Bisegmented Double-stranded RNA Genome Research Articles

Replication of single viruses across the kingdoms, Fungi, Plantae, and Animalia

It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.

A novel deltapartitivirus from red clover.

The family Partitiviridae has five genera, among which is the genus Deltapartitivirus. We report here the complete genome sequence of a deltapartitivirus from red clover, termed "red clover cryptic virus 3" (RCCV3). RCCV3 has a bisegmented double-stranded (ds) RNA genome. dsRNA1 and dsRNA2 are 1580 and 1589 nucleotides (nt) in length and are predicted to encode an RNA-directed RNA polymerase (RdRP) and a capsid protein (CP), respectively. The RCCV3 RdRP shares the highest sequence identity with the RdRP of a previously reported deltapartitivirus, Medicago sativa deltapartitivirus 1 (MsDPV1) (76.5%), while the RCCV3 CP shows 50% sequence identity to the CP of MsDPV1. RdRP- and CP-based phylogenetic trees place RCCV3 into a clade of deltapartitiviruses. The sequence and phylogenetic analyses clearly indicate that RCCV3 represents a new species in the genus Deltapartitivirus. RCCV3 was detectable in all three tested cultivars of red clover.

A unified genotypic classification of infectious bursal disease virus based on both genome segments

ABSTRACT Infectious bursal disease virus (IBDV) of chickens is a birnavirus with a bi-segmented double-stranded RNA genome, the segments designated as A and B. We performed phylogenetic analysis using a 366-bp fragment of segment A (nt 785–1150) and a 508-bp fragment of segment B (nt 328–835) of IBDV. A total of 463 segment A and 434 segment B sequences from GenBank, including the sequences of eight recent Bangladeshi isolates, were used in the analysis. The analysis revealed eight genogroups of segment A under serotype 1, designated as A1 (classical), A2 (US antigenic variant), A3 (very virulent), A4 (dIBDV), A5 (atypical Mexican), A6 (atypical Italian), A7 (early Australian) and A8 (Australian variant), and a single genogroup under serotype 2, designated as A0. On the other hand, segment B could be categorized into five genogroups irrespective of serotype, these being B1 (classical-like), B2 (very virulent-like), B3 (early Australian-like), B4 (Polish & Tanzanian) and B5 (Nigerian). Segment B of serotype 2 strains clustered within genogroup B1. With the bi-segmented genome of IBDV, these differences would allow for a total of 45 possible assortments. Based on the combinations of segment A and segment B genogroups observed in 463 IBDV strains, a total of 15 genotypes could be recognized. Recent Bangladeshi IBDV strains, isolated in 2016, appeared to be segment reassortants having segment A of genogroup A3 (very virulent) and segment B of genogroup B3 (early Australian-like). An extended system of nomenclature of IBDV strains is proposed.

Development of an RT-qPCR assay for the specific detection of a distinct genetic lineage of the infectious bursal disease virus

ABSTRACTThe infectious bursal disease virus (IBDV) is a major health threat to the world’s poultry industry despite intensive controls including proper biosafety practices and vaccination. IBDV (Avibirnavirus, Birnaviridae) is a non-enveloped virus with a bisegmented double-stranded RNA genome. The virus is traditionally classified into classic, variant and very virulent strains, each with different epidemiological relevance and clinical implications. Recently, a novel worldwide spread genetic lineage was described and denoted as distinct (d) IBDV. Here, we report the development and validation of a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay for the specific detection of dIBDVs in the global poultry industry. The assay employs a TaqMan-MGB probe that hybridizes with a unique molecular signature of dIBDV. The assay successfully detected all the assessed strains belonging to the dIBDV genetic lineage, showing high specificity and absence of cross-reactivity with non-dIBDVs, IBDV-negative samples and other common avian viruses. Using serial dilutions of in vitro-transcribed RNA we obtained acceptable PCR efficiencies and determination coefficients, and relatively small intra- and inter-assay variability. The assay demonstrated a wide dynamic range between 103 and 108 RNA copies/reaction. This rapid, specific and quantitative assay is expected to improve IBDV surveillance and control worldwide and to increase our understanding of the molecular epidemiology of this economically detrimental poultry pathogen.

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.

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 and Molecular Characterization of Porcine Picobirnavirus in Feces of Domestic Pigs from Kolkata, India

Picobirnaviruses (PBVs) are small, non-enveloped, 35-41nm virion with bisegmented double-stranded RNA genome. PBVs are widespread and were detected in feces of humans and a wide variety of animals. Domestic pig, one of the ubiquitous farm animal reported incessant association with a variety of viral zoonoses. The objective of our study is to find out the incidence of PBV infection in healthy domestic pigs. The study was conducted by collecting feces of healthy/asymptomatic pigs from a piggery located in an urban slum at Kolkata, India to detect PBV infections. All the 11 fecal samples were tested by polyacrylamide gel electrophoresis and reverse transcription-polymerase chain reaction assay. In this study, we report the first incidence of detection and molecular characterization of porcine PBV (BG-Por-2/2010 and BG-Por-7/2010) in feces of domestic pigs from India using the human PBV genogroup I specific primer pair: PicoB25(+) and PicoB43(-). Sequence comparison and phylogenetic analysis of partial RNA-dependent RNA polymerase gene of genome segment 2 revealed genetic relatedness to hitherto reported porcine, murine and human genogroup I PBVs from different geographical regions. This warrants a stringent global surveillance to study the potential zoonotic and emerging PBV infections.

Different Domains of the RNA Polymerase of Infectious Bursal Disease Virus Contribute to Virulence

BackgroundInfectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV has a bi-segmented double-stranded RNA genome. Segments A and B encode the capsid, ribonucleoprotein and non-structural proteins, or the virus polymerase (RdRp), respectively. Since the late eighties, very virulent (vv) IBDV strains have emerged in Europe inducing up to 60% mortality. Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood.Methodology, Principal FindingsStrain 88180 belongs to a lineage of pathogenic IBDV phylogenetically related to vvIBDV. By reverse genetics, we rescued a molecular clone (mc88180), as pathogenic as its parent strain. To study the molecular basis for 88180 pathogenicity, we constructed and characterized in vivo reassortant or mosaic recombinant viruses derived from the 88180 and the attenuated Cu-1 IBDV strains. The reassortant virus rescued from segments A of 88180 (A88) and B of Cu-1 (BCU1) was milder than mc88180 showing that segment B is involved in 88180 pathogenicity. Next, the exchange of different regions of BCU1 with their counterparts in B88 in association with A88 did not fully restore a virulence equivalent to mc88180. This demonstrated that several regions if not the whole B88 are essential for the in vivo pathogenicity of 88180.Conclusion, SignificanceThe present results show that different domains of the RdRp, are essential for the in vivo pathogenicity of IBDV, independently of the replication efficiency of the mosaic viruses.

Identification and pathogenicity of a natural reassortant between a very virulent serotype 1 infectious bursal disease virus (IBDV) and a serotype 2 IBDV

Infectious bursal disease virus (IBDV) causes an economically important, immunosuppressive disease in chickens. There are two serotypes of the virus that contain a bi-segmented double-stranded RNA genome. In December 2008, the first very virulent (vv)IBDV was identified in California, USA and in 2009 we isolated reassortant viruses in two different locations. Genome segment A of these reassortants was typical of vvIBDV serotype 1 but genome segment B was most similar to IBDV serotype 2. The CA-K785 reassortant caused 20% mortality in chickens but no morbidity or mortality in commercial turkey poults despite being infectious. There have been previous reports of natural reassortants between vvIBDV and other serotype 1 strains, but a natural reassortant between IBDV serotypes 1 and 2 has not been described. The apparent reassorting of California vvIBDV with an endemic serotype 2 virus indicates a common host and suggests vvIBDV may have entered California earlier than originally thought.

Crystal Structure of a Viral Protease Intramolecular Acyl-enzyme Complex: INSIGHTS INTO cis-CLEAVAGE AT THE VP4/VP3 JUNCTION OF TELLINA BIRNAVIRUS

Viruses of the Birnaviridae family are characterized by their bisegmented double-stranded RNA genome that resides within a single-shelled non-enveloped icosahedral particle. They infect birds, aquatic organisms, and insects. Tellina virus 1 (TV-1) is an Aquabirnavirus isolated from the mollusk Tellina tenuis. It encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is cleaved by the self-encoded protease VP4 to yield capsid precursor protein pVP2, peptide X, and ribonucleoprotein VP3. Here we report the crystal structure of an intramolecular (cis) acyl-enzyme complex of TV-1 VP4 at 2.1-Å resolution. The structure reveals how the enzyme can recognize its own carboxyl terminus during the VP4/VP3 cleavage event. The methyl side chains of Ala830(P1) and Ala828(P3) at the VP4/VP3 junction point into complementary shallow and hydrophobic S1 and S3 binding pockets adjacent to the VP4 catalytic residues: nucleophile Ser738 and general base Lys777. The electron density clearly shows that the carbonyl carbon of Ala830 is covalently attached via an ester bond to the Oγ of Ser738. A highly ordered water molecule in the active site is coordinated in the proper position to act as the deacylating water. A comparative analysis of this intramolecular (cis) acyl-enzyme structure with the previously solved intermolecular (trans) acyl-enzyme structure of infectious pancreatic necrosis virus VP4 explains the narrower specificity observed in the cleavage sites of TV-1 VP4.

Picobirnaviruses encode a protein with repeats of the ExxRxNxxxE motif

Picobirnaviruses possess a bisegmented double-stranded RNA genome. While the segment 2 encodes the RNA-dependent RNA polymerase, the segment 1 displays two open reading frames (ORFs). ORF2 was recently shown to code the capsid precursor and ORF1 product has not been characterized. In this study, we show that the three ORF1 sequences available in databases and representing three phylogenetically distant picobirnaviruses (two from human and one from rabbit hosts) encode proteins of various sizes (106–224 residues and without proline and cysteine) harbouring a particular sequence motif (ExxRxNxxxE) repeated four to ten times, depending on the virus species. Several algorithms predicted the three proteins to be mainly unfolded in the domains containing the repeats. The glycine-rich 25–40 amino acid long C-terminal domains containing hydrophobic residues with a periodicity of 3–4 residues are predicted structurally different of the upstream domains containing the motif repetitions. The ExxRxNxxxE sequence was not previously identified as a short linear motif in eukaryotic and prokaryotic proteins. Its function remains elusive.

Genogroup I picobirnavirus in diarrhoeic foals: Can the horse serve as a natural reservoir for human infection?

Picobirnaviruses (PBV) are small, non-enveloped viruses with a bisegmented double-stranded RNA genome. In this study a PBV strain, PBV/Horse/India/BG-Eq-3/2010, was identified in the faeces of a 10 month old weaned female foal with diarrhoea in January 2010 from Kolkata, India. Surprisingly, sequence comparison and phylogenetic analysis of a short stretch of the RNA dependent RNA polymerase gene revealed close genetic relatedness (> 98% nucleotide identity) to a human genogroup I PBV strain (Hu/GPBV1) detected earlier from the same part of India. Our observations together with earlier findings on genetic relatedness between human and animal PBV warrant further studies on zoonotic potential.

Development and validation of four real-time quantitative RT-PCRs specific for the positive or negative strands of a bisegmented dsRNA viral genome

Four tagged quantitative Real-Time RT-PCRs (qRT-PCRs) were developed to quantify the positive and negative strands of segments A and B of the bisegmented double-stranded RNA (dsRNA) genome of infectious bursal disease virus (IBDV, family Birnaviridae, genus Avibirnavirus). The qRT-PCRs were validated using single-stranded RNAs corresponding to each genomic strand (A+, B+, A−, B−). Specific quantitation proved possible from 5×107 to 5×102 copies of the template per reaction, with excellent reproducibility and linearity. The methods detected similar amounts of A+ and A− and of B+ and B− in a purified dsRNA viral genome preparation, thus corroborating the accuracy of quantitation. The qRT-PCRs were used to quantify the four strands in CsCl purified virus fractions and in samples collected during propagation of IBDV in cell culture. Purified virus fractions contained similar amounts of A− and B− strands, but also a large and unexplained excess of A+ and even more B+ strands. Results of the in vitro kinetic study showed an early accumulation of positive strands and a more delayed and lower accumulation of the A− and B− strands, both in similar amounts. These results suggest that minus strand synthesis occurs in IBDV after the equimolar packaging of A+ and B+ strands.

Genogroup I picobirnaviruses in pigs: evidence for genetic diversity and relatedness to human strains

Picobirnaviruses (PBVs) are small, non-enveloped viruses with a bisegmented double-stranded RNA genome. Their pathogenic potential, ecology, and evolutionary features are largely unexplored. Here, we describe the molecular analysis of porcine PBVs identified in the intestinal content of dead pigs. Six of 13 positive samples were cloned and then subjected to single-strand conformation polymorphism analysis and nucleotide sequencing. All clones belonged to genogroup I PBVs and almost all clones clustered on separate branches from human strains. A single strain shared a notably close genetic relationship with a Hungarian human PBV strain (89.9 nt and 96.4% aa identity). Genetic diversity was also observed among strains identified in mixed infections. Single point mutations and deleterious mutations within highly related strains suggested that PBVs exist as quasispecies in the swine alimentary tract. Clones with complete sequence identities originating from different animals suggested effective animal-to-animal transmission of the virus. Our findings indicate that infection with genogroup I PBVs is common in pigs.

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.

Synthesis of Reassortant Infectious Bursal Disease Virus in Chickens Injected Directly with Infectious Clones from Different Virus Strains

Abstract The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, containing a bisegmented double-stranded RNA genome, encodes four structural viral proteins, VP1, VP2, VP3, and VP4, as well as a non-structural protein, VP5. In the present paper, the segment A from two IBDV strains, field isolate ZJ2000 and attenuated strain HZ2, were inserted into one NaeI site by site-directed silent mutagenesis and subcloned into the eukaryotic expression plasmid pCI under the control of the human cytomegalovirus (hCMV) immediate early enhancer and promoter to construct the recombinant plasmids pCI-AKZJ2000 and pCI-AKHZ2, respectively. Each of the two recombinants was combined with another recombinant pCI plasmid containing the marked segment B of strain HZ2 (pCI-mB), and injected intramuscularly into nonimmunized chickens. Two chimeric IBDV strains were recovered from the chickens. Two out of eight chickens in each of two groups showed the bursal histopathological change. The reassortant virus derived from pCI-AKZJ2000/pCI-mB can infect chicken embryos and shows relatively low virulence. We have developed a novel virus reverse genetic approach for the study of IBDV. The results also form the basis for investigating the role of VP1 in viral replication and pathogenecity.

Synthesis of Reassortant Infectious Bursal Disease Virus in Chickens Injected Directly with Infectious Clones from Different Virus Strains

The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, containing a bisegmented double-stranded RNA genome, encodes four structural viral proteins, VP1, VP2, VP3, and VP4, as well as a non-structural protein, VP5. In the present paper, the segment A from two IBDV strains, field isolate ZJ2000 and attenuated strain HZ2, were inserted into one NaeI site by site-directed silent mutagenesis and subcloned into the eukaryotic expression plasmid pCI under the control of the human cytomegalovirus (hCMV) immediate early enhancer and promoter to construct the recombinant plasmids pCI-AKZJ2000 and pCI-AKHZ2, respectively. Each of the two recombinants was combined with another recombinant pCI plasmid containing the marked segment B of strain HZ2 (pCI-mB), and injected intramuscularly into non-immunized chickens. Two chimeric IBDV strains were recovered from the chickens. Two out of eight chickens in each of two groups showed the bursal histopathological change. The reassortant virus derived from pCI-AKZJ2000/pCI-mB can infect chicken embryos and shows relatively low virulence. We have developed a novel virus reverse genetic approach for the study of IBDV. The results also form the basis for investigating the role of VP1 in viral replication and pathogenecity.

Infectious Pancreatic Necrosis Virus RNA Cleavage In Vitro by Hammerhead Ribozymes and Enhancement of Ribozyme Catalysis by Oligonucleotide Facilitators.

Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus, has a bisegmented double-stranded RNA genome consisting of a 3.2-kb A segment and a 2.9-kb B segment. To determine the function of IPNV's viral proteins and to study the effects of viral RNA cleavage by hammerhead ribozymes, we cloned and sequenced the IPNV E1S strain of the A segment. After sequencing, we continued to study the virus pathogens inhibited by ribozyme cleavage and analyzed the cleavage of the virus RNA in vitro. The templates (VP2, 1220 bp) for in vitro transcription of S569 and S969 (substrates 569 and 969 bp in length) were synthesized by polymerase chain reaction. The DNA templates of hammerhead ribozymes targeted different sites in the partial sense RNA of IPNV. These templates were chemically synthesized RNAs prepared by runoff transcription of amplification products or synthetic DNA templates containing a T7 RNA polymerase promoter, and were used to characterize several properties of the cleavage reaction at 25 degrees C in 12 mM Mg(2+). Under this condition (25 degrees C, 12 mM Mg(2+)), the hammerhead ribozymes formed an estimated fraction of product during the reaction of only 30% in cleaving long RNA substrates in vitro. Short DNA facilitators (12 or 24-mers) that bind adjacent to either the 3' or 5' end of the ribozyme enhanced the rate of cleavage of the long RNA substrates containing 569 and 969 nucleotides, respectively, in trans. The hammerhead ribozymes with 3'-end facilitators reacted more efficiently (i.e., 65%).

A comparison of two RNA isolation methods for double-stranded RNA of infectious bursal disease virus

Infectious bursal disease virus (IBDV), a member of the birnaviridae family, contains a bisegmented double-stranded RNA (dsRNA) genome. The segments are linked covalently at 5′ termini by a large (90 kDa) viral genomic protein that migrates similar to viral RNA dependent RNA polymerase of IBDV. A proteinase K digestion based approach and acid-guanidium-phenol-chloroform (AGPC) RNA extraction method were used to extract dsRNA of IBDV from infected bursae. After extraction, dsRNA of IBDV was purified by precipitation with lithium chloride. The yield and purity of dsRNA of IBDV extracted by AGPC method was lower than that of proteinase K digestion based approach. This observation correlates with the presence of a genome-linked protein in IBDV. Although dsRNA obtained by both methods are suitable for reverse transcription-polymerase chain reaction (RT-PCR) amplification of at least up to 1201 base pairs (bp) of cDNA, dsRNA extracted by the proteinase K digestion method is more suitable than that by AGPC method for the amplification of longer fragments (1958 bp) of IBDV cDNA by PCR.

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.