previously published picobirnavirus sequences. Among all viruses, enrichment of the RBS motif is only observed in viral families that infect prokaryotes and not in eukaryotic infecting viral families. These results will enable future studies to more accurately understand the biology of picobirnaviruses. [ABSTRACT FROM AUTHOR]

Emerging viral infections can be identified by using a viral metagenomics approach for clinical human material. Diarrhea samples of patients with unexplained gastroenteritis from the Netherlands were analyzed by using viral metagenomics. Novel circular DNA viruses, bufaviruses, and genogroup III picobirnaviruses were identified. These data expand our knowledge of the human virome.

In this study 113 diarrhoeic faecal samples obtained from buffalo (n = 68) and cattle (n = 45) calves under 1 years of age were analysed in order to determine the presence of rotavirus infection and the frequency of picobirnavirus excretion. Eleven (9.73%) samples positive for group A rotavirus were identified through RNA-polyacrylamide gel electrophoresis (RNA-PAGE), while 4 (3.53%) samples showed a bisegmented genome with a typical picobirnavirus pattern. This is the first report of picobirnavirus in cattle and buffalo calves from Western India.

We report here molecular characterization of complete genomic segment-2 of picobirnavirus (PBV) strains PBV/Cat/KNA/K40/2014 and PBV/Dog/KNA/RVC7/2015 detected in a cat ( Felis catus ) and a dog ( Canis lupus familiaris ), respectively, on the Caribbean island of St. Kitts. To obtain the full-length nucleotide (nt) sequence of gene segment-2 of the canine and feline PBV strains, the 5′- and 3′- portions of gene segment-2 containing an overlapping region were amplified using a non-specific primer-based amplification method with modifications. The complete gene segment-2 of feline PBV strain K40 and canine PBV strain RVC7 was 1784 nt and 1689 nt long, respectively, and encoded a putative RNA-dependent RNA polymerase (RdRp) of 534 amino acid (aa) and 531 aa, respectively. The complete gene segment-2 of strains K40 and RVC7 exhibited a high degree of genetic diversity between themselves, and with those of PBVs from other host species. On the other hand, both the canine and feline PBV strains retained the 5′- and 3′- end nucleotide sequences and the three domains of putative RdRp that are conserved in PBVs. To our knowledge, this is the first report on molecular characterization of complete gene segment-2 of PBV strains detected in cats and dogs, allowing us to study the features of putative RdRps of PBVs in these host species, and providing important insights into the genetic makeup and evolution of feline and canine PBV strains. PBVs were detected for the first time in cats and dogs from the Caribbean region. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Picobirnavirus (PBV) is an emerging causal agent of gastrointestinal and respiratory infections in humans and animals. Herein, a RNA polymerase gene based RT-PCR diagnostic assay was developed for initial detection of PBVs viral RNA in stool samples. Furthermore, sensitivity, specificity and validation of this assay were accomplished using field samples. The ORF2 (RdRp) coding region was chosen as the target, with more consensus portions within genus in the family Picobirnaviridae, tested in the genogroups I and II (GGI and GGII) of the virus. The RdRp nucleotide sequences from human and animal PBVs were retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov) for primer designing. In this study, a total of 54 stool samples from animals (cattle calves (n=27), piglets (14) and goat kids (13)) suffering with diarrhea were screened. Expected size PCR amplicon of 275 bp was seen in 14 cattle calves, 12 piglets and 3 goat kids. The field validation of RT-PCR assay revealed presence of PBV viral RNA in 53.7% samples (29/54). The described genus specific assay could be applied for the primary detection of PBV infection in animals and, in combination with the genogroup specific PCRs, for the confirmation of new and emerging PBVs in animals. [ABSTRACT FROM AUTHOR]

During 2014–2015, 270 fecal samples were collected from non-diarrheic, captive and wild African green monkeys (AGMs) on the island of St. Kitts, Caribbean region. By RNA-PAGE, picobirnaviruses (PBVs) were detected in sixteen captive AGMs. By RT-PCR and sequencing of partial gene segment-2, PBVs in 15 of these 16 samples were assigned to genogroup-I. The full-length nucleotide (nt) sequence of gene segment-2 of one of the genogroup-I PBV strains, strain PBV/African green monkey/KNA/016593/2015, was obtained using a non-specific primer-based amplification method with modifications. Gene segment-2 of strain 016593 was 1707 bp long, and encoded a putative RNA-dependent RNA polymerase (RdRp) of 538 aa. Furthermore, the nearly complete gene segment-2 sequences of three other AGM PBV strains were determined using primers designed from gene segment-2 sequence of 016593. The gene segment-2 of the 4 AGM PBV strains were almost identical to each other, and exhibited a high degree of genetic diversity (maximum nt and deduced aa sequence identities of 66.4% and 65.3%, respectively) with those of PBVs from other host species. The 5′- and 3′- (except for one mismatch) end nt sequences and the three domains of RdRps were retained in the AGM PBV strains. To our knowledge, this is the first report on detection, and molecular characterization of complete gene segment-2 of PBVs in vervet monkeys. PBVs were detected for the first time from the Caribbean region. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The picobirnaviruses (Picobirnaviridae, Picobirnavirus, PBVs) are currently thought to be animal viruses, as they are usually found in animal stool samples. However, no animal model or cell culture for their propagation has yet been found. In 2018, a hypothetical assumption about PBVs belonging to prokaryotic viruses was put forward and experimentally substantiated. This hypothesis is based on the presence of Shine-Dalgarno sequences in the genome of all PBVs before three reading frames (ORF) at the ribosomal binding site, with which the prokaryotic genome is saturated, while in the eukaryotic genome such regions occur with low frequency. The genome saturation with the Shine-Dalgarno sequences, as well as the preservation of this saturation in the progeny, according to scientists, allows us to attribute PBVs to prokaryotic viruses. On the other hand, there is a possibility that PBVs belong to viruses of eukaryotic hosts - fungi or invertebrates, since PBV-like sequences similar to the genome of fungal viruses from the families of mitoviruses and partitiviruses have been identified. In this regard, the idea arose that, in terms of reproduction mode, PBVs resemble fungal viruses. The divergence of views on the true PBV host(s) has sparked discussions among scientists and required further research to elucidate their nature. The review highlights the results of the search for a PBV host. The reasons for the occurrence of atypical sequences among the PBV genome sequences that use an alternative mitochondrial code of lower eukaryotes (fungi and invertebrates) for the translation of viral RNA-dependent RNA polymerase (RdRp) instead of the standard genetic code are analyzed. The purpose of the review was to collect arguments in support of the hypothesis about the phage nature of PBVs and to find the most realistic explanation of the reasons for identifying non-standard genomic sequences for PBVs. Based on the hypothesis about the genealogical relationship of PBVs with RNA viruses from other families with similar segmented genomes, such as Reoviridae, Cystoviridae, Totiviridae and Partitiviridae, virologists support the assumption of a decisive role in the origin of atypical PBV-like reassortment strains between PBVs and viruses of the listed families. The collected arguments given in this review indicate a high probability of a phage nature of PBVs. The data presented in the review show that the belonging of PBV-like progeny to prokaryotic or eukaryotic viruses is determined not only by its genome saturation level with a prokaryotic motif, standard or mitochondrial genetic code. The primary structure of the gene encoding the viral capsid protein responsible for the presence or absence of specific proteolytic properties of the virus that determine its ability for independent horizontal transmission into new cells may also be a decisive factor., Competing Interests: The authors declare no conflict of interest., (Copyright © AUTHORS.)

The emergence of culture- and sequence-independent metagenomic methods has not only provided great insight into the microbial community structure in a wide range of clinical and environmental samples but has also proven to be powerful tools for pathogen detection. Recent studies of the food microbiome have revealed the vast genetic diversity of bacteria associated with fresh produce. However, no work has been done to apply metagenomic methods to tackle viruses associated with fresh produce for addressing food safety. Thus, there is a little knowledge about the presence and diversity of viruses associated with fresh produce from farm-to-fork. To address this knowledge gap, we assessed viruses on commercial romaine and iceberg lettuces in fields and a produce distribution center using a shotgun metagenomic sequencing targeting both RNA and DNA viruses. Commercial lettuce harbors an immense assemblage of viruses that infect a wide range of hosts. As expected, plant pathogenic viruses dominated these communities. Sequences of rotaviruses and picobirnaviruses were also identified in both field-harvest and retail lettuce samples, suggesting an emerging foodborne transmission threat that has yet to be fully recognized. The identification of human and animal viruses in lettuce samples in the field emphasizes the importance of preventing viral contamination on leafy greens starting at the field. Although there are still some inherent experimental and bioinformatics challenges in applying viral metagenomic approaches for food safety testing, this work will facilitate further application of this unprecedented deep sequencing method to food samples. [ABSTRACT FROM AUTHOR]