The genome of Uukuniemi virus consists of three unique RNA segments

The three RNA species isolated from virions of Uukuniemi virus, a proposed member of the newly defined Bunyaviridae family, have been characterized by analysis of ³²P-labeled ribonuclease T1 oligonucleotides separated on two-dimensional polyacrylamide gels. Each RNA species contains unique oligonucleotides not present in the two others, indicating that the genome of this virus is segmented. Each segment appears to contain a unique primary sequence with little or no overlapping among the segments. The complexities of the RNA segments as calculated from the radioactivity in unique oligonucleotides of defined lengths are about 8000 (L RNA), 3500 (M) and 1900 (S) nucleotides. Since these values are similar to the molecular weights determined by other methods, each size class of RNA corresponds to a single molecular species. The presence of a 5′ terminal pppAp … structure in each RNA segment confirms indications from electron microscopy that the apparently circular RNA segments are not covalently closed. The absence of either a 5′ terminal “cap” or 3′ terminal poly(A) supports the concept that Uukuniemi virus is a negative strand virus.     

Immunofluorescent detection of double-stranded RNA in cells infected with reovirus, infectious pancreatic necrosis virus, and infectious bursal disease virus

Urea treatment of ethanol-fixed virus-infected cells exposed nucleic acid antigens for immunofluorescence. Three double-stranded (ds) RNA-containing viruses showed bright fluorescence using antibodies against dsRNA. Three single-stranded RNA-containing viruses showed less intense fluorescence with anti-dsRNA. Four out of five cell lines persistently infected with various RNA-containing viruses showed no dsRNA detectable by immunofluorescence.     

Genomic structure of the large RNA segment of infectious bursal disease virus.

The larger RNA segment of infectious bursal disease virus (IBDV: Australian strain 002-73) has been characterized by cDNA cloning and nucleotide sequence analysis. We believe IBDV is the first birnavirus to be sequenced and so have confirmed the coding region by N-terminal amino acid sequence analysis of intact viral proteins and several tryptic peptide fragments. The large RNA segment encodes in order the 37-kDa, 28-kDa and 32-kDa proteins within a continuous open reading frame and the primary translation product appears to be subsequently processed into the mature viral proteins. The large protein precursor is still processed into the 32-kDa host protective immunogen when expressed as a fusion protein in E. coli. These results are in marked contrast to the predictions from in vitro translation data that birnavirus genomes are expressed as polycistronic templates. We can now propose that birnaviruses, in particular IBDV, possess monocistronic segments and that the precursor is proteolytically processed in vivo. The sequence data presented for the 32-kDa host protective immunogen may provide the basic information needed for the production of an effective subunit vaccine against this commercially important virus.     

Capped and conserved terminal structures in human rotavirus genome double-stranded RNA segments.

Both 3'- and 5'-terminal structures of human rotavirus genome double-stranded RNA segments were determined. RNAs were labeled at the 3'-termini with [32P]pCp by incubation with RNA ligase and at the 5'-termini with [32P]phosphate by polynucleotide kinase or, in the case of 5' caps, with 3H by chemical modification with [3H]NaBH4. Examination of radiolabeled termini released by digestion with several base-specific RNases revealed that rotavirus RNA segments are base paired end-to-end and contain the same terminal structures: (formula; see text)     

Intracellular interference of infectious bursal disease virus

A search for dominant-negative mutant polypeptides hampering infectious bursal disease virus (IBDV) replication has been undertaken. We have found that expression of a mutant version of the VP3 structural polypeptide known as VP3/M3, partially lacking the domain responsible for the interaction with the virus-encoded RNA polymerase, efficiently interferes with the IBDV replication cycle. Transformed cells stably expressing VP3/M3 show a significant reduction (up to 96%) in their ability to support IBDV growth. Our findings provide a new tool for the characterization of the IBDV replication cycle and might facilitate the generation of genetically modified chicken lines with a reduced susceptibility to IBDV infection.     

Evidence that the genome of hepatitis A virus consists of single-stranded RNA.

Nucleic acid was extracted from purified hepatitis A virus, radiolabeled with 125I, and shown to consist of single-stranded RNA which sediments at 35S and contains sequences of polyadenylic acid. These findings are consistent with hepatitis A virus being a member of the genus Enterovirus within the family Picornaviridae.     

Structural and growth characteristics of infectious bursal disease virus.

The infectious bursal disease virus is not enveloped and has a diameter of 60 nm and a density of about 1.32 g/ml. It contains two pieces of single-stranded RNA with molecular weights close to 2 X 10(6). The capsid is made up of four major polypeptides with molecular weights of 110,000, 50,000, 35,000, and 25,000. The virus replicates in chicken embryo fibroblasts rather than in epitheloid cells. After an eclipse period of 4 h, virus production reaches a maximum about 12 h later. The virus has no structural or biological similarities with defined avian reoviruses, and it cannot be classified in one of the established taxonomic groups.     

Polymorphism of the migration of double-stranded RNA genome segments of avian reoviruses

A number of field isolates of avian reovirus were characterized by analysis of the migration pattern of their genomic double-stranded RNA (dsRNA) segments upon polyacrylamide gel electrophoresis. Comparison of the various isolates has demonstrated (i) no relationship between serotype and migration of any individual dsRNA segment, (ii) marked polymorphism of migration patterns of all dsRNA segments among isolates of the same serotype as well as among different serotypes, (iii) no correlation between genotype and disease state, (iv) less marked variability in migration pattern from isolates within a restricted geographic locale compared to isolates from distant locales, (v) the presence of a single genotype in local outbreaks of disease, and (vi) the relative invariant migration of several dsRNA segments among the avian reoviruses, one of which (S1) may serve to distinguish the avian from the mammalian reoviruses.     

Characteristics of bursal T lymphocytes induced by infectious bursal disease virus

Infectious bursal disease virus (IBDV) is an avian lymphotropic virus that causes immunosuppression. When specific-pathogen-free chickens were exposed to a pathogenic strain of IBDV (IM), the virus rapidly destroyed B cells in the bursa of Fabricius. Extensive viral replication was accompanied by an infiltration of T cells in the bursa. We studied the characteristics of intrabursal T lymphocytes in IBDV-infected chickens and examined whether T cells were involved in virus clearance. Flow cytometric analysis of single-cell suspensions of the bursal tissue revealed that T cells were first detectable at 4 days postinoculation (p.i.). At 7 days p.i., 65% of bursal cells were T cells and 7% were B cells. After virus infection, the numbers of bursal T cells expressing activation markers Ia and CD25 were significantly increased (P<0.03). In addition, IBDV-induced bursal T cells produced elevated levels of interleukin-6-like factor and nitric oxide-inducing factor in vitro. Spleen and bursal cells of IBDV-infected chickens had upregulated gamma interferon gene expression in comparison with virus-free chickens. In IBDV-infected chickens, bursal T cells proliferated in vitro upon stimulation with purified IBDV in a dose-dependent manner (P<0.02), whereas virus-specific T-cell expansion was not detected in the spleen. Cyclosporin A treatment, which reduced the number of circulating T cells and compromised T-cell mitogenesis, increased viral burden in the bursae of IBDV-infected chickens. The results suggest that intrabursal T cells and T-cell-mediated responses may be important in viral clearance and promoting recovery from infection.     

Different domains of the RNA polymerase of infectious bursal disease virus contribute to virulence

BACKGROUND: Infectious 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 FINDINGS: Strain 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, SIGNIFICANCE: The 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.     

Modulation of macrophages by infectious bursal disease virus

Infectious bursal disease is one of the most important naturally occurring viral diseases of chickens worldwide. The causative agent, infectious bursal disease virus (IBDV), belongs to the family Birnaviridae. This virus causes an acute, highly contagious and immunosuppressive disease in chickens. The virus infects and destroys actively dividing IgM-bearing B cells. Although B cells are the principal targets for IBDV, recent data show that the virus also infects macrophages. IBDV-infected macrophages produce various cytokines and chemokines which may play an important role in the protection and/or pathogenesis of IBDV. In this review, the modulatory effects of IBDV on macrophages will be discussed.     

The double-stranded RNA genome of yeast virus L-A encodes its own putative RNA polymerase by fusing two open reading frames

The L-A double-stranded RNA virus of Saccharomyces cerevisiae encodes its major coat protein (80 kDa) and a minor single-stranded RNA binding protein (180 kDa) that has immunological cross-reactivity with the major coat protein. The sequence of L-A cDNA clones revealed two open reading frames (ORF), ORF1 and ORF2. These two reading frames overlap by 130 base pairs and ORF2 is in the -1 reading frame with respect to ORF1. Although the major coat protein of the viral particles is encoded by ORF1, the 180-kDa protein is derived from the entire double-stranded RNA genome by fusing ORF1 and ORF2, probably by a -1 translational frameshift. Within the overlapping region is a sequence similar to that producing a -1 frameshift by "simultaneous slippage" in retroviruses. The coding sequence of ORF2 shows a pattern characteristic of viral RNA-dependent RNA polymerases of icosahedral (+)-strand RNA viruses. Thus, the 180-kDa protein is analogous to gag-pol fusion proteins.     

Multiple double-stranded RNA segments are associated with virus particles infecting Trichomonas vaginalis.

Previous studies demonstrated that some isolates of the sexually transmitted protozoan Trichomonas vaginalis are infected with a nonsegmented, double-stranded RNA (dsRNA) virus. A reexamination of the total dsRNA extracted from several virus-harboring isolates indicated the presence of at least three dsRNAs with sizes ranging from 4.8 to 4.3 kbp. The double-stranded nature of each of the three segments was determined by hybridization experiments using riboprobes of opposite polarities obtained from cDNA generated to each of the segments. All three segments were present in agar clones originating from single organisms of T. vaginalis isolates, suggesting that the three segments were not the result of a mixed population of trichomonads harboring different sizes of dsRNA. The three segments were associated with CsCl-purified virus particles, as evidenced by electron microscopy, and RNAse treatment of the preparation containing virus particles did not destroy the dsRNAs. Finally, the individual dsRNA segments were purified for use as probes to determine whether the three dsRNAs shared any sequence homology. Each end-labeled dsRNA segment did not cross-hybridize to any of the other two segments, a finding consistent with the hybridization of labeled cDNAs to only the segments from which they were derived. These results show that the coding capacity of the dsRNA virus may be at least three times greater than that estimated earlier and illustrates further the complexity of this virus-parasite interrelationship.     

Recombinant infectious bursal disease virus carrying hepatitis C virus epitopes

The delivery of foreign epitopes by a replicating nonpathogenic avian infectious bursal disease virus (IBDV) was explored. The aim of the study was to identify regions in the IBDV genome that are amenable to the introduction of a sequence encoding a foreign peptide. By using a cDNA-based reverse genetics system, insertions or substitutions of sequences encoding epitope tags (FLAG, c-Myc, or hepatitis C virus epitopes) were engineered in the open reading frames of a nonstructural protein (VP5) and the capsid protein (VP2). Attempts were also made to generate recombinant IBDV that displayed foreign epitopes in the exposed loops (P(BC) and P(HI)) of the VP2 trimer. We successfully recovered recombinant IBDVs expressing c-Myc and two different virus-neutralizing epitopes of human hepatitis C virus (HCV) envelope glycoprotein E in the VP5 region. Western blot analyses with anti-c-Myc and anti-HCV antibodies provided positive identification of both the c-Myc and HCV epitopes that were fused to the N terminus of VP5. Genetic analysis showed that the recombinants carrying the c-Myc/HCV epitopes maintained the foreign gene sequences and were stable after several passages in Vero and 293T cells. This is the first report describing efficient expression of foreign peptides from a replication-competent IBDV and demonstrates the potential of this virus as a vector.     

Complete genome sequence of a double-stranded RNA virus from avocado

A number of avocado (Persea americana) cultivars are known to contain high-molecular-weight double-stranded RNA (dsRNA) molecules for which a viral nature has been suggested, although sequence data are not available. Here we report the cloning and complete sequencing of a 13.5-kbp dsRNA virus isolated from avocado and show that it corresponds to the genome of a new species of the genus Endornavirus (family Endornaviridae), tentatively named Persea americana endornavirus (PaEV).     

In vitro expression and monoclonal antibody of RNA-dependent RNA polymerase for infectious bursal disease virus

VP1, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV), has been suggested to play an essential role in the replication and translation of viral RNAs. In this study, we first expressed the complete VP1 protein gene in Escherichia coli (E. coli), and then the produced polyclonal antibody and four monoclonal antibodies (mAbs) to recombinant VP1 protein (rVP1) were shown to bind the IBDV particles in chicken embryo fibroblast and Vero cells. The epitopic analysis showed that mAbs 1D4 and 3C7 recognized respectively two distinct antigenic epitopes on the rVP1 protein, but two pair of mAbs 1A2/2A12 and 1E1/1H3 potentially recognized another two topologically related epitopes. Immunocytochemical stainings showed that VP1 protein formed irregularly shaped particles in the cytoplasm of the IBDV-infected cells. These results demonstrated that the mAbs to rVP1 protein could bind the epitopes of IBDV particles, indicating that the rVP1 protein expressed in E. coli was suitable for producing the mAb to VP1 protein of IBDV, and that the cytoplasm could be the crucial site for viral genome replication of IBDV.     

Genetic reassortment of infectious bursal disease virus in nature

Infectious bursal disease virus (IBDV), a double-stranded RNA virus, is a member of the Birnaviridae family. Four pathotypes of IBDV, attenuated, virulent, antigenic variant, and very virulent (vvIBDV), have been identified. We isolated and characterized the genomic reassortant IBDV strain ZJ2000 from severe field outbreaks in commercial flocks. Full-length genomic sequence analysis showed that ZJ2000 is a natural genetic reassortant virus with segments A and B derived from attenuated and very virulent strains of IBDV, respectively. ZJ2000 exhibited delayed replication kinetics as compared to attenuated strains. However, ZJ2000 was pathogenic to specific pathogen free (SPF) chickens and chicken embryos. Similar to a standard virulent IBDV strain, ZJ2000 caused 26.7% mortality, 100% morbidity, and severe bursal lesions at both gross and histopathological levels. Taken together, our data provide direct evidence for genetic reassortment of IBDV in nature, which may play an important role in the evolution, virulence, and host range of IBDV. Our data also suggest that VP2 is not the sole determinant of IBDV virulence, and that the RNA-dependent RNA polymerase protein, VP1, may play an important role in IBDV virulence. The discovery of reassortant viruses in nature suggests an additional risk of using live IBDV vaccines, which could act as genetic donors for genome reassortment.