Vv Infectious Bursal Disease Virus Research Articles

Isolation and molecular characterization of very virulent infectious bursal disease virus from Egypt

In Egypt, the poultry industry is plagued by the significant economic losses caused by the infectious bursal disease virus (IBDV). In the present study, a total of 30 diseased commercial chicken flocks aged 23–41 days old in three Egyptian governorates (Fayoum, Beni-Suef, and Minya) having symptoms consistent with IBDV were examined. The collected bursal samples (n=30, ten pooled bursae for each flock) were screened by RT-PCR to amplify the IBDV VP2 gene. Results revealed that 20 out of 30 samples were positive, as indicated by the amplification of 620 bp of the VP2 gene. The twenty positive samples were inoculated into specific pathogen-free embryonated chicken eggs (SPF-ECEs) via the chorioallantoic-membrane (CAM) route to isolate the suspected virus. Eight samples were successfully isolated and were subjected to a partial sequence of VP2 (targeting the HVR). The nucleotide sequences and phylogenetic analysis revealed that the examined isolates exhibited amino acids A222, I256, I294, and S299, which are extremely conserved among very virulent (vv)IBDV. The amino acid sequence analysis of the eight isolates revealed a high degree of similarity (97-100%) in between and 90-100% similarity compared to other Egyptian vvIBDV strains. In contrast, lower similarity was observed with attenuated IBDV vaccine strains D78, Lukert, and Bursavac (91-93%, 91-93%, and 92-94%, respectively). In conclusion, this study underscores the importance of ongoing surveillance of the IBDV situation in the field, as well as the need for further research to investigate the efficacy of current vaccination strategies to curb IBDV infection.

Differential Modulation of Innate Antiviral Profiles in the Intestinal Lamina Propria Cells of Chickens Infected with Infectious Bursal Disease Viruses of Different Virulence.

Infectious bursal disease virus (IBDV) is one of the most important infectious diseases of poultry around the world. Gut-associated lymphoid tissues (GALT) are the first line of defense of the host against the infection. The purpose of this study was to investigate the role of innate immune antiviral signaling triggered by Toll-like receptor 3 (TLR3), as well as macrophage activation and cytokine response in the intestinal lamina propria (ILP) cells after the oral challenge of IBDV in relation to IBDV virulence and disease pathogenesis. The results showed that the expression levels of TLR3, IRF7, IFN-α/β and the corresponding downstream antiviral factors OAS, PKR and Mx were all upregulated in the SPF chicken ILP cells at 8 h post-infection (hpi) and 12 hpi. Similarly, macrophages were activated, with the initial macrophage M1 activation observed at 8 hpi, but then it rapidly shifted to a non-protective M2-type. Both Th1 (IFN-γ, TNF-α, IL-12) and Th2 (IL-4 and IL-10) types of cytokines were differentially upregulated during the early stage of infection; however, the Th1 cytokines exhibited stronger activation before 8 hpi compared to those of the Th2 cytokines. Interestingly, differential regulations of gene expression induced by different IBDV strains with different virulence were detected. The HLJ0504-like very virulent (vv) IBDV strain NN1172 induced stronger activation of TLR3-IFN-α/β pathway, macrophages and the Th1/2 cytokines’ expression, compared to those induced by the attenuated strain B87 at 8 hpi and 12 hpi in the ILP cells. In conclusion, the innate antiviral response mediated by the TLR3-IRF7 pathway, macrophage activation and cytokine expression in the GALT cells at the early stage of IBDV infection was differentially modulated, and the HLJ0504-like vvIBDV strain triggered stronger activation than the attenuated vaccine strain, and that may play an important role in the progression of disease.

Quantitative Proteomics Analysis Revealed Compromised Chicken Dendritic Cells Function at Early Stage of Very Virulent Infectious Bursal Disease Virus Infection.

Chicken dendritic cells (DCs) have been demonstrated to be susceptible to infectious bursal disease virus (IBDV), a causative agent of acute and immunosuppressed disease in young chicks known as infectious bursal disease. Further functional characterization of IBDV-infected DCs of chickens is required to provide a better understanding on the influence of the virus on chicken bone marrow-derived dendritic cells (BM-DCs) following very virulent (vv) IBDV infection. Membrane proteins of BM-DCs were extracted and the proteins were further denatured and reduced before performing labeling with isobaric tags for relative and absolute quantitation. The differential expression protein profiles were identified and quantified using liquid chromatography coupled with tandem mass spectrometry, and later validated using flow cytometry and real-time reverse transcriptase PCR. The analysis has identified 134 differentially regulated proteins from a total of 283 proteins (cutoff values of ≤0.67, ≥1.5, and ProtScore >1.3 at 95% confidence interval), which produced high-yield membrane fractions. The entry of vvIBDV into the plasma membrane of BM-DCs was observed at 3 hr postinfection by the disruption of several important protein molecule functions, namely apoptosis, RNA/DNA/protein synthesis, and transport and cellular organization, without the activation of proteins associated with signaling. At the later stage of infection, vvIBDV induced expression of several proteins, namely CD200 receptor 1-A, integrin alpha-5, HSP-90, cathepsin, lysosomal-associated membrane protein, and Ras-related proteins, which play crucial roles in signaling, apoptosis, stress response, and antigen processing as well as in secretion of danger-associated proteins. These findings collectively indicated that the chicken DCs are expressing various receptors regarded as potential targets for pathogen interaction during viral infection. Therefore, fundamental study of the interaction of DCs and IBDV will provide valuable information in understanding the role of professional antigen-presenting cells in chickens and their molecular interactions during IBDV infection and vaccination.

Genetic characterization of Infectious Bursal Disease Viruses isolated from the vaccinated broiler chicken flocks in Egypt during 2015-2016.

The present study was conducted to characterize the infectious bursal disease virus (IBDV) circulating in clinically diseased broiler chicken flocks with previous vaccination history during 2015-2016 in Egypt. IBDVs were isolated from 48 out of 63 of the investigated bursae from 10 flocks onto embryonated chicken eggs (ECEs) and verified by reverse transcriptase-poly- merase chain reaction (RT-PCR). Histopathologically, bursae lesions revealed some lymphocytes depletion as well as the presence of vesicles in the lining epithelium. The hyper variable region (HVR) of VP2 and VP1 genes of the 10 isolates (1 isolate/flock) were partially sequenced and subjected to comparative alignment and phyologenetic analysis. Phylogenetically, IBDV isolates were clustered into two distinct genetic lineages: variants of classical virulent (cv) and very viru- lent (vv) IBDV strains based on VP1 and VP2 amino acid (aa) sequences. Alignment analysis of HVR-VP2 aa sequences has demonstrated that the vvIBDV isolates have the conserved residues of the vvIBDV pathotype (A222, I242, and I256), while, the cvIBDV isolates have the same aa sequences of the classical attenuated vaccine strain (D78). Expected single point mutation occurred at position 253 (H253N). All previously characterized isolates were re-subjected to molecular analysis with VP1 protein due to its correlation with virulence and pathogenicity of IBDVs. vvIBDV isolates have the conserved tripeptide (TDN), while, the cvIBDV isolates have aa substitutions at conserved tripeptide including NEG at 145-147 amino acid. The present study has demonstrated that variants of classical virulent and very virulent IBDV circulated among vaccinated flocks in Egypt during 2015-2016.

Combination of quaternary ammonia and glutaraldehyde as a disinfectant against enveloped and non-enveloped viruses

Infectious bursal disease is a highly infectious immunosuppressive disease of chickens endemic in many poultry-producing areas around the world. The non-enveloped virus that causes the disease, infectious bursal disease virus (IBDV), is highly stable and resistant to inactivation by common disinfectants. Avian influenza viruses (AIV), on the other hand, are highly vulnerable to most disinfectants due to their phospholipid envelope, but still pose a major threat to the poultry industry, as the outbreaks in 2015 in the United States have shown. Several studies have evaluated the efficacy of disinfectants against both IBDV and AIV but failed to take into consideration factors that would affect the disinfectant efficacy once used in the field, such as organic material. Therefore, the aim of this study was to investigate the efficacy of a commercial combination of quaternary ammonia and glutaraldehyde as a disinfectant against IBDV and AIV in the presence of organic material commonly found in the commercial poultry industry: fecal matter alone, feathers/dust mixed with feces, and bedding material mixed with feces. After a 10-minute disinfectant contact time, each surface was swabbed and virus isolation attempted in embryonated specific-pathogen-free (SPF) chicken eggs. The non-enveloped very virulent (vv) IBDV was reisolated from spiked feces and shavings treated with the disinfectant. This was confirmed by RT-PCR detection of the virus. In contrast, no viral isolate or RT-PCR product was obtained from the samples collected from spiked feathers/dust treated with the disinfectant. Finally, no low pathogenic (LP) AI was re-isolated from the samples treated with the disinfectant indicating that, under laboratory conditions, the combination of quaternary ammonia and glutaraldehyde was partially and completely effective in the inactivation of vvIBDV and LPAI, respectively. Not only the viral envelope, but also the presence of organic matter plays an important role in the viral resistance to disinfectants.

GENETIC CHARACTERIZATION OF INFECTIOUS BURSAL DISEASE VIRUS ISOLATES IN UKRAINE

The objective of the investigation was to characterize infectious bursal disease viruses (IBDV) circulating in commercial poultry farms in Ukraine between 2014 and 2016. IBDV genetic material was amplified directly from bursa. The nucleotide sequence for VP2 hypervariable region of 16 IBDVs were determined by RT-PCR method, sequenced and compared to well characterised IBDV isolates worldwide. Neighbor-joining method was used for phylogenetic analyses. In result of the studyUkrainian IBDVs represented two genetic lineages: very virulent (vv) IBDVs and classical IBDV closely related to attenuated vaccine stains. The nucleotide identity among UkrainianvvIBDVs ranged between 87.2% and 99,8%. Ukrainian vvIBDV strains clustered together with very virulent strains from other counties like: United Kingdom, Egypt, China, Netherlands and Spain. In conclusion this report demonstrates the circulation of vvIBDV in commercial poultry farms in Ukraine.

Genetic characterisation of infectious bursal disease virus isolates in Ethiopia

The objective of the investigation was to characterise infectious bursal disease viruses (IBDV) circulating in commercial and breeding poultry farms in Ethiopia between 2009 and 2011. The nucleotide and deduced amino acid sequence for VP2 hypervariable region of ten IBDVs were determined by RT-PCR, sequenced and compared to well characterised IBDV isolates worldwide. IBDV genetic material was amplified directly from bursa or cell passaged material. Phylogenetically, Ethiopian IBDVs represented two genetic lineages: very virulent (vv) IBDVs or variants of the classical attenuated vaccine strain (D78). The nucleotide identity between Ethiopian vvIBDVs ranged between 0% and 2.6%. Ethiopian vvIBDVs are clustered phylogenetically with the African IBDV genetic lineage, independent of the Asian/European lineage. This report demonstrates the circulation of vvIBDV in commercial and breeding poultry farms in Ethiopia.

Molecular characterization and phylogenetic analysis of infectious bursal disease viruses isolated from chicken in South China in 2011

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus that causes immunosuppressive disease in young chickens. Thousands of cases of IBDV infection are reported each year in South China, and these infections can result in considerable economic losses to the poultry industry. To monitor variations of the virus during the outbreaks, 30 IBDVs were identified from vaccinated chicken flocks from nine provinces in South China in 2011. VP2 fragments from different virus strains were sequenced and analyzed by comparison with the published sequences of IBDV strains from China and around the world. Phylogenetic analysis of hypervariable regions of the VP2 (vVP2) gene showed that 29 of the isolates were very virulent (vv) IBDVs, and were closely related to vvIBDV strains from Europe and Asia. Alignment analysis of the deduced amino acid (aa) sequences of vVP2 showed the 29 vv isolates had high uniformity, indicated low variability and slow evolution of the virus. The non-vvIBDV isolate JX2-11 was associated with higher than expected mortality, and had high deduced aa sequence similarity (99.2 %) with the attenuated vaccine strain B87 (BJ). The present study has demonstrated the continued circulation of IBDV strains in South China, and emphasizes the importance of reinforcing IBDV surveillance.

Molecular Evidence of Very Virulent Infectious Bursal Disease Viruses in Chickens in Ethiopia

Infectious bursal disease virus (IBDV) is an important immunosuppressive pathogen of chickens worldwide. The introduction and evolution of IBDV in most African countries, especially in Ethiopia, remains unclear. We have investigated IBDV isolates obtained from commercial broilers, indigenous chickens, and pullets. The hypervariable region of the virus protein (VP) 2 and the 5' two-thirds of VP1 of 11 IBDV isolates were characterized by RT-PCR and further sequencing. All isolates were identified as very virulent (vv) IBDV based on the predicted amino acid (aa) sequences of the VP2 protein. Interestingly, the sequence analysis of the 5' two-thirds of VP1 indicated that the Ethiopian IBDV strains have aa residues typical for vvIBDV and for attenuated IBDV strains. Among all IBDV strains included in this study for phylogenetic comparison of VP2 nucleotide sequences, Ethiopian strains form a cluster within the vvIBDV lineage. We have also shown that Ethiopian IBDV strains have mutations in the VP1 region. Their roles in IBDV virulence may require further in vivo studies. As depicted in this study, the nucleotide and aa sequence analysis of VP1 in addition to VP2 is necessary to obtain a clear picture of the molecular evolution of IBDV.

Molecular epidemiology of infectious bursal disease viruses isolated from Southern China during the years 2000–2010

We performed a molecular epidemiology study of infectious bursal disease virus (IBDV) from six provinces in southern China by analyzing IBDVs isolated during the years 2000-2010. Sequence analysis of hypervariable regions of the VP2 gene (vVP2) in the genome of these isolates revealed that the majority of these viruses (45/59) were characterized as vv (very virulent) IBDV genotype, 12 out of 59 isolates were avirulent IBDV genotype and two from Guangxi were intermediate IBDV genotype. Phylogenetic analysis revealed that 45 vvIBDV genotype isolates have divided into five groups, all displaying strong divergence from the currently used vaccine strains. In all isolates, 14 non-critical amino acid substitutions were found in vVP2. The isolates from 2006 to 2010 had more substitutions (11 sites) than the isolates from 2000 to 2005 (7 sites). This study demonstrates that there were different genotypes of IBDV prevailing in six provinces of southern China. The mutations in vVP2 were common, which might be one of the reasons for the evolution of the IBDVs. Therefore, in regards to IBDV prevention, it is vital to have continuous monitoring of the genetic variability (long-term tracking of viral evolution) to provide optimal protection against IBDV.

Diversity of Genome Segment B from Infectious Bursal Disease Viruses in the United States

Several phylogenetic lineages of the infectious bursal disease virus (IBDV) genome segment B have been identified. Although this genome segment has been shown to contribute to virulence, little is known about the genetic lineages that exist in the United States. The nucleotide genome segment B sequences of 67 IBDV strains collected from 2002 to 2011 in the United States were examined. Although they were from nine different states, a majority (47) of these viruses were from California. A 722-base pair region near the 5' end of genome segment B, starting at nucleotide 168 and ending at 889, was examined and compared to sequences available in GenBank. The nucleotide sequence alignment revealed that mutations were frequently observed and that they were uniformly spaced throughout the region. When the predicted amino acids were aligned, amino acids at positions 145, 146, and 147 were found to change frequently. Six different amino acid triplets were observed and the very virulent (vv) IBDV strains (based on presence of vvIBDV genome segment A sequence) all had the triplet T145, D146, and N147. None of the non-vvIBDV strains had this TDN triplet. Phylogenetic analysis of the 67 nucleotide sequences revealed four significant genome segment B lineages among the U.S. viruses. One of these included the genome segment B typically found in vvIBDV and three contained non-vvIBDV genome segment B sequences. When the available sequences in GenBank were added to the analysis, two additional lineages were observed that did not contain U.S. viruses; one included viruses from China and the other contained viruses from the Ivory Coast. Although the samples tested do not represent all poultry producing regions in the United States, serotype 1 viruses from states outside California all belonged to one genome segment B lineage. The other three lineages observed in the United States were populated with viruses exclusively found in California, except the serotype 2 lineage, where the type strain was a serotype 2 virus from Ohio. The data provide further evidence for the importance of genome segment B identification during routine molecular diagnosis of all IBDV strains.

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

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

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

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

Sequence Analysis of Both Genome Segments of Three Croatian Infectious Bursal Disease Field Viruses

In order to determine the mutations responsible for virulence, three Croatian field infectious bursal disease viruses (IBDV), designated Cro-Ig/02, Cro-Po/00, and Cro-Pa/98 were characterized. Coding regions of both genomic segments were sequenced, and the nucleotide and deduced amino acid sequences were compared with previously reported full-length sequenced IBDV strains. Phylogenetic analysis, based on the nucleotide and deduced amino acid sequences of polyprotein and VP1, was performed. Eight characteristic amino acid residues, that were common to very virulent (vv) IBDV, were detected on polyprotein: 222A, 256I, 294I, 451L, 685N, 715S, 751D, and 1005A. All eight were found in Cro-Ig/02 and Cro-Po/00. C-Pa/98 had all the characteristics of an attenuated strain, except for glutamine on residue 253, which is common for vv, classical virulent, and variant strains. Between less virulent and vvIBDV, three substitutions were found on VP5: 49 G --> R, 79 --> F, and 137 R --> W. In VP1, there were nine characteristic amino acid residues common to vvwIBDV: 146D, 147N, 242E, 390M, 393D, 511S, 562P, 687P, and 695R. All nine residues were found in A-Ig/02, and eight were found in B-Po/00, which had isoleucine on residue 390. Based on our analyses, isolates Cro-Ig/02 and Cro-Po/00 were classified with vv IBDV strains. C-Pa/98 shared all characteristic amino acid residues with attenuated and classical virulence strains, so it was classified with those.

Development of Differential RT-PCR Assays and Molecular Characterization of the Complete VP1 Gene of Five Strains of Very Virulent Infectious Bursal Disease Virus

Several antigenic and pathogenic subtypes of infectious bursal disease virus (IBDV) have been identified worldwide. Simple and quick differential diagnostic assays are vital for implementing control and prevention strategies for infectious bursal disease (IBD). Currently reverse transcriptase-polymerase chain reaction/restriction fragment length polymorphism analysis (RT-PCR/RFLP) and real-time RT-PCR detection have been used. However, the RT-PCR/RFLP analysis is time consuming and not feasible for assaying large numbers of samples, and the real-time PCR is expensive. The reliable indicator for very virulent (vv) IBDV (vvIBDV) remains in vivo pathogenicity testing because of the lack of a virulence marker. In this study simple RT-PCR assays were developed for differentiating various types of IBDV. Two sets of primers specific for serotype 2 and vvIBDV were designed based on the sequences of segment A and B of IBDVs. Initially a total of 25 previously characterized virus strains including 11 serotype 1 classics, 4 serotype 1 variants, and 5 serotype 1 vv and 5 serotype 2 strains were used to validate the differential RT-PCR assays. The results indicated that primer set 1 specifically amplified a 415 bp RT-PCR product for the serotype 2 viruses, and primer set 2 specifically amplified a 715 RT-PCR product for vvIBDV except for two vv Taiwan strains. To further confirm the specificity of primer set 2 for the vvIBDVs, 20 field samples suspected to be vvIBDVs from different geographic locations around the world were tested. All but one suspected vv Korean strain (91108) tested positive by this primer set. To understand the molecular basis for the failure to detect these viruses with primer set 2, a complete VP1 gene of two vv Taiwan (PT and IL) strains along with two vv Turkey (OA and OE) and one vv Holland (Hol) strain was sequenced and phylogenetically analyzed with 11 other strains retrieved from GenBank. The results showed that PT and IL were closely related to the classic strain IM with nucleotide (nt) similarities of 98.6% and 97.7%, respectively. The two Taiwan strains clustered together with the classic and variant IBDVs in the unrooted neighbor-joining phylogenetic tree, indicating independent evolution of these strains from the rest of the vv isolates. The RT-PCR assays developed in this study could differentiate vvIBDVs from classic strains and serotype 1 from serotype 2 IBDVs with a high degree of sensitivity and specificity and are fast, simple, and inexpensive.

A Very Virulent Genotype of Infectious Bursal Disease Virus Predominantly Associated with Recurrent Infectious Bursal Disease Outbreaks in Tunisian Vaccinated Flocks

Outbreaks of infectious bursal disease (IBD) still continue to afflict the Tunisian poultry industry even in those flocks where the vaccination program is strictly applied. To characterize the viruses that circumvent protection provided by vaccination, field isolates of infectious bursal disease virus (IBDV) obtained from vaccinated flocks that have repeatedly experienced IBDV outbreak episodes were analyzed from bursal samples by reverse transcription coupled with polymerase chain reaction and dideoxynucleotide sequencing of the VP2 hypervariable region. Although sequence data were obtained from samples collected from three distinct flocks over a period of 3 years, only limited sequence variation has been observed. The few nucleotide changes were silent and the deduced amino acid sequences were identical. Thus, the virus population that predominates in the field seems to represent a homogeneous antigenic pool. Compared with the VP2 sequences of several IBDV strains, this predominant pool was found to be closely related to the very virulent (vv) IBDV viruses described in Europe and Asia. Sequence and phylogenetic analysis of the precursor polyprotein coding sequence of a representative Tunisian isolate further confirmed its assignment to the vv genotype. The deduced amino acid sequence of the whole polyprotein of the Tunisian isolate was found to be identical to a South Korean IBDV strain. Alignment of the polyprotein amino acid sequence of 35 IBDV strains identified additional mutations outside the VP2 variable domain and which occur frequently in vv strains. Based on this comparative analysis, the set of amino acid residues that should represent a typical vv profile involves Ala222, Ile242, Ile256, Ile294, Leu451, Tyr680, N685, Ser715, Asp751, Val990, and Ala1005. Such a combination of amino acid changes was observed for the majority of vvIBDV strains that define a distinct phylogroup.

Virus strains from a flock exhibiting unusually high mortality due to infectious bursal disease

To characterise infectious bursal disease viruses (IBDVs) isolated from commercial broiler flocks exhibiting unusually high mortality due to infectious bursal disease (IBD). An IBD outbreak occurred in mid 1999 on two broilers farms (A and B) in northern New South Wales amongst chickens 28 to 38 days of age, with a sharp rise in mortality of 2.5%. Initial histopathological diagnosis indicated acute IBD. Since acute IBD caused by classical pathogenic and very virulent (vv) IBDVs is exotic to Australia, samples from both farms A and B were obtained and used for virus characterisation. Tissue homogenates were made from six bursae collected from farm B. One histological sample from farm A was also used. Nucleotide sequencing of the hypervariable region (HVR) within the VP2 gene of IBDVs was determined and the deduced amino acid sequences compared with previously characterised Australian and overseas IBDVs. The phylogenetic relationship between IBDVs from farm B and IBDVs from Australia and overseas was then determined. Pathogenicity of one isolate, N2/99 from farm B, was compared with 3 other local IBDVs, as well as with three pathogenic overseas strains in 3-week-old specific pathogen-free (SPF) chickens. Initial histopathological characterisation of a sample of bursa from a bird on farm A showed widespread acute lymphoid necrosis, follicular haemorrhage and stromal oedema, indicative of acute IBD. Subsequent analysis using reverse transcriptase polymerase chain reaction (RT-PCR), followed by nucleotide sequencing of the same bursal sample, as well as 6 samples from nearby farm B, showed that the IBDVs involved were similar in sequence to Australian vaccine strains and not to classical pathogenic or vvIBDVs. One isolate, N2/99 from farm B, was only marginally more pathogenic than other local IBDVs. It induced mild clinical signs in 30% of chicks and no mortality. In comparison, vvIBDV CS89 and classical pathogenic 52/70 strains induced severe clinical signs in 100% and 80% of chickens, respectively with mortalities of 27% and 12%, respectively. The results illustrated the value of nucleotide sequencing as a method for discrimination of local and exotic types of IBDV.

Assessment of genetic, antigenic and pathotypic criteria for the characterization of IBDV strains

The aim of this work was the selection and comparison of representative infectious bursal disease virus (IBDV) strains. Nine strains of IBDV, isolated at different times and from different geographic regions of Europe and China, were characterized. Batches of all strains were prepared following standardized protocols and checked for the absence of contaminating viruses. Criteria used for their characterization were: (i) the nucleotide sequence of the VP2 variable region, (ii) binding to a panel of neutralizing monoclonal antibodies in antigen capture enzyme-linked immunosorbent assays, and (iii) virulence in specific pathogen free chickens after infection with a standardized number of median embryo infective doses. Based on the first two criteria, two of nine strains were classified as classical virulent (cv) IBDV (F52/70, Cu-1wt), and five as very virulent (vv) IBDV (849VB, 96108, HK46, GX, Harbin). Remarkably, although a clear-cut difference was demonstrable between European cvIBDV (F52/70 and Cu-1wt) and vvIBDV (849VB and 96108) strains, there was a continuum in the pathogenicity of Chinese vvIBDVs. Our results indicate the probable existence of differences in virulence within IBDV lineages determined on the basis of antigenic typing using monoclonal antibodies and the alignment of the VP2 sequences. This indicates limitations in the analysis of IBDV pathotypes based on the VP2 variable region and emphasizes that these criteria may not be sufficient for the classification of IBDV strains.

Characterization of infectious bursal disease virus isolates from Indonesia indicates the existence ofvery virulent strains with unique genetic changes

Sequencing of the hypervariable region of viral protein VP2 of infectious bursal disease virus (IBDV) isolates obtained from non-vaccinated chickens in Indonesia showed that the majority (16/17) were closely related topublished very virulent (vv)IBDV strains. Four isolates contained identical amino acid sequences to Asian andEuropean wIBDVs, sharing w-specific amino acid residues 222(Ala), 256(Πe), and 294(Πe). Eight isolatesdiffered by one amino acid at position 222(Ala->Ser); however, this change did not alter the pathogenicity orantigenicity of these strains. Two isolates, with amino acid substitutions at positions 272(Π→Thr) and279(Asp→Asn), did not cause clinical disease or mortality, and were therefore considered to be naturallyoccurring, attenuated mutants of vvIBDV. The results illustrate variability that might occur among vvIBDVstrains.

Antigenic and molecular characterization of recent infectious bursal disease virus isolates in China.

Eleven infectious bursal disease virus (IBDV) strains isolated recently from China were compared with the early classical virulent strain CJ801, the chicken embryo fibroblast-adapted (CEF) variant strain GZ902, and the attenuated vaccine strains BJ836, BK912, and LM to discern the evolutionary characteristics of IBDV in China at both antigenic and genetic levels. Virus neutralization (VN) assay showed that all ten very virulent (vv) IBDV strains belong to the same subtype as attenuated strains, whereas the other variant isolate strain BX could be attributed to other subtype of the variant strain GZ902. Antigen-capture ELISA (AC-ELISA) determined by a panel of monoclonal antibodies (Mabs) against classical and variant strains showed further that among these vv strains, nine strains except for strain NC had no reaction with neutralizing Mab B69. The vv strains SC and YV had no reaction with non-neutralizing Mabs 2B8 and 2C4, respectively, whose epitopes were located in classical IBDV strains. On the other hand, there is no alteration in antigenic epitopes located in the variant strain BX as that of the variant GZ902. Sequence comparison of the highly variable region (HVR) of the VP2 proteins showed that these vv strains had 98.6-100.0% identities to European and Asian vv strains at amino acid level. For the vv strains NC, SC, and YV, all had one amino acid substitution at the major hydrophilic domains, indicating that new vv strains are evolving. In addition, the vv strains DMS and NC had amino acid residue 279N as well, showing that the substitution of amino acid at this position might not be related to the virulence of IBDV. The variant strain BX had one amino acid substitution in the two major hydrophilic domains and two unique amino acids 249K and 254S as the other early variant strains, and shared 97.3% of amino acid identity to the variant strain VarE. Phylogenetic analysis suggests that the recent Chinese vvIBDVs and the previous European and Asian vv strains still belong to a genetic group and the variant strain BX to the other genetic group, which is more closely related to the European classical virulent strain F52/70 and the American classical virulent strain STC than to the early Chinese classical virulent strain CJ801, showing that the recent vv and variant strains that spread widely in the country might be derived from Europe and America than from early Chinese classical virulent strains.