Picobirnavirus Research Articles

Molecular characterization of complete genomic segment-2 of picobirnavirus strains detected in a cat and a dog

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 1784nt and 1689nt 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.

Detection of picobirnaviruses in vervet monkeys (Chlorocebus sabaeus): Molecular characterization of complete genomic segment-2

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 1707bp long, and encoded a putative RNA-dependent RNA polymerase (RdRp) of 538aa. 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.

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.

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.

High Diversity of Genogroup I Picobirnaviruses in Mammals.

In a molecular epidemiology study using 791 fecal samples collected from different terrestrial and marine mammals in Hong Kong, genogroup I picobirnaviruses (PBVs) were positive by RT-PCR targeting the partial RdRp gene in specimens from five cattle, six monkeys, 17 horses, nine pigs, one rabbit, one dog, and 12 California sea lions, with 11, 9, 23, 17, 1, 1, and 15 sequence types in the positive specimens from the corresponding animals, respectively. Phylogenetic analysis showed that the PBV sequences from each kind of animal were widely distributed in the whole tree with high diversity, sharing 47.4–89.0% nucleotide identities with other genogroup I PBV strains based on the partial RdRp gene. Nine complete segment 1 (viral loads 1.7 × 104 to 5.9 × 106/ml) and 15 segment 2 (viral loads 4.1 × 103 to 1.3 × 106/ml) of otarine PBVs from fecal samples serially collected from California sea lions were sequenced. In the two phylogenetic trees constructed using ORF2 and ORF3 of segment 1, the nine segment 1 sequences were clustered into four distinct clades (C1–C4). In the tree constructed using RdRp gene of segment 2, the 15 segment 2 sequences were clustered into nine distinct clades (R1–R9). In four sea lions, PBVs were detected in two different years, with the same segment 1 clade (C3) present in two consecutive years from one sea lion and different clades present in different years from three sea lions. A high diversity of PBVs was observed in a variety of terrestrial and marine mammals. Multiple sequence types with significant differences, representing multiple strains of PBV, were present in the majority of PBV-positive samples from different kinds of animals.

Picobirnavirus: A Putative Emerging Threat to Humans and Animals

Picobirnavirus: A Putative Emerging Threat to Humans and Animals

Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus.

During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5’-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.

SIV Infection-Mediated Changes in Gastrointestinal Bacterial Microbiome and Virome Are Associated with Immunodeficiency and Prevented by Vaccination.

AIDS caused by simian immunodeficiency virus (SIV) infection is associated with gastrointestinal disease, systemic immune activation, and, in cross-sectional studies, changes in the enteric virome. Here we performed a longitudinal study of a vaccine cohort to define the natural history of changes in the fecal metagenome in SIV-infected monkeys. Matched rhesus macaques were either uninfected or intrarectally challenged with SIV, with a subset receiving the Ad26 vaccine, an adenovirus vector expressing the viral Env/Gag/Pol antigens. Progression of SIV infection to AIDS was associated with increased detection of potentially pathogenic viruses and bacterial enteropathogens. Specifically, adenoviruses were associated with an increased incidence of gastrointestinal disease and AIDS-related mortality. Viral and bacterial enteropathogens were largely absent from animals protected by the vaccine. These data suggest that the SIV-associated gastrointestinal disease is associated with the presence of both viral and bacterial enteropathogens and that protection against SIV infection by vaccination prevents enteropathogen emergence.

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.

Electrophoretic RNA genomic profiles of Brazilian Picobirnavirus (PBV) strains and molecular characterization of a PBV isolated from diarrheic calf

Picobirnavirus (PBV) belongs to the family Picobirnaviridae. PBV are a group of emerging non-enveloped viruses, with a bisegmented double-stranded RNA genome that can infect a wide range of hosts. This study reports the occurrence of PBV in fecal samples from five Brazilian dairy cattle herds. From the 289 stool samples of individual calves analyzed by silver-stained polyacrylamide gel electrophoresis (ss-PAGE) the PBV was detected in 8.3 % (24/289), of which 10.2% (18/176) had diarrheic consistency. Of the 24 positive samples in ss-PAGE, 5 (20.8%) of them showed a small electrophoretic profile and 19 (79.2%) samples had large profile. From the 24 positives samples by ss-PAGE, 15 (62.5%) were successfully amplified (201bp) using GI specific primers targeting the RdRp gene of PBV. The analysis of nucleotide identity matrix revealed that the bovine PBV strain identified in this study, showed the highest nucleotide identity (81%) with PBV strain detected in turkey (MD-2010/HM803965). This is the first nucleotide sequence of a bovine PBV strain in the American continent and the first detection of small genome profile of PBV-like strains in bovine hosts.

Detection and evolutionary analysis of picobirnaviruses in treated wastewater.

Wastewater contains numerous viruses. In this study, picobirnaviruses (PBVs) were detected in the stream of a wastewater treatment plant in Changsha, Hunan province, China, and evolutionary analysis of the isolated PBVs was performed. The phylogenetic tree revealed that the PBVs were highly divergent and could be classified into six distinct groups according to their hosts. Among these groups, pairwise comparison of the six groups revealed that the nucleotide distance of group 4 (bootstrap value = 0.92; nucleotide identity = 94%) was the largest. Thus, group 4 might represent a new division of PBVs. Comprehensive analysis of the obtained PBV sequences to investigate their evolutionary history and phylodynamics revealed that group 5 (PBVs from monkey) exhibited maximum polymorphism (K = 30.582, S = 74, η = 98, Pa = 47) and lowest nucleotide substitutions per site per year (6.54E-3 subs per site per year), except group 4. Maximum clade credibility tree indicated that group 5 appeared earlier than the other groups. In conclusion, this study detected PBVs in treated wastewater in China, and identified a new PBV group. Furthermore, among these PBVs, group 5 was found to survive longer and present a balance between PBVs and their monkey host.

Prevalence and complete genome characterization of turkey picobirnaviruses

The “light turkey syndrome” (LTS), in which birds weigh less than their standard breed character at the marketing time, is believed to be a consequence of viral enteritis at an early age (3–5weeks) from which the birds never fully recover. In a previously published study, we collected fecal pools from 2, 3, 5 and 8week old turkey poults (80 pools from LTS farms and 40 from non-LTS farms) and examined them for the presence of astro-, rota-, reo-, and coronaviruses. To determine the presence of additional enteric viruses, we analyzed a fecal pool by Illumina sequencing and found picobirnavirus (PBV). Segments 1 and 2 of this virus shared 45.8%aa and 60.9–64.5%aa identity with genogroup I of human PBV, respectively. Primers based on RNA-dependent RNA polymerase and capsid genes were designed for detection and molecular characterization of PBVs in the 120 fecal pools described above. From LTS farms, 39 of 80 (48.8%) pools were PBV positive while 23 of 40 (57.5%) were positive from non-LTS farms. The phylogenetic analysis of 15 randomly selected strains divided them into four subgroups within genogroup I (subgroups 1A–D). Nine strains were in subgroup IA showing 69.9–76.4%nt identity with human PBV GI strainVS111 from the Netherlands. Strains in subgroup IB (n=2) had 91.4–91.7%nt identity with chicken PBV GI strain AVE 42v1 from Brazil. Two strains in subgroup IC had 72.3–74.2%nt identity with chicken PBV strain AVE 71v3 from Brazil. In subgroup ID, two strains showed 72.4–81.8%nt identity with chicken PBV GI strain AVE 57v2 from Brazil. Subgroup IC and ID were the most divergent. Five of the 15 strains were typed using capsid gene primers. They showed 32.6–33.4%nt and 39.5–41.3%aa identity with VS10 human PBV strain. These results indicate co-circulation of divergent strains of PBVs among Minnesota turkeys.

Maintenance of picobirnavirus (PBV) infection in an adult orangutan (Pongo pygmaeus) and genetic diversity of excreted viral strains during a three-year period

The present work provide data about the maintenance of picobirnavirus (PBV) infection during adulthood in a mammalian host. For this purpose PBV infection was studied in an adult orangutan (Pongo pygmaeus) by PAGE/SS, RT-PCR and nucleotide sequencing. PBV infection in the animal was asymptomatic and was characterized by interspaced silent and high/ low active viral excretion periods. The PBV strains excreted by the studied individual were identified as genogroup I and revealed a nucleotide identity among them of 64–81%.The results obtained allowed to arrive to a deeper understanding of the natural history of PBV infection, which seems to be characterized by new-born, juvenile and adult asymptomatic hosts which persistently excrete closely related strains in their feces. Consequently, picobirnaviruses could be considered frequent inhabitants of the gastrointestinal tract, leaving the question open about the molecular mechanisms governing persistent and asymptomatic coexistence within the host and the potential host suitability to maintain this relationship.

Divergent picobirnaviruses in human feces.

The near-complete genomes of two picobirnaviruses (PBVs) in diarrheal stool samples, human picobirnaviruses D and E (HuPBV-D and -E), were genetically characterized. Their RNA-dependent RNA polymerase (RdRp) protein sequences had <66% identities to known PBVs. Due to a single nucleotide insertion, the open reading frame 2 (ORF2) in segment 1 of HuPBV-D was interrupted by a stop codon. A small stem-loop structure overlying the stop codon may result in translational readthrough into the rest of ORF2.

Viral metagenomics analysis demonstrates the diversity of viral flora in piglet diarrhoeic faeces in China

To investigate the diversity of viral flora, we used metagenomics to study the viral communities in a pooled faecal sample of 27 diarrhoeic piglets from intensive commercial farms in China. The 15 distinct mammalian viruses identified in the pooled diarrhoeic sample were, in order of abundance of nucleic acid sequence, Porcine epidemic diarrhea virus (PEDV), sapovirus, porcine bocavirus-4 (PBoV-4), sapelovirus, torovirus, coronavirus, PBoV-2, stool-associated single-stranded DNA virus (poSCV), astrovirus (AstV), kobuvirus, posavirus-1, porcine enterovirus-9 (PEV-9), porcine circovirus-like (po-circo-like) virus, picobirnavirus (PBV) and Torque teno sus virus 2 (TTSuV-2). The prevalence rate of each virus was verified from diarrhoeic and healthy piglets by PCR assay. A mean of 5.5 different viruses were shed in diarrhoeic piglets, and one piglet was in fact co-infected with 11 different viruses. By contrast, healthy piglets shed a mean of 3.2 different viruses. Compared with samples from healthy piglets, the co-infection of PEDV and PBoV had a high prevalence rate in diarrhoea samples, suggesting a correlation with the appearance of diarrhoea in piglets. Furthermore, we report here for the first time the presence of several recently described viruses in China, and the identification of novel genotypes. Therefore, our investigation results provide an unbiased survey of viral communities and prevalence in faecal samples of piglets.

Animal picobirnavirus.

Picobirnavirus (PBV) is a small, non-enveloped, bisegmented double-stranded RNA (dsRNA) virus of vertebrate hosts. The name 'Picobirnavirus' derives from the prefix 'pico' (latin for 'small') in reference to the small virion size, plus the prefix 'bi' (latin for 'two') and the word 'RNA' to indicate the nature of the viral genome. The serendipitous discovery of PBV dates back to 1988 from Brazil, when human fecal samples collected during the acute gastroenteritis outbreaks were subjected for routine rotavirus surveillance by polyacrylamide gel electrophoresis (PAGE) and silver straining (S/S). The PAGE gels after silver staining showed a typical 'two RNA band' pattern, and it was identified as Picobirnavirus. Likewise, the feces of wild black-footed pigmy rice rats (Oryzomys nigripes) subjected for PAGE assay by the same research group in Brazil reported the presence of PBV (Pereira et al., J Gen Virol 69:2749-2754, 1988). PBVs have been detected in faeces of humans and wide range of animal species with or without diarrhoea, worldwide. The probable role of PBV as either a 'primary diarrhoeal agent' in 'immunocompetent children'; or a 'potential pathogen' in 'immunocompromised individuals' or an 'innocuous virus' in the intestine remains elusive and needs to be investigated despite the numerous reports of the presence of PBV in fecal samples of various species of domestic mammals, wild animals, birds and snakes; our current knowledge of their biology, etiology, pathogenicity or their transmission characteristics remains subtle. This review aims to analyse the veterinary and zoonotic aspects of animal Picobirnavirus infections since its discovery.

Kingdom-Agnostic Metagenomics and the Importance of Complete Characterization of Enteric Microbial Communities

Advanced sequencing techniques have shown that bacteria are not the only complex and important microbes in the human intestine. Nonbacterial organisms, particularly the virome and the mycobiome, are important regulators of intestinal immunity and inflammation. The virome is mucosal and systemic; it can alter the host response to bacteria and interact with host genes and bacteria to contribute to disease pathogenesis. The human mycobiome is also complex and can contribute to intestinal inflammation. We review what has recently been learned about the nonbacterial and nonarchaeal microbes in the gastrointestinal tract, discussing their potential effects on health and disease and analytical approaches for their study. Studies of associations between the microbiome and intestinal pathology should incorporate kingdom-agnostic approaches if we are to fully understand intestinal health and disease.

Epidemiology, phylogeny, and evolution of emerging enteric Picobirnaviruses of animal origin and their relationship to human strains.

Picobirnavirus (PBV) which has been included in the list of viruses causing enteric infection in animals is highly versatile because of its broad host range and genetic diversity. PBVs are among the most recent and emerging small, nonenveloped viruses with a bisegmented double-stranded RNA genome, classified under a new family “Picobirnaviridae.” PBVs have also been detected from respiratory tract of pigs, but needs further close investigation for their inhabitant behavior. Though, accretion of genomic data of PBVs from different mammalian species resolved some of the ambiguity, quite a few questions and hypotheses regarding pathogenesis, persistence location, and evolution of PBVs remain unreciprocated. Evolutionary analysis reveals association of PBVs with partitiviruses especially fungi partitiviruses. Although, PBVs may have an ambiguous clinical implication, they do pose a potential public health concern in humans and control of PBVs mainly relies on nonvaccinal approach. Based upon the published data, from 1988 to date, generated from animal PBVs across the globe, this review provides information and discussion with respect to genetic analysis as well as evolution of PBVs of animal origin in relation to human strains.

Detection of Genogroup I Picobirnavirus in Diarrhoeic Faecal Samples of Calves from West Bengal, India

Picobirnaviruses (PBVs) have recently been classified into the Picobirnaviridae family. They are small, non-enveloped viruses with bi-segmented, double-stranded (ds) RNA genomes. Identification of PBVs has been done using RNA-PAGE and reverse transcription PCR (RT-PCR). In this study, two (2.25%) calves were found positive for large genome profile picobirnavirus. Both RNA-PAGE positive picobirnavirus samples showed amplicons of approximate 201 bp using specific primer pair viz. PicoB25 (+) and PicoB43(-) for genogroup I picobirnavirus by RT-PCR.

Novel bisegmented virus (picobirnavirus) of animals, birds and humans

Picobirnaviruses (PBVs) are novel group of small, nonenveloped, bisegmented and double stranded RNA viruses. PBVs have been identified in the faeces of a broad range of hosts by several international research groups. Since attempts to culture PBV in vitro have not been made to date and no animal model of infection and disease exists. Laboratory diagnosis relies upon electron microscopy, the detection of the double stranded RNA bisegmented genome by polyacrylamide gel electrophoresis and reverse transcription polymerase chain reaction. PBVs have been identified in both normal and diarrheic faeces. Although their pathogenicity is still unclear, their potential needs further investigation.