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PubMedDate not supplied

Infectious bursal disease virus: further characterization with evidence for a single-stranded RNA virus.

Infectious bursal disease virus (IBDV) that had been adapted to grow and was then cloned in chick embryo fibroblast (CEF) cell culture was examined for its physicochemical properties, the cellular site of virus replication, and the nature of its viral RNA. The IBDV was an RNA virus, acid-stable, absolutely resistant to chloroform, and moderately thermolabile. It appeared to replicate only in the cytoplasm, as shown by virus-specific antigens restricted to the cytoplasm of infected cells. The viral RNA was composed of single-stranded RNA, as evidenced by flame-red fluorescence on acridine-orange staining and an absence of specific fluorescence in infected cells on immunofluorescent staining with antiserum specific for double-stranded RNA. The IBDV virion had a hexagonal outline with an average diameter of 62 nm and possessed a single layer of capsid composed of hollow capsomeres without envelope. The buoyant density as determined in a continuous sucrose gradient was 1.178 g/cm3. The IBDV was found to possess morphologial and physicochemical properties different from those of any established RNA virus group.

Animals↗

PubMed2006

Transcriptional profiles of chicken embryo cell cultures following infection with infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease in chickens and causes a significant economic loss for the poultry industry. Little is understood about the mechanism involved in the host responses to IBDV infection. For better understanding the IBDV-host interaction, we measured steady-state levels of transcripts from 28 cellular genes of chicken embryo (CE) cell cultures infected with IBDV vaccine stain Bursine-2 during a 7-day infection course by use of the quantitative real-time RT-PCR SYBR green method. Of the genes tested, 21 genes (IRF-1, IFN 1-2 promoter, IFNAR-1, IRF-10, IFN-gamma, 2',5'-OAS, IAP-1, caspase 8, TRAIL-like, STAT-3, IL-6, IL-8, MIP-3 alpha, MHC-I, MHC-II, TVB, GLVR-1, OTF, IL-13R alpha, ST3GAL-VI and PGK) showed an increased expression. The remaining seven genes (IFNAR-2, IFN-alpha, NF-kappaB subunit p65, BLRcp38, DDX1, G6PDH and UB) showed a constant expression or only slight alteration. Apparently, the host genes involved in pro-inflammatory response and apoptosis, interferon-regulated proteins, and the cellular immune response were affected by IBDV infection, indicating involvement in the complex signaling pathways of host responses to the infection. This study thus contributes to the understanding of the pathogenesis of IBD and provides an insight into the virus-host interaction.

Animals↗

PubMed2002

Phylogenetic analysis of Newcastle disease virus in Taiwan.

The virulent forms of Newcastle disease virus cause a devastating disease of poultry. Between 1998 and 2000, sporadic outbreaks of Newcastle disease occurred in Taiwan despite vaccination. The causes of the failure of the vaccination remain unclear. The purpose of this study was to investigate the possible factors causing these outbreaks by serologic and virologic methods. Anti-Newcastle disease virus hemagglutination-inhibition titers were measured for serum samples obtained from a breeder farm and a broiler farm. The serologic data showed continued presence of virulent Newcastle disease viruses in the field during inter-outbreak periods. Phylogenetic analysis demonstrated that the field virulent Newcastle disease viruses were genetically similar and were grouped into genotype VIIa. Efficacy testing by virulent Newcastle disease virus challenge revealed that the vaccines used were effective for protecting chickens from infections. This investigation demonstrated that the Newcastle disease virus strain can spread quickly and widely throughout a large geographic area, and that the sporadic cases originate from virulent Newcastle disease viruses present in the field.

Animals↗

PubMed2003

Characterization of the RNA-binding activity of VP3, a major structural protein of Infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is the agent of an immune-depressive disease affecting the poultry industry worldwide. Infection of IBDV leads to expression of five mature virus-encoded proteins. Proteolytic processing of the virus-encoded polyprotein generates VP3 which coats the inner surface of the IBDV capsid. In this report, we describe the characterization of the RNA-binding activity of VP3. For these studies, the VP3 coding region was fused to a histidine tag and expressed in insect cells using a recombinant baculovirus. The histidine-tagged VP3 was affinity-purified and used to study its ability to bind RNA molecules using three complementary methods: (i) Northwestern blotting; (ii) binding of VP3 protein-RNA complexes to nitrocellulose membranes; and (iii) electrophoretic mobility shift assays. The results demonstrated that VP3 efficiently bound ssRNA and dsRNA. Under the experimental conditions used in this study, the formation of VP3-RNA complexes did not depend upon the presence of specific RNA sequences. A series of histidine-tagged VP3 deletion mutants spanning the whole VP3 coding region were generated. The use of these mutants revealed that the VP3 RNA-binding domain layed in a highly conserved 69 aa stretch close to the N-terminus of the protein.

Amino Acid Sequence↗

PubMedDate not supplied

Restriction fragment length polymorphisms in the VP2 gene of infectious bursal disease viruses.

Infectious bursal disease viruses (IBDVs) were examined for restriction fragment length polymorphisms in a fraction of the VP2 gene with the use of the reverse transcriptase/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay. The restriction enzymes BstNI and Mbol were used to obtain RFLP results. A third enzyme, StyI, was tested, but its utility for differentiation of IBDV strains was limited. Thirteen vaccine viruses and five IBDV strains that were previously characterized were placed into five molecular groups. Two groups contained viruses described as being classic strains, and two groups contained viruses described as being variant strains. The fifth group contained both classic and variant strains. The RFLP observed for the serotype 2 IBDV strain OH was unlike any of the RFLPs observed in viruses in the five molecular groups. Seven IBDV strains from commercially reared chickens in the United States, Mexico, Puerto Rico, and Thailand were tested in the RT/PCR-RFLP assay to determine if they were similar to the commercial IBDV vaccine strains tested. These viruses were selected because they were associated with lesions in the bursa of chickens that should have been protected by maternal antibodies or active immunity. Each of the viruses tested contained a unique RFLP compared with the IBDV strains and vaccine viruses examined in this study and, thus, did not fit into any of the five molecular groups. These viruses also were distinguishable from each other.

Animals↗

PubMed2003

Recombinant Newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication.

Newcastle disease virus (NDV) was examined for its suitability as a vector for the expression and delivery of foreign genes for vaccination and gene therapy. A reporter gene encoding human secreted alkaline phosphatase (SEAP) was inserted as an additional transcription unit at four different positions in the NDV genome, between the NP and P, M and F, and HN and L genes and behind the L gene. Eight infectious recombinant NDV (rNDV) viruses, four in the non-virulent strain NDFL and four in the virulent derivative NDFLtag, were generated by reverse genetics. SEAP expression levels, replication kinetics and virus yield were examined. Replication kinetics of the rNDV viruses in primary chicken embryo fibroblasts showed that the insertion of an additional gene resulted in a delay in the onset of replication. This effect was most prominent when the gene was inserted between the NP and P genes. With the exception of the strain that carried the SEAP gene behind the L gene, all recombinant strains expressed high levels of SEAP, both in cell culture and in embryonated chicken eggs. In embryonated eggs, the rNDV viruses showed a 2.6- to 5.6-fold (NDFL) or 2.1- to 8.1-fold (NDFLtag) reduction in yield compared with the parent strains. These results show that foreign genes can be inserted at different positions in the NDV genome without severely affecting replication efficiency or virus yield.

Alkaline Phosphatase↗

PubMed2004

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.

Amino Acid Sequence↗

PubMed2000

Swine vesicular disease virus. Pathology of the disease and molecular characteristics of the virion.

Swine vesicular disease is a highly contagious disease of pigs that is caused by an enterovirus of the family Picornaviridae. The virus is a relatively recent derivative of the human coxsackievirus B5, with which it has high molecular and antigenic homology. The disease is not severe, and affected animals usually show moderate general weakening and slight weight loss that is recovered in few days, as well as vesicular lesions in the mucosa of the mouth and nose and in the interdigital spaces of the feet. However, the similarity of these lesions to those caused by foot-and-mouth disease virus has led to the inclusion of this virus in list A of the Office International des Epizooties. The disease has been eradicated in the European Union except in Italy, where it is considered endemic in the south. Nevertheless, as occasional outbreaks still appear and must be eliminated rapidly, European countries are on the alert and farms are monitored routinely for the presence of the virus. This circumstance has led to a considerable effort to study the pathology of the disease and the molecular biology and antigenicity of the virus, andto the development of optimized methods for the diagnosis of the infection.

Animals↗

PubMedDate not supplied

Lysis of chicken lymphocytes by infectious bursal disease viruses.

Infectious bursal disease virus types 1 and 2 were able to induce direct lysis of chicken bursal cells, thymus cells, and peripheral blood lymphocytes in chromium-release assays. These two viruses were unable to lyse two established lymphoblastoid cell lines, although IBDV-1 was capable of multiplying in MSB-1 cells.

Animals↗

PubMed1985

Increase of virus yields and releases of Borna disease virus from persistently infected cells.

Borna disease virus grows to low titres in persistently infected cells with an infectious particle to cell ratio of 0.01 to 0.05. Inclusion of n-butyrate in the growth medium enhances infectivity yields up to 1 log. This effect is time and concentration dependent. In hypertonic medium with an excess of NaCl, KCl or Na2SO4 up to 50% of the total infectious virus yield is released from the cells. Released supernatant virus (buoyant density in sucrose rho = 1.22 g/cm3) is more heat stabile than cell-bound virus (rho = 1.18 g/cm3). The access to cell-free (released) virus opens new possibilities for the characterization of this neurotropic agent.

Animals↗

PubMed1984

Immunoglobulin classes of Aleutian disease virus antibody.

Aleutian disease virus (ADV) persistently infects mink and causes marked hypergammaglobulinemia. Immunoglobulin class-specific antisera were used to define the total immunoglobulin of each class by radial immunodiffusion and the immunoglobulin class of ADV-specific antibody by immunofluorescence in experimentally and naturally infected mink. Electrophoretic gamma globulin closely reflects the immunoglobulin G (IgG) level in mink, and the majority of the increased immunoglobulin and ADV antibody in infected mink is IgG. IgM becomes elevated within 6 days after infection, reaches peak levels by 15 to 18 days, and returns to normal by 60 days after infection. The first ADV antibody demonstrable is IgM, and most mink have virus-specific IgM antibody for at least 85 days postinfection. Serum IgA levels in normal mink are not normally distributed, and ADV infection causes a marked elevation of IgA. Low levels of ADV-specific IgA antibody can be shown throughout the course of infection. Failure of large amounts of virus-specific IgG antibody to inhibit the reaction of virus-specific IgM and IgA antibodies suggests that the various classes of antibodies are directed against spatially different antigenic determinants. The IgM and IgA were shown not to be rheumatoid factors.

Aleutian Mink Disease↗

PubMed2000

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.

Animals↗

PubMed1998

Two proline-rich nuclear localization signals in the amino- and carboxyl-terminal regions of the Borna disease virus phosphoprotein.

Borna disease virus (BDV) uses a unique strategy of replication and transcription which takes place in the nucleus, unlike other known, nonsegmented, negative-stranded RNA viruses of animal origin. In this process, viral constituents necessary for replication must be transported to the nucleus from the cytoplasm. We report here the evidence that BDV P protein, which may play an important role in viral replication and transcription, is transported into the nucleus in the absence of other viral constituents. This transportation is accomplished by its own nuclear localization signals (NLSs), which are present in both N-terminal (29PRPRKIPR36) and C-terminal (181PPRIYPQLPSAPT193) regions of the protein. These two NLSs can function independently and both have several Pro residues as key amino acids.

Amino Acid Sequence↗

PubMed2000

Characterization of the P protein-binding domain on the 10-kilodalton protein of Borna disease virus.

The Borna disease virus (BDV) is the prototype member of the Bornaviridae, and it replicates in the cell nucleus. The BDV p24P and p40N proteins carry nuclear localization signals (NLS) and are found in the nuclei of infected cells. The BDV p10 protein does not have an NLS, but it binds with P and/or N and is translocated to the nucleus. Hence, p10 may play a role in the replication of BDV in the cell nucleus. Here, we show that the P-binding domain is located in the N terminus of p10 and that S(3) and L(16) are important for the interaction.

Amino Acid Sequence↗

PubMed1997

Characterization of Borna disease virus p56 protein, a surface glycoprotein involved in virus entry.

Borna disease virus (BDV) is a nonsegmented negative-stranded (NNS) RNA virus, prototype of a new taxon in the Mononegavirales order. BDV causes neurologic disease manifested by behavioral abnormalities in several animal species, and evidence suggests that it may be a human pathogen. To improve our knowledge about the biology of this novel virus, we have identified and characterized the product of BDV open reading frame IV (BVp56). Based on sequence features, BVp56 encodes a virus surface glycoprotein. Glycoproteins play essential roles in the biology of NNS RNA viruses. Expression of BVp56 resulted in the generation of two polypeptides with molecular masses of about 84 and 43 kDa (GP-84 and GP-43). GP-84 and GP-43 likely correspond to the full-length BVp56 gene and to its C terminus, respectively. Endoglycosidase studies demonstrated that both products were glycosylated and that this process was required for the stabilization of newly synthesized products. Moreover, our results suggested that GP-43 is generated by cleavage of GP-84 by a cellular protease. Subcellular localization studies demonstrated that GP-84 accumulates in the ER, whereas GP-43 reaches the cell surface. Both BVp56 products were found to be associated with infectious virions, and antibodies to BVp56 had neutralizing activity. Our findings suggest that BVp56 exhibits a novel form of processing for an animal NNS RNA virus surface glycoprotein, which might influence the assembly and budding of BDV.

Animals↗

PubMed2006

Evaluation of FTA paper and phenol for storage, extraction and molecular characterization of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is an important poultry pathogen and is distributed world wide that can cause immune suppression and lesions of the bursa of Fabricius. The main component of the virus, VP2, is not only responsible for the bird's immune response, but is important for the molecular identification of this virus as well. The nucleic acid of the virus must be adequately preserved to be analyzed by reverse-transcriptase PCR (RT-PCR) and sequenced for the molecular characterization of the field strain. Phenol inactivation has been the standard for IBDV tissue collection and international shipment; however, there have been some reports of interference with molecular detection capabilities when using phenol. Phenol is also a hazardous chemical and must be handled and shipped carefully. The ability to use the Flinders Technology Associates filter paper (FTA card) for inactivation of several avian pathogens has been proven previously, however no work has been published on its use in IBDV nucleic acid detection. Bursas from experimentally infected birds was imprinted on FTA cards, and then placed in phenol. Samples were evaluated and compared based on molecular detection capabilities between the two inactivation methods. The nucleic acid of the virus was detected in 85% of the FTA card inactivated samples compared to 71% in the phenol inactivated samples. Sequence analysis was performed on samples inactivated by both methods and no differences were found. When comparing the RNA stability at different temperatures, euthanized IBDV infected birds were held at two different temperatures before sampling. No differences were detected for FTA sampling; however, for tissues in phenol the nucleic acid was only detectable up to 2 h post-mortem in the tissues held at 4 degrees C prior to sampling. These findings indicate that the FTA card is an efficient and reliable alternative collection method for molecular detection and characterization of IBDV.

Animals↗

PubMed2001

Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus.

Infectious bursal disease viruses (IBDVs), belonging to the family Birnaviridae, exhibit a wide range of immunosuppressive potential, pathogenicity, and virulence for chickens. The genomic segment A encodes all the structural (VP2, VP4, and VP3) and nonstructural proteins, whereas segment B encodes the viral RNA-dependent RNA polymerase (VP1). To identify the molecular determinants for the virulence, pathogenic phenotype, and cell tropism of IBDV, we prepared full-length cDNA clones of a virulent strain, Irwin Moulthrop (IM), and constructed several chimeric cDNA clones of segments A and B between the attenuated vaccine strain (D78) and the virulent IM or GLS variant strain. Using the cRNA-based reverse-genetics system developed for IBDV, we generated five chimeric viruses after transfection by electroporation procedures in Vero or chicken embryo fibroblast (CEF) cells, one of which was recovered after propagation in embryonated eggs. To evaluate the characteristics of the recovered viruses in vivo, we inoculated 3-week-old chickens with D78, IM, GLS, or chimeric viruses and analyzed their bursae for pathological lesions 3 days postinfection. Viruses in which VP4, VP4-VP3, and VP1 coding sequences of the virulent strain IM were substituted for the corresponding region in the vaccine strain failed to induce hemorrhagic lesions in the bursa. In contrast, viruses in which the VP2 coding region of the vaccine strain was replaced with the variant GLS or virulent IM strain caused rapid bursal atrophy or hemorrhagic lesions in the bursa, as seen with the variant or classical virulent strain, respectively. These results show that the virulence and pathogenic-phenotype markers of IBDV reside in VP2. Moreover, one of the chimeric viruses containing VP2 sequences of the virulent strain could not be recovered in Vero or CEF cells but was recovered in embryonated eggs, suggesting that VP2 contains the determinants for cell tropism. Similarly, one of the chimeric viruses containing the VP1 segment of the virulent strain could not be recovered in Vero cells but was recovered in CEF cells, suggesting that VP1 contains the determinants for cell-specific replication in Vero cells. By comparing the deduced amino acid sequences of the D78 and IM strains and their reactivities with monoclonal antibody 21, which binds specifically to virulent IBDV, the putative amino acids involved in virulence and cell tropism were identified. Our results indicate that residues Gln at position 253 (Gln253), Asp279, and Ala284 of VP2 are involved in the virulence, cell tropism, and pathogenic phenotype of virulent IBDV.

Amino Acid Sequence↗

PubMed1976

Radioimmunossay for detection of antigen and antibodies to Newcastle disease virus.

When Newcastle disease virus (NDV) is treated with NP-40 and ether a membrane fraction of 150,000 m.w. is obtained. This fraction which is composed of two polypeptides with m.w. of 56,000 and 76,000 was used in a radioimmunoassay (RIA). The assay was developed for both antigen and antibody and was found to be reproducible, specific, and highly sensitive. Titers of 1:51,200 were determined by RIA as compared to 1:4 by agar gel diffusion and 1:200 by hemaglutination inhibition (HI). As little as 5 ng of viral protein were detected by RIA inhibition technique. Labeled antigen could be stored in the presence of serum, KCI and Triton X-100 at -20 degrees C for as long as 6 weeks and retained similar reactivity as fresh reagent.

Animals↗