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Genomic characterisation of the large segment of a rabbit picobirnavirus and comparison with the atypical picobirnavirus of Cryptosporidium parvum.

The 2362 base pair sequence of the larger of the two double stranded RNA genome segments of a rabbit strain of picobirnavirus (PBV) has a major open reading frame (ORF) of 591 amino acids and two smaller ORFs of 55 and 155 amino acids. A clone of the segment did not hybridise with other viral bisegmented ds RNAs from faecal samples. There is no relationship in sequence or organisation between this PBV sequence and the bisegmented dsRNAs found associated with Cryptosporidium parvum. This suggests that there are at least two distinct classes of bisegmented dsRNA viruses or viral-like agents in faeces.

Animals↗

Detection and characterization of a novel bisegmented double-stranded RNA virus (picobirnavirus) from rabbit faeces.

In two separate studies rabbits were fed orally with human and rabbit "picobirnaviruses". Polyacrylamide gel electrophoresis (PAGE) of nucleic acid extracted from faecal samples collected from inoculated rabbits revealed the presence of discrete equimolar bands, typical of picobirnaviruses, in several specimens. The genome profiles detected in both studies differed significantly from that of the inoculum suggesting that passage of the inoculated picobirnaviruses had not taken place and that the bands were a co-incidental finding. The presence of rabbit picobirnaviruses was confirmed by characterization of the genome bands, as dsRNA by enzyme digestion and by their co-sedimentation in caesium chloride (CsCl) gradients with 32 nm virus particles at a buoyant density of 1.39 g/ml. Picobirnavirus genome segments varied in size in a range between 2.3-2.6 kilo base pairs (kbp) and 1.6-1.9 kbp for the slow and fast migrating bands, respectively. Immune electron microscopy of the picobirnavirus particles revealed round or slightly hexagonal particles with a smooth surface and a mean diameter of 30.7 nm. In one rabbit, an immune response, temporally associated with picobirnaviruses excretion, was demonstrated by immune electron microscopy (IEM) supporting the view that picobirnaviruses may be vertebrate viruses. Two antigenically distinct picobirnavirus strains were defined by IEM.

Animals↗

Molecular epidemiology of human picobirnaviruses among children of a slum community in Kolkata, India.

Picobirnaviruses are a group of unclassified, non-enveloped, small spherical viruses, 35-41 nm in diameter without any apparent surface morphology. They have characteristic bisegmented double stranded RNA genome of two types namely large profile (2.3-2.6 kbp for the larger and 1.5-1.9 kbp for the smaller segment, respectively) or small profile (1.75 and 1.55 kbp for segments 1 and 2, respectively). Human picobirnaviruses (n=12 positives; 2/56 diarrhoeic children and 10/607 non-diarrhoeic children) with large (n=11) or small (n=1) genome pattern were observed in faecal specimens of children from a slum community by silver stained PAGE gels. Faecal specimen from four asymptomatic cases (P597_02_IND, K135_02_IND, A373_03_IND, A356_03_IND) and one diarrhoeic case (K135_03_IND) had genogroup I picobirnaviruses (1-CHN-97 like) showing amplicons within the 201 bp region, with primers PicoB25-PicoB43, targeting the conserved domain of RNA-dependent RNA polymerase (RdRp) gene. It was interesting to note that only the PBV strain P597_02_IND from Kolkata with large genome was closely related to a reported strain (similarity with 2-GA-91 from USA was 87% at the nucleotide level and 90% at the amino acid level). Sequence analysis showed three conserved amino acid domains as well as a highly conserved D-S-D motif, characteristic of RNA-dependent RNA polymerase gene of bisegmented, double stranded RNA viruses. Sequence data of the picobirnavirus A356_03_IND indicated strong heterogeneity with all other picobirnavirus strains sequenced till date. After nearly a decade a genogroup II picobirnavirus strain (R227_03_IND) was isolated from a diarrhoea case in the community, with small genome profile and amplified with specific primers PicoB23-PicoB24; but the sequence data showed that it was divergent from the hitherto reported prototype strain 4-GA-91 of genogroup II human picobirnaviruses.

Amino Acid Sequence↗

Detection of picobirnavirus in HIV-infected patients with diarrhea in Argentina.

Diarrhea due to enteric pathogens is an important complication of advanced HIV infection. Picobirnaviruses are agents recently linked with human enteritis. In total, 197 fecal samples collected from HIV-infected and noninfected patients with and without diarrhea were investigated for the presence of rotavirus and picobirnavirus by polyacrylamide gel electrophoresis. Picobirnavirus was detected in 8.8% of 57 HIV-infected patients with diarrhea, but it was detected in neither those without diarrhea (p<.018) nor in the group of subjects uninfected with HIV (p<.022). All genomic electropherotypes of picobirnavirus strains had a wide pattern. Picobirnavirus genome segments varied in size between 2.4 and 2.7 and 1.6 and 1.9 kbp for the slow and fast migrating bands, respectively. Rotaviruses were not detected in any of the clinical groups studied. Two methods for the extraction of nucleic acid-phenol/chloroform and guanidinium thiocynate (GTC)/silica-were compared. Detection of picobirnavirus by polyacrylamide gel electrophoresis was 2.5 times more sensitive following guanidinium thiocynate RNA extraction. This investigation offers preliminary results about the circulation of picobirnavirus in HIV-infected patients in Córdoba, Argentina.

Adult↗

Cloning of human picobirnavirus genomic segments and development of an RT-PCR detection assay.

Nearly full-length genomic segments 2 and a partial-length genomic segment 1 of human picobirnavirus were cloned and sequenced. The clones were derived from viruses obtained from human immunodeficiency virus (HIV)-infected patients in Atlanta, Georgia (strains 3-GA-91 and 4-GA-91) and a nonHIV-infected person from China (strain 1-CHN-97). The picobirnavirus genomic segments lacked sequence similarities with other viral sequences in GenBank and EMBL. Comparison of genomic segment 1 from a human and a rabbit picobirnavirus identified a region of 127 nucleotides with 54.7% identity. The genomic segments 2 of the 4-GA-91 and 1-CHN-97 strains had 41.4% nucleic acid identity and 30.0% amino acid similarity and contained amino acid motifs typical of RNA-dependent RNA polymerase genes. Reverse transcription-PCR detection assays were developed with primers targeted to the genomic segments 2 of strains 4-GA-91 or 1-CHN-97. Picobirnaviruses related to the China strain were the predominant viruses detected in stool samples from people in four countries on three continents. Picobirnaviruses were detected in samples from two outbreaks of gastroenteritis in long-term elder care facilities but were not determined to be the primary pathogen. Our findings support the view that picobirnaviruses constitute a distinct family of viruses.

Amino Acid Sequence↗

Sequence heterogeneity among human picobirnaviruses detected in a gastroenteritis outbreak.

Human picobirnaviruses characterised in this study were serendipitously detected in a non-bacterial gastroenteritis outbreak when specimens were examined for the presence of human rotaviruses using polyacrylamide gel electrophoresis. Of ten stool samples sent for virological examination, two, three, and one specimens were positive for human caliciviruses, picobirnaviruses, and both viruses, respectively. Partial sequences of the RNA-dependent RNA polymerase gene were determined for three picobirnavirus-positive samples. The sequence identity among these three strains was 60% to 65% for the nucleic acid and 64% to 70% for the deduced amino acid sequences. Phylogenetic analysis revealed that each of the three strains clustered with strains identified in geographically separate areas. In contrast, human calicivirus strains co-incidentally identified, showed complete nucleotide sequence identity. These findings demonstrate a lack of common exposure to or point of source for picobirnavirus infection, suggesting that the outbreak was caused by human caliciviruses. Further studies are needed to determine the etiologic role and to establish the taxonomic basis of picobirnaviruses.

Adult↗

Complete nucleotide sequences of two RNA segments of human picobirnavirus.

Picobirnaviruses are unclassified, non-enveloped, spherical, small viruses with a genome comprising two double-stranded RNA segments. Only incomplete sequence data on picobirnaviruses are available so far. By cloning involving single primer amplification, full-length cDNAs were prepared corresponding to RNA segments 1 and 2 of a picobirnavirus (strain Hy005102) isolated from a stool specimen from an infant with acute non-bacterial gastroenteritis in Thailand, and the complete nucleotide sequences were determined. RNA segments 1 and 2 are 2,525 and 1,745 base pairs in length, respectively. RNA segment 1 encodes two open reading frames (ORFs) of 224 and 552 amino acids, and RNA segment 2 codes for a single ORF of 534 amino acids. On comparison with a part of the nucleotide sequences of the RNA segment, 2 of the other published picobirnavirus strains, the Thai strain was found to be related most closely to one of the US strains.

Base Sequence↗

Detection of Genogroup I and II human picobirnaviruses showing small genomic RNA profile causing acute watery diarrhoea among children in Kolkata, India.

Picobirnaviruses (PBVs) with bisegmented small RNA genome profile (1.75 and 1.55kbp for segment 1 and 2, respectively) were detected from 1999 to 2003 in faecal specimens of acute watery diarrhoea cases, largely children (n=20) and an adult in Kolkata, India. Varying degrees of dehydration necessitated their visit to hospital for further treatment and management of acute watery diarrhoea. PBV was associated with rotavirus (n=3) or astrovirus (n=3) and with both in one case. No co-infection with norovirus, sapovirus or adenovirus was detected in the picobirnavirus positive cases. No co-infection with parasites (Cryptosporidium spp., Giardia spp., Entamoeba spp., helminths) or bacteria (Vibrio spp., Shigella spp., Escherichia coli) was detected among the picobirnavirus positive cases. There was a single instance of co-infection with Salmonella spp. (n=1). PBVs not associated with serious diarrhoea illness and showing large genome profile (2.3-2.6 and 1.5-1.9kbp for segment 1 and 2, respectively) have earlier been reported in adult individuals and recently among children from a slum community in Kolkata, India. The short genome profile PBVs associated with acute watery diarrhoea may be another emerging diarrhoeagenic virus in Kolkata, India. Molecular characterization using reported primers PicoB25-PicoB43 for Genogroup I and PicoB23-PicoB24 for Genogroup II in RT-PCR showed the presence of Genogroup I PBVs (n=6) and Genogroup II PBVs (n=5), while some could not be amplified (n=3) with these primers. Sequence analysis of Genogroup I amplicons indicated remarkable sequence heterogeneity. After more than a decade, four PBV positives of Genogroup II were detected during this study. Phylogenetic analysis showed varying degree of genetic diversity amongst PBV strains from Kolkata and other countries.

Adult↗

Identification of picobirnavirus from faeces of Italian children suffering from acute diarrhea.

Polyacrylamide gel electrophoresis of nucleic acid extracted from stool samples of diarrhoeic children revealed in 3 out of 690 (0.43%) specimens two electrophoretic bands with a migration pattern characteristic of picobirnavirus ds-RNA. In none of the 92 control children were similar bands detected. No other potential enteric pathogens were found in the patients with picobirnavirus infection.

Child↗

Detection of a picobirnavirus associated with Cryptosporidium positive stools from humans.

A picobirnavirus with an atypical genome profile was detected by polyacrylamide gel electrophoresis (PAGE) in 37% (20/54) of human faecal samples also containing oocysts of Cryptosporidium typical of C. parvum. This virus shares many of the characteristics of the previously described picobirnaviruses, but has a significantly smaller genome (1.75 and 1.55 Kbp).

Animals↗

Identification of picobirnavirus, viruses with bisegmented double stranded RNA, in rabbit faeces.

Picobirnaviruses are a novel group of viruses recently found in the faeces of several species of vertebrates. Examination by polyacrylamide gel electrophoresis of rabbit faecal samples collected in one animal facility revealed the viruses in 23 (11 per cent) of 211 samples. Further analysis by electron microscopy and caesium chloride isopycnic centrifugation confirmed the presence of picobirnaviruses in the samples. The oral inoculation of three newly weaned rabbits with purified viruses resulted in the excretion of a virus with an electropherotype similar to the inoculum, by two of the three inoculated animals. Maximal viral shedding was detected 13 days after inoculation. No sign of diarrhoea was observed either in the inoculated animals or in the virus excreting animals surveyed. No antibody activity could be detected in the paired serum samples taken from the inoculated animals.

Animals↗

[Picobirnavirus].

Picobirnavirus is named after the small birnavirus which contains two double-stranded RNA segments as a genome. However, their properties are quite different to each other. Although the virus has been detected mainly from the stools of gastroenteritis patients and several mammals and birds, the pathogenicity of the virus has not been established. Characterizations of the virus are hampered due to the lack in the system for multiplication of the virus in cultured cells or experimental animals. Recently, complete nucleotide sequences of two RNA segments of a human picobirnavirus detected in Thailand were determined.

Animals↗

Picobirnavirus, a novel group of undescribed viruses of mammals and birds: a minireview.

Picobirnavirus, a novel group of viruses recently detected in children and several species of animals including chickens, are different from the existing members of the family Birnaviridae. Picobirnavirus (PBV) is the tentatively proposed name for the group of these viruses. These viruses are 30-40 nm in diameter and have icosahedral symmetry with triangulation number (T) equal to 3. Their buoyant density in CsCl is 1.4 g/ml. Their genome is bi- or trisegmented double-stranded RNA (dsRNA) with segment lengths of 2.6 and 1.9 kbp for bisegmented and 2.9, 2.4 and 0.9 kbp for trisegmented genomes. The electrophoretic migration profile has considerable heterogeneity. PVBs are detected in diarrhoeic as well as non-diarrhoeic animals, hence, their potential needs further investigation.

Animals↗

Detection and characterisation of bisegmented double-stranded RNA viruses (picobirnaviruses) in human faecal specimens.

The prevalence of picobirnaviruses (PBVs) in human stools was investigated by polyacrylamide gel electrophoresis (PAGE) analysis of 832 faecal specimens collected between 1982 and 1993 from patients in various clinical groups. Similar prevalences (9-13%) were detected in patients with or without gastroenteritis and throughout the age range of 3 to > 65 years. Two methods for the extraction of nucleic acid, a phenol/chloroform method and a guanidinium thiocynate (GTC)/silica method, were compared. Detection of PBVs by PAGE was three times more sensitive following RNA extraction by the GTC/silica method. Characterisation of three strains was carried out. Segment sizes ranged from 1.625 to 1.95 kilo base pairs (Kbp) and 2.2 to 2.5 Kbp for the fast and slow migrating bands, respectively. The nuclic acid was shown to be double-stranded RNA (dsRNA) by nuclease digestion. PBV-like particles were detected by electron microscopy in two PAGE-positive stools. Virion diameters ranged from 35 to 41 nm and a buoyant density of 1.38-1.4 g/ml in caesium chloride (CsCl) was demonstrated. These findings suggest that PBVs are widespread in humans in the United Kingdom. However, no disease association could be demonstrated.

Adolescent↗

Identification of a bisegmented double-stranded RNA virus (picobirnavirus) in calf faeces.

To determine the incidence of rotavirus infection among dairy herds in the State of São Paulo, Brazil, 576 faecal samples obtained from calves aged 1-45 days with and without diarrhoea, reared on 63 dairy cattle farms, were analyzed. Polyacrylamide gel electrophoresis (PAGE) identified 28 samples positive for group A rotavirus, while four samples, two diarrhoeic and two non-diarrhoeic, showed a bisegmented genome with a typical picobirnavirus pattern. Electron microscopy revealed spherical virus particles with a diameter of 37 nm and without a defined surface structure. The present study is the first report of a bisegmented virus identified in cattle in Brazil.

Animals↗

Molecular diversity of partial-length genomic segment 2 of human picobirnavirus.

OBJECTIVES: This study was carried out in order to evaluate the efficacy of the recently developed picobirnavirus (PBV) sets of primers and to establish the phylogenetic relationships of Argentine strains with PBV strains isolated in China and the USA. METHODS: Thirteen fecal specimens tested as positive for PBV by polyacrylamide gel electrophoresis were analyzed by reverse transcription-polymerase chain reaction assays using primers target to the genomic segments 2 of PBV strains isolated in China and the USA. The amplicons were sequenced and analyzed. RESULTS: Primers derived from the China strain produced amplicons in only 4 of the 13 specimens (30.76%). No sample was revealed as positive with the primers derived from the US strain. DNA sequencing of polymerase chain reaction products differed in nucleic acid and amino acid sequences by 13.9-42.28% and 18.1-51.1%, respectively. Despite this strain diversity, three domains of conserved nucleotide sequences as well as the amino acid motif D-S-D typical of RNA-dependent RNA polymerase gene of double-strand RNA viruses were identified. Comparatively, these conserved regions were also identified in homologous PBV strains from the USA and China. Phylogenetic analysis showed no time or geographic clustering. CONCLUSIONS: These findings provide evidence that PBV may represent an emerging heterogeneous group of viruses.

Adolescent↗

[Detection of picobirnaviruses by electrophoresis of RNA in polyacrylamide gel].

Double-segment profiles typical of picobirnavirus (PBV) were detected (during 1994-2001) in nucleic acids extracted from feces of children (3 cases) and calf (1 case) with diarrhea by using the method of electrophoresis. The human genomic PBV segments migrated in the polyacrylamide gel (PAAG) as dsRNA segments sized 1.7 and 2.4 kbp for small and large segments, respectively; the similar calf sizes were 1.5 and 2.6 kbp. PBVs were detected in various places of the Nizhny Novgorod Region and at different time periods. It was for the first time that the PBV circulation was proven to be present in Russia's territory. However, their association with diarrhea was not reliably established, and the pathogenic PBV potential needs further investigations.

Child↗