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PubMedDate not supplied

Cloning and expression of the VP2 gene of an infectious bursal disease virus.

A serotype I infectious bursal disease virus (IBDV) strain HZ96 was isolated in Hangzhou, China, in 1996 and attenuated by adaptation to chicken embryo fibroblast cells. The VP2 gene of strain HZ96 was amplified by reverse transcription-polymerase chain reaction, cloned, and sequenced. Compared with the VP2 sequences of other IBDV strains, HZ96 is most related to two attenuated strains, CU-1 and PBG98, and two attenuated Chinese strains, Harbin and CJ801bkf. HZ96 shares nucleotide sequence homology 98.9% with CU-1 and PBG98, 98.5% with Harbin, and 98.6% with CJ801bkf. Most of the sequence variations observed between HZ96 and other strains are located in the middle variable region from nucleotides 637 to 996. Similar to other attenuated IBDVs, HZ96 has unique substitutions at residues 279 (Asp to Asn) and 284 (Ala to Thr), suggesting that these two substitutions may be directly related to adaptation of the virus to cell culture and attenuation of its virulence. As part of our effort to develop a submit vaccine for IBDV, the VP2 gene of HZ96 was cloned into a heat-inducible expression vector and expressed in Escherichia coli system. A protein band with expected molecular weight of 52 kD was detected by direct protein staining and western blotting.

Amino Acid Sequence↗

PubMed2000

Development of probes for differentiation of infectious bursal disease virus strains of various virulence by dot-blot hybridization.

Two different radio-labeled nucleic acid probes, prepared from reverse transcription-polymerase chain reaction (RT-PCR) amplified variable region of VP2 and VP1 gene sequences of a highly virulent infectious bursal disease virus (IBDV), were tested for their ability to detect field isolates of IBDV directly in clinical bursal tissue specimens and vaccine strains of IBDV in tissue cultures. The VP2 gene probe was able to detect both field isolates and vaccine strains of IBDV under high as well as low stringency while the VP1 gene probe could differentiate under high stringency field isolates from vaccine strains, hybridizing only with RNA of field isolates. The sensitivity of both the probes was found to be 4 ng of purified viral RNA.

Animals↗

PubMed2001

Selection of an infectious bursal disease virus mutant with increased immunogenicity following passage under humoral immune pressure.

It is generally known that the pathogenicity of infectious bursal disease virus (IBDV) strains decreases following passage in cell culture. However, there is no information about the effect of passage under immune pressure on the phenotypic and molecular properties of IBDV. In the present study, a small plaque mutant virus with poor neutralization capability, but showing similar growth characteristics as the parental virus strain, QC2, was isolated after serial passage in Vero cells in presence of IBDV serotype 1 chicken polyclonal antiserum. This mutant virus showed reduced pathogenicity but enhanced immunogenicity compared to the parental virus. Sequence analysis of the non-coding regions of the genome revealed 4 and 3 nucleotide changes in the 3' non-coding regions of segments A and B, respectively, and none in the 5' non-coding regions. Restriction enzyme analysis of selected coding regions of the IBDV genome in both viruses revealed a loss of the PstI site in the VP2 region of the mutant virus. Selection of such mutant viruses by passaging under immune pressure may offer an improved method for developing safer and more effective attenuated vaccine strains against infectious bursal disease of chickens.

Animals↗

PubMedDate not supplied

The in vivo and in vitro effects of chicken interferon alpha on infectious bursal disease virus and Newcastle disease virus infection.

The in vitro and in vivo effects of chicken interferon alpha on infectious bursal disease virus (IBDV) infection were investigated in this study. A cDNA of interferon alpha was first cloned from a Chinese strain chicken Shiqi by reverse transcription-polymerase chain reaction. The deduced amino acid sequence has one amino acid substitution with chicken interferon alpha 1 at residue 65 (N to S) and two amino acid substitutions with chicken interferon alpha 2 at residues 50 (N to S) and 58 (P to L), respectively. A prokaryotic expression system was employed to produce a large quantity of recombinant protein. Recombinant interferon was purified in a one-step process, and an optimal refolding process was devised. About 51% recombinant protein from inclusion bodies was refolded, and the final yield of the recombinant interferon reached 24.66 mg/liter culture. The recombinant interferon suppressed IBDV plaque formation in a dose-dependent manner and ameliorated IBDV and Newcastle disease virus infection in both specific-pathogen-free (SPF) and commercial chickens. The antiviral effect of interferon alpha is more significant in commercial chickens than in SPF chickens, and the route of administration affects the efficacy of interferon therapy. This is the first reported study of the effects of interferon alpha on IBDV infection.

Amino Acid Sequence↗

PubMedDate not supplied

In situ localization of infectious bursal disease virus-binding cells by a biotin-streptavidin system.

A biotin-streptavidin system was established to directly visualize infectious bursal disease virus (IBDV)-binding cells in cell culture or in fresh tissues. The cells or tissue sections were first incubated with a biotinylated, purified IBDV strain GZ911 and then with a streptavidin-beta-galactosidase conjugate. In the presence of the enzyme substrate X-gal, IBDV-binding cells were labeled in blue color. By applying this method to frozen tissue sections, virus-binding sites were localized in situ in the bursa, spleen, and kidney tissue sections, whereas no positive cells were detected in the thymus tissue sections. Chicken embryo fibroblasts, Vero cells, MOP-8 cells, 293-EBNA cells, PANC-1 cells, and HuTu 80 cells were found to bind to the virus. However, the binding of the virus to MDA-MB-231 cells and SVG p12 cells was undetectable. This method can be employed for the expressional cloning of IBDV receptor and can be applied to studies on other avian viruses.

Animals↗

PubMedDate not supplied

Viral and bacterial agents associated with experimental transmission of infectious proventriculitis of broiler chickens.

Proventriculitis of broilers can be reproduced by oral inoculation of day-old chicks with a proventricular homogenate from affected 3-wk-old broilers. The objective of the following studies was to isolate from this homogenate viral and bacterial isolates that could produce proventriculitis. A monoclonal antibody to infectious bursal disease virus (IBDV) was used to precipitate virus from the homogenate. A primary chicken digestive tract cell culture system was also used to isolate virus from a 0.2-microm filtrate of the homogenate, and a bacterium was also isolated from the homogenate. In trial 1, day-old birds were orally inoculated with either proventriculus homogenate or monoclonal antibody immunoprecipitated IBDV (MAB-IBDV). At 4, 7, 14, and 21 days postinfection (PI), 12 birds from each treatment group were subjected to necropsy. In trial 2, day-old birds were orally inoculated with either infectious proventriculus homogenate, suspect virus isolated in cell culture and propagated in embryo livers and spleens, or a bacterial isolate. Twelve birds from each treatment were subjected to necropsy at days 7, 14, 21, and 28 PI. In trial 3, treatments were maintained in negative pressure isolation chambers, and an additional treatment included virus plus bacterial isolate. Twenty-four birds from each treatment were subjected to necropsy at day 21 PI. In trial 1, infectious homogenate decreased body weight and relative gizzard weights at 4, 7, 14, and 21 days PI. Proventriculus relative weight was increased at days 7, 14, and 21 PI, and proventriculus lesion scores were increased at days 14 and 21 PI. Bursa/spleen weight ratios were decreased at day 14, and feed conversion was increased at days 4 and 21. The MAB-IBDV treatment decreased proventriculus and gizzard relative weights at day 4 PI, increased proventriculus lesion scores and bursa/spleen weight ratios at day 14, and decreased heterophil/lymphocyte ratios at day 21. In trial 2, all infected birds had significantly higher mean relative proventriculus weights at 21 days PI and had higher 4-wk mean proventriculus scores as compared with both control groups. In trial 3, birds treated with homogenate and birds treated with both suspect virus and the bacterial isolate had significantly higher proventriculus lesion scores; higher relative weights of proventriculus, gizzard, liver, and heart; lower body weights; and lower relative bursa weights compared with the saline control group. These studies suggest that infectious proventriculitis has a complex etiology involving both viral and bacterial infection.

Animals↗

PubMed2001

Nucleotide sequence and phylogenetic analysis of a segment of a highly virulent strain of infectious bursal disease virus.

The complete nucleotide sequences encoding precursor polyprotein (VP2-VP3-VP4) and VP5 of a highly virulent (hv) infectious bursal disease virus (IBDV), UPM97/61 was determined. Comparison of the deduced amino acid sequences with the published ones revealed 8 common amino acid substitutions, which were found only in the hv IBDV including the UPM97/61 strain. Three of the amino acid substitutions (222 Ala, 256 Ile and 294 Ile) were used as a marker for determining hv IBDV strains. The other five substitutions (685 Asn, 715 Ser, 751 Asp, 990 Val and 1005 Ala) were also conserved in hv IBDV strains isolated in various countries. UPM97/61 strain demonstrated also 8 unique amino acid substitutions of which 3 were in VP2, 4 in VP3 and 1 in VP4. There was 1 unique amino acid substitution in VP5 at position 19 (Asp-->Gly) not found in other strains. However, all the strains have a conserved 49 Arg. The amino acid sequence of UPM97/61 strain differed by 1.09% from the Japanese (OKYM) and Hong Kong (HK46) strains, and by 1.48% from the Israeli (IBDVKS) and European (UK661) strains. Hence, UPM97/61 is more closely related to the hv strains from Asia. However, phylogenetic analysis indicated that the origin of UPM97/61 might be the same as that of other hv strains isolated from other parts of the world.

Amino Acid Sequence↗

PubMed2001

Amplification and Cloning by Long RT-PCR of Full-length Genome of Larger Segment of Chicken Infectious Bursal Disease Virus.

To develop the genetic rescue techniques for infectious bursal disease virus (IBDV), Birnaviridae family, the full-length cDNA of the larger segment of the chicken IBDV was amplified and cloned by long RT-PCR. A comparison of four purification and extraction methods of RNA from IBDV infected chicken embryoid fibroblasts (CEF) showed that the ultracentrifugation followed by proteinase Kdigestion extracted dsRNA more effectively. Then reverse transcription was carried out at 50 degrees using Superscipt II enzyme, followed by RNase H digestion. Amplification of single stranded cDNA in a single step resulted in the synthesis of the full-length segment A of 3 259 bp. The amplified product was cloned and sequenced, identifying that it was an IBDV. This method is superior to other methods based on amplifying different parts of the genome many times, therefore the cloning procedure was simplified.

Journal Article↗

PubMed2000

[Production of avian infectious bursitis virus by in vivo DNA genomic transcription].

Generation of infectious particles of infectious bursal disease virus (IBDV) has been essentially performed by transfecting susceptible cells with in vitro synthesized cRNAs of genomic segments A and B. In the present study, we have explored the possibility to proceed directly in vivo without taking into account the extra-genomic vector-derived sequences. For this purpose, cDNAs of segments A and B were subcloned into an eucaryotic expression vector under the control of the immediate early CMV promoter. Upon transfection of primary culture of chicken embryonic fibroblasts with both constructs, a cytopathic effect (CPE) typical of that produced by IBDV could be observed, indicating that the 5'- and 3'- vector-derived extra-nucleotides did not interfere with the replication and packaging process. Finally, in order to develop a GFP-based packaging assay, we first tried to express this fluorescent protein in the context of the IBDV polyprotein encoded by the genomic segment A. Our initial results indicate that the presence of IBDV specific sequences upstream of the GFP polypeptide dramatically decreased the fluorescence of the latter protein.

Animals↗

PubMed2004

Detection of Infectious bursal disease virus by ELISA using an antipeptide antibody raised against VP3 region.

Antigenic determinant analysis was carried out on VP3, one of major immunogenic proteins of Infectious bursal disease virus (IBDV) using computer algorithms. Altogether 17 peptides were synthesized for predicted putative regions and were tested for their reactivity with IBDV-positive polyclonal sera as well as with antisera to other common avian viruses to confirm specificity and to rule out cross reactivity. Of 17 peptides tested, three were selected and synthesized in multiple antigenic peptide (MAP) format. The immunization of rabbits with the three MAPs resulted in high humoral immune response. The purified antipeptide antibodies were screened against native IBDV antigen and the respective titers were determined. Out of the three antisera to MAPs that raised against the MAP3, spanning the amino acids (aa) 974-995 region on the VP3 protein had a very high titer (2048) and reacted specifically with IBDV. Thus, the antiserum to MAP3 detected native virus in enzyme-linked immunosorbent assay (ELISA), revealing the presence of a potential antigenic determinant on the C-terminus of the protein. This study proved that an antipeptide antibody could be used as a safe and specific tool for the diagnosis of IBD in chickens.

Animals↗

PubMed2005

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.

Animals↗

PubMed2004

[Expression of the infectious bursal disease virus polyprotein in Vero cells using attenuated Salmonella typhimurium as transgenic carrier].

To examine if polyprotein gene (VP2/VP4/VP3) of Infectious Bursal Disease Virus (IBDV) could be delivered into mammalian cells and expressed using attenuated Salmonella typhimurium as vector. The IBDV polyprotein gene was amplified by RT-PCR and inserted in to pCI, an eukaryotic expression plasmid. The resulting recombinant pCI-VP2/VP4/VP3 was transformed by electroporation into attenuated Salmonella typhimurium strain ZJ111 (dam- and phoP-), which was then use to transfect the Vero cells. Gene specific RT-PCR revealed that VP2/VP4/VP3 was transcribed into mRNA in the Vero cells. Indirect immunofluorscence assay, SDS-PAGE and Western-blot analysis showed that VP2/VP4/VP3 was expressed and the product was immuno-reactive with anti-IBDV serum. This work provides essential precondition for developing a new oral DNA vaccine against IBDV.

Animals↗

PubMed1991

[Effect of viral structure and replication characteristics on the pathogenesis of infectious bursal disease].

Infectious bursal disease (IBD) is a highly contagious disease of young chickens, which occurs world-wide, and is responsible for severe losses in poultry industries. In birds surviving an acute infection, lymphoid cells in the bursa of Fabricius are destroyed, resulting in B-cell-dependent immunodeficiency. This causes increased susceptibility to diseases by otherwise harmless agents. The decisive role in the pathogenesis of IBD is played by the bursa, representing the target organ of the aetiological agent, infectious bursal disease virus (IBDV). By adaptation of IBDV to chicken embryo cells, we obtained several variants of a pathogenic wild type strain. These variants had altered abilities for replication in actively dividing B lymphocytes and, consequently, had altered pathogenic properties. An IBDV isolate from turkeys, non-pathogenic for chickens, was used to create reassortant virus strains. The virological and the biological characterization of these IBDV variants is reported.

Animals↗

PubMedDate not supplied

Variability in a commercially available enzyme-linked immunosorbent assay system. I. Assay variability.

Three experiments were conducted to characterize the variation in enzyme-linked immunosorbent assay (ELISA) kits for infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV). Expt. 1 was carried out to determine the variation in assay results when the same pools of low-, medium-, and high-titered serum were assayed. Significant variation occurred among separate lots and among test plates within the same lots for the IBV and IBDV assays. In most cases, variability between days and among technicians was not significant. Coefficients of variation were larger than is acceptable for immune-type assays. In the IBDV assay with high-titered serum, most of the wells in the plates reached maximum absorbance and were not capable of detecting titers above 1:8000-1:9000. Expt. 2 was conducted to determine the effects of varying the length of the ortho-phenylene-diamine (OPD) incubation time upon assay results. Either 7-, 12-, or 15-minute OPD incubation times were used. Incubation time significantly affected mean titer at all combinations of assay types and times, except determinations on the low-titered IBV samples. Expt. 3 was conducted to determine the effects of three different dilution methods on observed IBDV titer. The use of non-standard dilutions had significant effects on observed titer. In the medium- and high-titered samples, the use of two different dilution methods at 1:5000 rather than 1:500 resulted in titers that were three to four times those observed at the 1:500 dilution.

Animals↗

PubMed2006

[Inhibitory effect of new antimicrobial substance by Bacillus subtilis fmbJ on Newcastle disease virus and infectious Bursal disease virus in vitro].

The resistance effect on Newcastle disease virus (NDV) and Infectious Bursal Disease Virus(IBDV) in vitro of a new antimicrobial substance (AS), which produced by a Bacillus subtilis strain named B. subtilis fmbJ. Results showed that the TD50 and TD0 value of this AS on Chicken Embryo Fibroblasts cell (CEF) were 128.95mg/L and 25.79mg/L, respectively. This AS could strongly inhibit the cytopathic effects of cell induced by NDV as well as IBDV, and increase the survival rate of cell remarkably. This AS could inhibit the function of NDV and IBDV, and it could defend against the infection and inhibit multiplication of NDV and IBDV, and the effect was the same as the antiviral medicine Ribavirin. It had lower toxicity to CEF cell, therefore we would study it further that it was as antiviral medicine.

Animals↗

PubMedDate not supplied

Isolation of infectious bursal disease virus from turkeys with arthritic and respiratory symptoms in commercial farms in Quebec.

Infectious bursal disease viruses (IBDVs) were isolated from turkeys showing symptoms of arthritis and respiratory disease in commercial poultry farms in the province of Quebec, Canada. Synovial fluids collected from hock joints of arthritic birds and peripheral blood leukocytes obtained from the birds with respiratory problems were used for virus isolation in embryonated chicken eggs, and Vero and BGM-70 cell cultures. The infected cells were evaluated for the presence of IBDV by indirect immunofluorescence assay using monoclonal antibodies. The viruses were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of viral genome and by electron microscopy. Although one of these turkey isolates tested was neutralized by serotype 1-specific commercial chicken antisera, preliminary results indicated that there are antigenic differences between the Quebec isolate, IBDV QT-1, and the existing strains of IBDV belonging to serotype 1.

Animals↗

PubMedDate not supplied

Infectious bursal disease virus variant from commercial Leghorn pullets.

An infectious bursal disease virus (IBDV) was isolated from 39-to-43-day-old commercial leghorn pullets suspected of having infectious bursal disease (IBD). These chickens had been vaccinated with a commercial live IBDV vaccine at 28 and 35 days of age. An isolate designated IN was recovered using specific-pathogen-free (SPF) chickens and the BGM-70 established cell line. Experimental studies using SPF chickens vaccinated with either inactivated vaccines made from the vaccine strain used in the problem flock or a standard-type vaccine indicated no protection against the IN isolate. However, two variants and another standard-type vaccine induced protection against the IN isolate. Cross-neutralization tests indicated that the IN isolate differed antigenically from commercial vaccine strains and was related to the variant IBDV strains recently isolated from broilers. To our knowledge, this is the first report of a variant IBDV recovered from commercial layer chickens in the United States.

Animals↗

PubMedDate not supplied

Experimental induction of hemorrhagic-aplastic anemia in chickens. I. Etiology.

Exposure to infectious bursal disease virus (IBDV) at 1 day old followed by inclusion body hepatitis virus (IBHV) inoculation at 36 days produced typical lesions of hemorrhagic-aplastic anemia syndrome (HAS). The lesions included severe anemia, widespread hemorrhages, and dermatitis. HAS could not be induced in the first 4 weeks of life in chickens inoculated at one day old with IBHV alone or in combination with IBDV. It was concluded that the immunosuppressive effects of IBDV failed to alter the pathogenicity of IBHV in chicks less than 4 weeks old. This resistance was considered to be age-related. Subcutaneous inoculation of day-old chicks with IBDV produced a more severe infection than did oral exposure. Serial passage of IBHV in day-old chicks had no significant effect on the viral pathogenicity.

Adenoviridae Infections↗