Infectious Bursal Disease Virus RNA Research Articles

Development and Evaluation of the Biological Adjuvant Bivalent Vaccine for Preventing Newcastle Disease and Infectious Bursal Disease

Newcastle disease (ND) and infectious bursal disease (IBD) are both one of the most economically important infectious diseases, which cause high infection rate and mortality of chickens and restrict the development of the poultry industry. Currently, vaccination is the most effective method to prevent ND and IBD. In this study, the biological adjuvant bivalent vaccine (rClone30-VP2L-chGM-CSF) was developed for preventing ND and IBD. Furthermore, the immune and protective effects of rClone30-VP2L-chGM-CSF were evaluated in specific-pathogen-free chickens. The experimental results indicated that chickens immunized with rClone30-VP2L-chGM-CSF vaccine significantly improved anti-IBDV and anti-NDV antibody titer; the level of CD4+ T, CD8+ T, B+, MHC-II+, and monocyte/macrophagocyte+ cells; the concentrations of IL-1β, IL-4, IL-17, IFN-α, IFN-β, and IFN-γ; and the splenocyte proliferation rate. Anti-NDV antibody titer reached the theoretical protection value of 4log2 at 7 days after immunization in chickens of rClone30-VP2L-chGM-CSF group; however, which at 14 days after immunization in chickens of rClone30-VP2L and rClone30. These results showed that chickens immunized with rClone30-VP2L-chGM-CSF stimulated stronger immune response than those with the rClone30-VP2L and rClone30. Chickens were challenged with virulent IBDV BC6/85, which were protected in the rClone30-VP2L-chGM-CSF group. Furthermore, IBDV RNA was not measured, and there appeared to be little apoptosis in the bursa of Fabricius in the rClone30-VP2L-chGM-CSF group. Therefore, rClone30-VP2L-chGM-CSF is a promising vaccine candidate against infectious bursal disease virus (IBDV) and newcastle disease virus (NDV), and it provides an idea for developing other poultry vaccines.

Examination of macroscopic and microscopic lesions in IBDV-infected organs and molecular characterization of IBDV VP1 gene fragments obtained from commercial broiler farms in Indonesia.

Infectious bursal disease (IBD) is an infectious immunosuppressive disease that affects young chickens. Instead of strict biosecurity practices, vaccination is used to control IBD. However, the disease has not been effectively managed. Variations in the observed clinical symptoms lead to confounding diagnoses. The study aimed to obtain pathological lesion data from chickens suspected of IBD virus (IBDV) infection by gross pathology, confirm IBDV infection through molecular diagnostics, and genotype the VP1 gene fragments of circulating IBDV in the field. The bursa of Fabricius, thymus, spleen, proventricular-ventricular junction, thigh muscles, and kidneys samples were collected from chickens suspected of IBDV infection from four commercial broiler farms in Central Java and The Yogyakarta Special Region Province between 2021 and 2022. The collected samples were examined histopathologically. Infectious bursal disease virus RNA was extracted from the bursa of Fabricius and VP1 gene was identified by reverse-transcriptase polimerase chain reaction (RT-PCR). The RT-PCR positive sample were sequenced and analyzed in Mega X for homology search and phylogenetic tree analysis. Macroscopic pathological lesions in the bursa of Fabricius were demonstrated by enlarged edema and thickened plica, presence of gelatinous exudate, hemorrhage, atrophy, and caseous exudate in the lumen. Moreover, the thymus had atrophy and small gray foci were observed in the spleen. Petechiae or hemorrhage was detected on the thigh muscle, and the kidney was dull and pale. Hemorrhage in the proventricular-ventricular junction was distinct. The histopathological examination of the bursa of Fabricius showed follicular vacuolization, edema, heterophilic infiltration, follicular atrophy, congestion, and hemorrhage. The thymus and spleen showed the presence of multifocal necrosis. Hemorrhage was observed in thigh muscle and mucosal part of proventricular-ventricular junction. Vacuolization was seen in renal tubules (nephrosis). Reverse transcriptase-PCR of 26 bursa of Fabricius samples from chickens suspected of IBDV infection showed four negative and 22 positive samples. Phylogenetic analysis of the VP1 gene fragment has indicated very virulent IBD (vvIBD) and belonged to B2 genotype. Infectious bursal diseases virus infection in broiler chicken generated macroscopic and microscopic primary lesions in the bursa of Fabricius and thigh muscle. Other organs such as the spleen, thymus, proventricular-ventricular junction, and kidney, were also involved. Molecular analysis of the VP1 gene confirmed the causative agent and grouped the virus into vvIBD and B2 genotype. All samples were collected from vaccinated birds therefore, the efficacy of available vaccine is required for urgent evaluation. Since most studies only focused on VP1, further exploration on VP2 gene is suggested notably for new-generation vaccines. Monitoring clinical signs' transformation over time could assist field diagnostics.

The Interaction between B87 Vaccine Strain and BC6/85 of Infectious Bursa Disease Virus in SPF Chickens.

This study was initiated to determine the interaction between two infectious bursal disease virus (IBDV) strains in the early stages of infection by detection and quantification of IBDV RNA in lymphoid and non-lymphoid tissues. SPF chickens were inoculated with single infection or dual infection by the mild strain B87 followed by the pathogenic strain BC6/85 at 0, 1, 2, and 3 days post-inoculation (dpi) with B87. Real-time RT-PCR assays were developed to examine the viral loads of the tissues collected at various time intervals. The results reveal that B87 could delay the time point of positive detection of the BC6/85 strain in the bursa of Fabricius from 1 dpi to 3 dpi, indicating that B87 interfered with the replication of BC6/85. The interference occurred when BC6/85 was inoculated at 2 dpi and 3 dpi with the B87 strain. Moreover, BC6/85 could affect the proliferation and duration of B87 in SPF chickens. The rates of positive detection for B87 decreased significantly during dual infection. The investigation of the interaction between the two strains is important for the implementation of appropriate control measures.

Development and evaluation of a recombinant VP2 neutralizing epitope antigen vaccine candidate for infectious bursal disease virus.

Infectious bursal disease (IBD) is one of the most economically important infectious diseases. Currently, vaccination is the most effective method to prevent IBD. Medium-virulence vaccines can damage the bursa of Fabricius and result in immunosuppression. Therefore, it is essential to develop a safe and effective vaccine against infectious bursal disease virus (IBDV). In this study, the five neutralizing epitopes of the IBDV VP2 protein were confirmed by neutralizing single chain variable fragment antibodies. Then, the neutralizing epitopes antigen (NEA) protein was constructed with five neutralizing epitopes and expressed by pET-27b. Furthermore, the immune effect and protective immunity of the NEA protein with the following adjuvants were evaluated in specific-pathogen-free chickens: oil emulsion adjuvant (OEA), double emulsion adjuvant (DEA), granulocyte-macrophage colony-stimulating factor (GM-CSF) adjuvant and complete Freund's adjuvant (CFA). The experimental results demonstrated that chickens immunized with NEA vaccines elicited stronger humoral and/or cellular immune responses and inflammatory responses than those in the NEA protein group. Chickens were protected in OEA, CFA and GM-CSF adjuvant groups, which were challenged with virulent IBDV BC6/85. Furthermore, IBDV RNA was not measured, and there appeared to be little apoptosis in the bursa of Fabricius based on TUNEL histology and the expression of Bax and Bcl-2 in the OEA, CFA and GM-CSF adjuvant groups. Based on the experimental results, the advantages and disadvantages of adjuvants and industrial production methods, GM-CSF was found to be the optimal adjuvant. Therefore, NEA with GM-CSF adjuvant is a promising vaccine candidate against IBDV, and it provides a framework for developing other vaccines against infectious viral diseases.

An optimized, highly efficient, self-assembled, subvirus-like particle of infectious bursal disease virus (IBDV)

Infectious bursal disease virus (IBDV) causes immunosuppression in young chickens, leading to increased susceptibility to other diseases and a reduction in the immune response to other vaccines. Thus, IBDV results in great economic losses to the poultry industry. The most effective method of prevention is vaccination. However, medium-virulence vaccines can cause bursal pathological damage and immunosuppression. Here, we describe a safer, self-assembled, subvirus-like particle (sVP) vaccine without a complex purification process. The IBD-VP2 gene was cloned into Pichia pastoris, and the expressed protein self-assembled into T=1 sVPs (∼23nm). Immunization experiments showed that the sVP vaccine elicited high IBDV-neutralizing antibodies in each group, and all birds survived challenge with very virulent IBDV (vvIBDV). Additionally, IBDV RNA was not detected, and sterile immunity was achieved. In conclusion, the IBD-sVP is a suitable candidate for a recombinant subunit vaccine against IBDV.

Further evidence for very virulent infectious bursal disease virus in vaccinated chickens in Nigeria.

Reverse transcription polymerase chain reaction (RT-PCR) and partial sequencing of the VP2 hypervariable region was performed on clinical samples from two infectious bursal disease (IBD) outbreaks in Plateau state, Nigeria. IBD virus RNA was detected in all four bursa of Fabricius samples. Nucleotide sequencing and analysis of the four samples revealed high similarity to previous IBDV sequences from northern and southern Nigeria. The deduced amino acid sequences were compared to reference IBDV strains retrieved from the GenBank; virulence markers A222, I256, and I294 were conserved in both outbreak and reference sequences. Amino acid residue S254 was conserved in the outbreak viruses and previous viruses from northern Nigeria. Phylogenetic analysis revealed that all four viruses were very virulent IBDVs. These viruses clustered with vv2-1 variant viruses from Oyo and Ogun states and less closely with vv2-2 isolates from Tanzania. The nucleotide identity of the sequences in this study ranged from 99.6 to 100 % with each other. These findings are further evidence of IBD outbreaks in vaccinated chicken flocks in Nigeria.

Effective multiple oral administration of reverse genetics engineered infectious bursal disease virus in mice in the presence of neutralizing antibodies.

Despite spectacular successes in hepatitis B and C therapies, severe hepatic impairment is still a major treatment problem. The clinically tested infectious bursal disease virus (IBDV) superinfection therapy promises an innovative, interferon-free solution to this great unmet need, provided that a consistent manufacturing process preventing mutations or reversions to virulent strains is obtained. To address safety concerns, a tissue culture adapted IBDV vaccine strain V903/78 was cloned into cDNA plasmids ensuring reproducible production of a reverse engineered virus R903/78. The therapeutic drug candidate was characterized by immunocytochemistry assay, virus particle determination and immunoblot analysis. The biodistribution and potential immunogenicity of the IBDV agent was determined in mice, which is not a natural host of this virus, by quantitative detection of IBDV RNA by a quantitative reverse transcriptase-polymerase chain reaction and virus neutralization test, respectively. Several human cell lines supported IBDV propagation in the absence of visible cytopathic effect. The virus was stable from pH 8 to pH 6 and demonstrated significant resistance to low pH and also proved to be highly resistant to high temperatures. No pathological effects were observed in mice. Single and multiple oral administration of IBDV elicited antibodies with neutralizing activities in vitro. Repeat oral administration of R903/78 was successful despite the presence of neutralizing antibodies. Single oral and intravenous administration indicated that IBDV does not replicate in mammalian liver alleviating some safety related concerns. These data supports the development of an orally delivered anti-hepatitis B virus/ anti-hepatitis C virus viral agent for human use.

Chicken melanoma differentiation-associated gene 5 (MDA5) recognizes infectious bursal disease virus infection and triggers MDA5-related innate immunity

The objective of the present study was to determine if chicken melanoma differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus (IBDV) infection to initiate and amplify an innate immune response in the chicken MDA5 (chMDA5) signaling pathway. Chicken embryo fibroblast DF-1 cells were infected with IBDV LP1 at a multiplicity of infection (MOI) of 0.5 or 10. In addition, knockdown and overexpression of chMDA5 were performed by transfecting DF-1 cells with chMDA5-targeting small interfering RNA (siRNA) or chMDA5-expressing DNA. The transfected cells were infected with IBDV LP1 at an MOI of 10. Cell culture supernatants and lysates were collected at 2, 8, 16 and 24 hours postinfection (hpi) for IBDV titer determination and RNA extraction, respectively. IBDV RNA loads and mRNA expression levels of chicken MDA5, interferon-β (IFN-β) promoter stimulator 1 (IPS-1), interferon regulatory factor-3 (IRF-3), IFN-β, double-stranded RNA-dependent protein kinase (PKR), 2′,5′-oligoadenylate synthetase (OAS), myxovirus resistance gene (Mx), and major histocompatibility complex class I (MHC class I) were determined by real-time RT-PCR. The IBDV titer increased up to 1.4 × 107 plaque-forming units (PFU)/mL at 24 hpi, and the IBDV RNA load reached 464 ng/μL at 24 hpi. The mRNA expression levels of chicken MDA5, IRF-3, IFN-β, PKR, OAS, Mx and MHC class I in IBDV-infected DF-1 cells exhibited significant (p < 0.05) upregulation up to 906-, 199-, 26,310-, 12-, 66,144-, 64,039- and 33-fold, respectively. Expressed chMDA5 from transfection and double-stranded RNA from IBDV infection were localized or colocalized in the cytoplasm of DF-1 cells at 16 hpi. When chMDA5 was knocked down in DF-1 cells, IBDV titers and RNA loads were significantly higher (p < 0.05) than those in DF-1 cells without chMDA5 knockdown at 24 hpi. The expression levels of chicken MDA5, IRF-3, IFN-β and MHC class I in chMDA5-knockdown DF-1 cells were significantly lower (p < 0.05) at 16 and 24 hpi. DF-1 cells overexpressing chMDA5 by transfection with chMDA5 expressing DNA had significantly lower (p < 0.05) IBDV titers and RNA loads at 16 and 24 hpi and showed significantly higher (p < 0.05) expression of chicken MDA5, IRF-3, IFN-β, PKR, OAS, Mx and MHC class I at 2 hpi. The results indicated that chicken MDA5 recognized IBDV infection and that this interaction resulted in the activation of chMDA5-related innate immune genes and upregulation of chicken MHC class I.

In vitro rapid clearance of infectious bursal disease virus in peripheral blood mononuclear cells of chicken lines divergent for antibody response might be related to the enhanced expression of proinflammatory cytokines

Infectious bursal disease (IBD) is an acute and highly contagious viral disease of young chickens caused by infectious bursal disease virus (IBDV). An effective way to control IBDV would be to breed chickens with a reduced susceptibility to IBDV infection. In the present work, we used chickens selected for high and low specific responses to sheep red blood cells (SRBC) (H and L, respectively) to assess the susceptibility of differential immune competent animals to IBDV infection. The peripheral blood mononuclear cells (PBMCs) of high SRBC line (HL) and low SRBC line (LL) were infected with IBDV and viral RNA loads were determined at different time post-IBDV infection. Chicken orthologues of the T helper 1 (Th1) cytokines, interferon-γ (IFN-γ) and interleukin-2 (IL-2); a Th2 cytokine, IL-10; a pro inflammatory cytokine, IL-6; the CCL chemokines, chCCLi2, chCCLi4 and chCCLi7; colony stimulating factor, GM-CSF; and a anti-inflammatory cytokine, transforming growth factor β-2 (TGFβ-2) were quantified. The expression of chCCLi2, chCCLi4 and chCCLi7 was significantly higher in L line as compared to H line. However, in H line the viral RNA loads were significantly lower than in L line. Therefore, the upregulated chemokines might be associated with the susceptibility to IBDV. The expression of IFN-γ, IL-2 and IL-6 was significantly higher in H line as compared to L line. We assume that the higher proinflammatory cytokines expression in H line might be related to the rapid clearance of virus from PBMCs. Significantly higher levels of IL-10 and TGFβ-2 mRNAs in L line might be related to the pathogenesis of IBDV. In conclusion, selection for antibody responses appears to influence the expression profiles of chemokines and cytokines against IBDV. Further, the selection for high SRBC response might improve the immuno-competence of chickens against IBDV.

Effect of maternally-derived antibodies on the performance and immunity of broilers induced by in ovo or post-hatching immunizations with a live vaccine against infectious bursal disease

The interference of low or high maternal antibodies titers on the attenuated infectious bursal disease (IBD) virus (IBDV) vaccine infection and its effects on the performance of broilers vaccinated at the 18th day of incubation (in ovo), at one day of age (subcutaneously-SC), or at 15 days of age (drinking water-DW) were investigated. After a series of three live vaccinations, breeders were given or not an IBD oil emulsion vaccine (IBD-OEV) prior to sexual maturity. At day 18 of incubation (in ovo), a commercial vaccine containing HVT and an intermediate IBDV strain or the single HVT vaccine was given. An intermediate IBDV vaccine was given SC at one day of age, or at 15 days of age via DW. The progeny of unvaccinated breeders presented higher neutralizing IBDVspecific antibody (IBDVab) titers at 25 and 40 days of age than those of the progeny of IBD-OEV breeders (p<0.05) at any broilers vaccination age and route. The lower IBDV RNA detection by RT-PCR in the bursa of Fabricius (BF) and the lower IBDV antibody titers in the serum of the groups vaccinated at one and 15 days of age derived from IBD-OEV breeders may indicate antibody-mediated IBDV neutralization. The inovo and one-day vaccinations did not interfere with performance, both in low and high antibody-titered progenies. The in-ovo vaccination against IBD is considered convenient and safe for industrial chickens, irrespective their maternal antibody levels.

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-γ

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-γ were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-γ (ChIFN-γ) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-γ did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.

A Practical Tissue Sampling Method Using Ordinary Paper for Molecular Detection of Infectious Bursal Disease Virus RNA by RT-PCR

A practical sampling method for bursal tissue using ordinary paper for molecular diagnosis of infectious bursal disease (IBD) was established. IBD virus-infected bursa was directly smeared on chromatography paper, filter paper, or stationery copy paper and was then fixed with absolute ethanol, Tris-HCl-saturated phenol, or phenol:chloroform:isoamyl alcohol (25:24:1). Flinders Technology Associates (FTA) card, which is designed for the collection of biological samples for molecular detection, was also used. After storage at 37 C for up to 30 days, total RNA directly extracted from the tissue fixed on the papers and FTA card were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of IBD virus (IBDV) RNA. In addition, the ability of each chemical used in the fixation and the FTA card to inactivate IBDV was evaluated. Regardless of the paper quality, storage period, and fixation method, IBDV RNA was consistently detected in all of the samples. IBDV in the bursal tissue was inactivated with phenol but not with ethanol or the unknown chemicals in FTA card. These results show that ordinary papers sustain the viral RNA, as does FTA card, but phenol fixation is superior to FTA card in inactivating IBDV. The new sampling method using ordinary paper with phenol fixation is safe, inexpensive, simple, and easy, and is thus suitable for conducting a global survey of IBD even where laboratory resources are limited. This practical method should contribute to the control of IBD worldwide.

Fast taqman multiplex assay to detect varicella zoster and herpes simplex viruses

A quantitative reverse transcription polymerase chain reaction (RT-PCR) protocol for assessing infectious bursal disease virus (IBDV) RNA levels in blood was developed using the ABI PRISM™ 7700 Sequence Detection System coupled with TaqMan® chemistry. To control for variations in sampling and processing between samples 28S rRNA was co-amplified in a multiplex reaction and used to quantify total RNA. Relative quantification and standardisation was achieved using a log10 dilution series of RNA extracted from IBDV stock. A linear relationship was observed between input RNA and cycle threshold values (CT) over 5 log10 dilutions for the IBDV-specific product and 6 log10 dilutions for the 28S rRNA-specific product. As a test of the assay it was used to determine whether differences in susceptibility to IBDV observed between inbred lines of chickens could be detected at the level of viral load in the blood. Viral RNA levels peaked 2 days post-infection when there was significantly less viral RNA in the blood of resistant line 61 chickens compared with the more susceptible Brown Leghorns (P=0.01). These results demonstrate that the course of IBDV infection can be monitored by quantifying IBDV RNA extracted from blood of infected chickens using TaqMan® technology.

Measuring infectious bursal disease virus RNA in blood by multiplex real-time quantitative RT-PCR

A quantitative reverse transcription polymerase chain reaction (RT-PCR) protocol for assessing infectious bursal disease virus (IBDV) RNA levels in blood was developed using the ABI PRISM™ 7700 Sequence Detection System coupled with TaqMan® chemistry. To control for variations in sampling and processing between samples 28S rRNA was co-amplified in a multiplex reaction and used to quantify total RNA. Relative quantification and standardisation was achieved using a log 10 dilution series of RNA extracted from IBDV stock. A linear relationship was observed between input RNA and cycle threshold values ( C T) over 5 log 10 dilutions for the IBDV-specific product and 6 log 10 dilutions for the 28S rRNA-specific product. As a test of the assay it was used to determine whether differences in susceptibility to IBDV observed between inbred lines of chickens could be detected at the level of viral load in the blood. Viral RNA levels peaked 2 days post-infection when there was significantly less viral RNA in the blood of resistant line 6 1 chickens compared with the more susceptible Brown Leghorns ( P=0.01). These results demonstrate that the course of IBDV infection can be monitored by quantifying IBDV RNA extracted from blood of infected chickens using TaqMan® technology.

Efficient Rescue of Infectious Bursal Disease Virus from Cloned cDNA: Evidence for Involvement of the 3′-Terminal Sequence in Genome Replication

To study the mechanism of replication of infectious bursal disease virus (IBDV), and to determine factors on the IBDV RNA which are involved in viral replication, we used cloned full-length cDNA of both the A- and B-segments to generate infectious IBDV. Infectious IBDV was rescued from plasmids that contained full-length IBDV cDNA behind a T7 promoter, by transfecting these plasmids into cells which were infected with a recombinant Fowlpox virus that expressed T7 RNA polymerase. By using the cDNA transfection system we evaluated the effect of the length of the 3′ terminus of the A-segment plus strand of IBDV. Although wild-type IBDV predominantly contains four cytosines at the 3′ terminus, no difference in virus yield was found when virus was rescued from cDNAs containing three to six adjacent cytosines. When the 3′ terminus was shorter than three cytosines the efficiency to generate infectious IBDV from cDNA was reduced, but IBDV could still be recovered reproducibly. The rescued viruses from cDNAs containing 3′-terminal deletions appeared to have a restored 3′-terminal sequence. The missing nucleotides are probably restored by using complementary bases of a stem-loop structure as template.

Amplification and cloning of infectious bursal disease virus genomic RNA segments by long and accurate PCR

Improved methods of reverse transcription, polymerase chain reaction (PCR) amplification, and cloning of full-length coding region of both strands of infectious bursal disease virus (IBDV) variant strain E genome were developed. Denaturation of IBDV RNA by heat in the presence of primers and use of a reverse transcriptase lacking RNase-H activity produced full-length coding region and partial non-coding region cDNA copies of the viral genomic segments. Digestion of the RNA component of RNA-cDNA hybrids by RNase-H followed by long and accurate PCR (LA-PCR) amplification of IBDV cDNA in a single step resulted in the synthesis of 3182 base-pairs (bp) of segment A and 2777 bp of segment B cDNA copies of IBDV genome. The resulting amplicons were successfully cloned and sequenced revealing their identity of IBDV. The LA-PCR method can be utilized for the amplification and cloning of the other IBDV strains or isolates and will greatly enhance the availability of sequence information or infectious cDNA copies of IBDV.

Simplified Sample Processing Combined with a Sensitive Nested Polymerase Chain Reaction Assay for Detection of Infectious Bursal Disease Virus in the Bursa of Fabricus

A rapid and sensitive protocol for the detection and amplification of infectious bursal disease virus (IBDV) RNA in the bursa of Fabricius was developed. By digestion with proteinase K and subsequent extraction with phenol and chloroform, IBDV RNA was efficiently released from a single bursa. IBDV RNA extraction time was shortened to 4 hr, compared with 24 hr with the traditional method, and only one bursa was needed instead of five. This more simplified procedure resulted in a significant reduction in costs due to less labor and the reduction in expensive chemicals and reagents. Four primers were selected from the sequence of a hypervariable region in VP2 genes. For the amplification of genomic IBDV RNA, the product (643 bp) of reverse transcriptase-polymerase chain reaction (RT-PCR) was reamplified and double checked by a nested PCR amplifying a 491-bp cDNA. The sensitivity of nested PCR was at least 100 times greater than RT-PCR as determined by dilution of the bursal homogenate. The fidelity of the nested PCR product was confirmed by Southern blotting, demonstrating specificity to the VP2 gene of IBDV. The simplified sample processing, shortened procedure time, and technical ease of this nested PCR render it more suitable for implementation in routine RT-PCR with restriction fragment length polymorphism testing for the detection and differentiation of IBDV RNA, especially for studies of IBDV infections of individual chickens.

Quantitative competitive polymerase chain reaction for detection and quantification of infectious bursal disease virus cDNA and RNA

A polymerase chain reaction (PCR)-based method to measure complementary DNA (cDNA) and RNA levels of infectious bursal disease virus (IBDV) was developed. Quantification was achieved by quantitative competitive PCR (QC-PCR) amplification. A competitor, a deletion mutant of the wild type IBDV cDNA, was 10-fold serially diluted and co-amplified with IBDV cDNA after being reversely transcribed from the viral RNA. After agarose gel electrophoresis, staining, and densitometric scanning, the bands on the digitized images were analyzed and quantified by computer-assisted image analysis. Complementary DNA of standard, as well as variant strains, of serotype l IBDV was detected and quantified using the same QC-PCR procedures. The assay could measure IBDV cDNA levels ranging from l μg to 45 fg and RNA levels ranging from 9 μg to 45 fg. The results indicated that QC-PCR is sensitive, easy to perform, and suitable for routine quantitation of IBDV cDNA or RNA levels.

Characterization of nonradioactive hybridization probes for detecting infectious bursal disease virus

Reverse transcription followed by the polymerase chain reaction was used to amplify a fragment of infectious bursal disease virus (IBDV) strain P3009 genome. The amplified DNA fragment was annealed into the plasmid pUC18 and used to transform Escherichia coli strain JM109. A clone that contained IBDV-specific nucleotide sequences was selected and designated pC23. The DNA fragment within pC23 was 320 base pairs in length and designated C23. Radiolabeled probes prepared from C23 hybridized to genome segment A of strain P3009 by a northern-blot hybridization assay. Biotin-labeled probes prepared from C23 and pC23 either by using nick translation (designated C23/ NT and pC23/NT, respectively) or by direct introduction of biotin molecules into C23 and pC32 (designated C23/BH and pC23/BH, respectively) were used in the dot blot hybridization assay for detecting IBDV strains. All four biotinylated probes detected three serotype l viruses and one serotype 2 IBDV. However, they did not cross-react with nucleic acids extracted from mockinfected cells or from seven unrelated avian viruses. Probe pC23/BH detected as little as 0.04 ng of IBDV RNA, while the other three probes were less sensitive and detected approximately 1 ng of IBDV RNA. In addition, the probe pC23/ BH detected IBDV RNA in bursa tissues from commercial broiler raising farms following the dot blot hybridization.

Molecular Detection of Infectious Bursal Disease Virus by Polymerase Chain Reaction

The polymerase chain reaction (PCR) technique was applied to the detection of infectious bursal disease virus (IBDV). Reverse transcription followed by the PCR was used to amplify a portion of IBDV genome. A set of primers that specify a 150-base-pair segment of IBDV genome was chosen from an Australian strain of IBDV. Standard challenge strain and variant strains A, D, E, G, and GLS-5 of IBDV serotype 1 and OH strain of serotype 2 from infected bursae were subjected to reverse transcription, followed by 30 cycles of PCR. A single band of the PCR product (DNA) of the expected size from each strain of IBDV was visible on polyacrylamide gels stained with ethidium bromide. Using the same primers, no PCR product was detected from genomic nucleic acids of turkey hemorrhagic enteritis virus, infectious bronchitis virus, reovirus, Salmonella enteritidis, Escherichia coli, and uninfected bursae. The PCR could be efficiently performed on serially diluted IBDV RNA and could detect 2 femtograms of IBDV RNA. The identity of the PCR products was confirmed by direct sequencing. The PCR is a specific and sensitive method for the detection of IBDV.