Search

Since the detection of antigenically atypical very virulent Infectious bursal disease viruses (vvIBDV) in Egypt in 1999, the country has been experiencing recurrent outbreaks with high mortality rates and typical gross lesions associated with typical vvIBDV. However, a significant change occurred in 2023, marked by a notable increase in reported subclinical IBDV cases. To evaluate the field situation, samples from 21 farms in 2023 and 18 farms from 2021 and 2022, all of which had experienced IBD outbreaks based on clinical diagnosis, were collected, and subjected to VP2-HVR sequencing. Phylogenetic analysis revealed that all samples collected in 2021 and 2022 clustered with classical virulent strains and vvIBDV. In 2023, one sample clustered with the Egyptian vvIBDV, another with classical virulent IBDV, and the rest with the novel variant IBDV (nVarIBDV) circulating in China. The alignment of deduced amino acid sequences for VP2 showed that all Egyptian classic virulent strains were identical to the Winterfield or Lukert strains, while vvIBDV strains exhibited two out of the three typical residues found in Egyptian vvIBDV, namely Y220F and G254S, but not A321T. Meanwhile, all Egyptian variant strains exhibited typical residues found in nVarIBDV. However, all Egyptian variants showed a mutation at position 321 (321V), which represents the most exposed part of the capsid and is known to have a massive impact on IBDV antigenicity, except for one sample that had 318G instead. This report highlights the emergence of a new variant IBDV in Egypt, clustered with the Chinese new variants, spreading subclinically in broiler farms across a wide geographic area. RESEARCH HIGHLIGHTS New variant IBDV which emerged in Egypt clustered with Chinese nVarIBDV.nVarIBDV spread subclinically across a wide geographic area.Mutation at 321 represents capsid's most exposed part, a defining feature.Antigenically modified vvIBDV still circulating in Egypt with typical lesions.

Infectious bursal disease (IBD) is one of the most important immunosuppressive diseases of young chickens, causing considerable economic losses to the poultry industry. More than 30 years ago, an antigenic variant (av) pathotype of the IBD virus (IBDV) was reported to originate in, and subsequently spread among, poultry farms in the USA. Recently, a novel avIBDV lineage was identified in China and was shown to exhibit clear differences in its pathogenicity as well as molecular characteristics compared with the previously isolated variant strains. In this study, we conducted a passive surveillance of chicken carcasses submitted to our research division from June-December 2019, and detected the IBDV strains by reverse transcription PCR. Five avIBDV strains were isolated, and their pathogenicity was determined by necropsy and molecular analysis. Additionally, a coinfection field case involving an avIBDV strain and a very virulent IBDV (vvIBDV) strain was identified. Multiple sequence alignment and phylogenetic analysis of partial viral protein 1 (VP1) and hypervariable region (hv) VP2 genes revealed that those strains originated from two different avIBDV lineages. The co-occurrence of two sub-groups of avIBDVs in South Korea confirms for the first time the evolution of antigenic variant IBDV strains, and highlights the urgency for the development of new strategies for IBDV intervention in South Korea. RESEARCH HIGHLIGHTS Five avIBDV strains were identified in South Korea by passive surveillance test in 2019.A coinfection between two IBDV strains from different genogroups was reported in a field case.By phylogenetic analysis, Korean avIBDVs belonged to two distinct lineages of antigenic variant genogroup.

Recently, multiple spillover events between domesticated poultry and wild birds have been reported for several avian viruses. This phenomenon highlights the importance of the livestock-wildlife interface in the possible emergence of novel viruses. The aim of the current study was to investigate the potential spillover and epidemiological links of infectious bursal disease virus (IBDV) between wild birds and domestic poultry. To this end, twenty-eight cloacal swabs were collected from four species of free-living Egyptian wild birds (i.e. mallard duck, bean goose, white-fronted goose and black-billed magpie). Genetic and phylogenetic analysis of three positive isolates revealed that the IBDV/USC-1/2019 strain clustered with previously reported very virulent IBDV (vvIBDV) Egyptian isolates. Interestingly, two other wild bird-origin isolates (i.e. IBDV/USC-2/2019 and IBDV/USC-3/2019) grouped with a vaccine strain that is being used in commercial poultry. In conclusion, our results revealed the molecular detection of vaccine and vvIBDV-like strains in Egyptian wild birds and highlighted the potential role of wild birds in IBDV epidemiology in disease-endemic regions.

Infectious bursal disease virus (IBDV) is the aetiological agent of a highly contagious chicken immunodeficiency disorder known as Gumboro disease, which cause severe economic loses to the poultry worldwide. The emergence of very virulent IBDV strains (vvIBDV) during the late 80s resulted in drastic changes to the epidemiology of IBDV with a dramatic increase in the mortality of the animals affected. Molecular studies determined that the emergence of the vvIBDV was a consequence of a genomic reorganization of IBDV known as reassortant event by which the virus combined two emergent genetic background vvIBDV for segment A and vvIBDV for segment B. In the current study, a retrospective analysis was conducted, and samples collected during acute outbreaks of Gumboro disease in Poland during 1992-2015 were submitted to sequencing and further molecular and phylogenetic analyses. The results obtained not only revealed a high genetic diversity for Polish IBDV strains but a new population of IBDV was identified. These novel reassortant strains with a unique genetic background contain the segment A from very virulent strains and segment B from an unidentified source, phylogenetically segregated and classified as 'transitional lineage'. The results obtained also showed the presence of this new lineage in Finland, evidencing the expansion of this new genomic reorganized viral strain in Europe representing an additional threat to the global situation of IBDV., (© 2020 Blackwell Verlag GmbH.)

Infectious bursal disease virus (IBDV), which infects young chickens, is one of the most important pathogens that harm the poultry industry. Evaluation of the immune status of birds before and after vaccination is of great importance for controlling the disease caused by this virus. Therefore, the development of low-cost and easy-to-manufacture test systems for IBDV antibody detection remains an urgent issue. In this study, three expression systems (bacteria, yeast, and human cells) were used to produce recombinant VP3 protein of IBDV. VP3 is a group-specific antigen and hence may be a good candidate for use in diagnostic tests. Comparison of the antigenic properties of the obtained polypeptides showed that the titres of antibodies raised in chickens against bacteria- or human-cell-derived recombinant VP3 were high, whereas the antibody level against yeast-derived recombinant VP3 was low. The results of an enzyme-linked immunosorbent assay (ELISA) of sera from IBDV-infected chickens demonstrated that the recombinant VP3 produced in E. coli would be the best choice for use in test systems.

Infectious bursal disease (IBD) is an immunosuppressive viral disease of chickens, associated with severe economic losses and major threats to poultry production worldwide. Disease prevention programs rely on unequivocal identification of the pathogen, as well as vaccination programs. This study developed a sensitive, one-step, real-time, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay using a hydrolysis probe system for infectious bursal disease virus (IBDV, VP1 gene) detection and quantification, which was compared to other routinely used diagnostic methods. The assay successfully detected IBD reference viruses and field isolates. The absence of cross-reactivity was detected with negative samples or with other avian viruses in the analytical specificity test. The detection limit of this assay was 70 RNA copies. RT-qPCR was more sensitive in the detection of serially diluted IBDV isolates compared to virus isolation. For clinical samples, the sensitivity and specificity values of RT-qPCR compared to enzyme-linked immunosorbent assay (ELISA) were 97.5% and 100%, respectively, and compared to histopathology, these values were 100% and 93.94%, respectively. RT-qPCR can provide a simple and reliable assay for IBDV surveillance programs and for evaluation of control strategies.

Infectious bursal disease virus (IBDV) is an immunosuppressive pathogen of poultry causing great economic losses to the poultry industry. In this study, the IBDV antibodies were detected in captured free-living wild birds in Zaria, Nigeria. One hundred and fifty free-living wild birds, comprising 30 birds each of 5 different species, were sampled over a period of 9 months. Blood samples were collected from each bird, and harvested sera were tested for IBDV antibodies using enzyme-linked immunosorbent assay. Results indicated IBDV seroprevalences in speckled pigeon (6.67%) and cattle egret (3.33%). In conclusion, the detection of IBDV antibodies in free-living wild birds in this study is indicative of previous natural exposure of these birds to the virus. These species of wild birds could therefore serve as carriers of these viruses and, consequently, transmit these viruses to chickens., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Infectious bursal disease virus (IBDV), the etiological agent of infectious bursal disease (IBD), is a variable RNA virus of Avibirnavirus. Some artificially attenuated vaccine strains of IBDV can adapt to cell culture of chicken embryo fibroblast (CEF) cell or its immortalized cell line DF1 in vitro while wild-type IBDV cannot. In this study, for the first time, a naturally occurring cell-adapted classic strain (genogroup 1) of IBDV named IBD17JL01 was identified in China. Animal experiments showed that IBD17JL01 could severely damage the central immune organ of infected chickens. Sequence analysis of the full-length genome revealed the peculiar molecular characteristics of IBD17JL01 with a few amino acid substitutions that might be involved in cell-tropism, antigenicity, and virulence of IBDV. Identification of this novel strain is beneficial to our understanding of the complexity of the epidemiology of IBDV. And the expansion of viral cell-tropism might increase the potential risk of the reassortment of different IBDVs including the live vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Recently, a new genotype of infectious bursal disease virus (IBDV), named ITA, was detected in IBD-vaccinated Italian broilers. Genome characterization revealed ITA to be a genetically different IBDV, belonging to genogroup 6 according to a recently proposed IBDV classification. The currently available clinical data do not allow any definition of the degree of pathogenicity of the ITA-IBDV isolates. In the present study, a pathogenicity trial was conducted by the oral inoculation of specific-pathogen-free (SPF) chickens. Birds were housed in poultry isolators and inoculated at 35 days of age with an ITA-IBDV isolate (35 birds) or a strain belonging to the G1a genogroup as a comparison (35 birds). Control birds (25 birds) were contextually mock-inoculated with sterile water. Birds were observed daily for clinical signs and at 0, 7, 14, 21 and 28 days post-inoculation (dpi) were bled for IBDV antibody detection. At 2, 4, 7, 14, 21 and 28 dpi, five birds from each of the inoculated groups, and three from the control group, were euthanized and subjected to a post-mortem examination; the bursa:body weight and thymus:body weight ratios were calculated. Microscopic lesions of the bursa and thymus were scored on the basis of lymphoid necrosis and/or depletion or cortex atrophy, respectively. Both viruses induced a subclinical course of disease, as neither clinical signs nor mortality were recorded during the study, even in the presence of typical IBDV gross and microscopic lesions. Bursal damage, measured by the bursa:body weight ratio, was more noticeable and precocious after ITA-IBDV inoculation. Histopathology scores of the bursa, indicative of rapid lymphoid depletion, confirmed the aggressiveness of the ITA-IBDV strain in this organ. This study showed that, although the ITA-IBDV strain tested causes infection with a subclinical course, it induces severe damage to lymphoid tissues. Therefore, its circulation in birds might be a threat for the poultry industry and may jeopardize the success of the production cycle., (© 2019 Blackwell Verlag GmbH.)

Bursa tissue samples from a pullet flock in New York State that was experiencing immune suppression related disease were sent to our laboratory in 2018. A very virulent infectious bursal disease virus (vvIBDV) was identified in those samples through molecular and pathogenicity studies and designated 1/chicken/USA/1054NY/18. Phylogenetic analyses of the hypervariable VP2 nucleotide sequence region indicated that this strain belonged to genogroup 3 which comprises the vvIBDV. Partial sequence data of the VP1 gene indicated this virus also had a VP1 typical of vvIBDV. While vvIBDV have previously been identified in the United States in California and Washington State, the 1054NY vvIBDV was most closely related to isolates from Ethiopia, suggesting it is a new introduction into the U.S. The 1054NY vvIBDV was used to challenge four-week old specific-pathogen-free (SPF) layer chicks where it caused 100% morbidity and 68.7% mortality within 4 days. Upon necropsy, gross pathological findings in infected SPF birds included small yellowish coloured bursas, some with haemorrhages on the serosal and mucosal surfaces. Microscopic lesions included inflammation, severe lymphocyte necrosis, atrophy of the follicles and follicular depletion of lymphocytes. RESEARCH HIGHLIGHTS A very virulent infectious bursal disease virus (vvIBDV) was detected in a pullet flock in New York state, USA. Nucleotide sequence analysis of the vvIBDV VP2 gene indicates it is not related to previous US vvIBDV isolates and appears to be a new introduction into the US. The New York vvIBDV caused 100% morbidity and 68.7% mortality in four-week-old specific-pathogen-free chicks.

The complete nucleotide sequence of the viral protein 2 (VP2) ORF was determined for 26 Vietnamese infectious bursal disease isolates collected from clinical outbreaks in vaccinated flocks from 1987 to 2018 and two commercial vaccine specimens. These sequences were compared for molecular classification with 42 reference strains representing all four main classes of serotype 1, including very virulent (vvIBDV), classical (cvIBDV), antigenic variant (avIBDV) and attenuated (atIBDV) strains, and serotype 2 strains. Amino acids at nine key positions in the VP2-HVR in 20 Vietnamese isolates, A222, I242, Q253, I256, D279, A284, I294, S299, A329, which are typical of the vvIBDV class, were found to be identical in all of the isolates. Eighteen of these isolates had a unique change at residue 212 (D212N) located in the P AB loop. Phylogenetic analysis revealed a distinct lineage/subclade with strong nodal support (96%) that included recent Chinese IBDV strains that were distinct from typical vvIBDVs. Six isolates contained the amino acid substitutions P222, V242, Q253, V256, D279, A284, I294, N299, A329, which are present in two vaccine strains derived from strain 2512 and these isolates were also closely related to the classical virulent STC strain. Data from this study show that there is considerable genetic diversity among vvIBDVs, which vary according to geographic region. Antigenic drift and differences in genetic characteristics between virulent strains and IBDV vaccine strains may be the cause of vaccine failure. Better antigenic matching of vaccines to the strains circulating in Vietnam is therefore required.

Between July and September 2017, samples collected from six unvaccinated chickens in Namibia were shown to be positive for infectious bursal disease virus (IBDV) by RT-PCR. Partial sequence and phylogenetic analysis of the VP1 and VP2 genes from six viruses revealed that they all belong to the very virulent pathotype (Genogroup 3) and are genetically very similar to IBDVs identified in neighbouring Zambia. This is the first molecular characterisation of IBDV in Namibia and has implications on the control and management of the disease in the country.

Fowl adenovirus serotype 4 (FAdV-4) is the pathogenic agent of hydropericardium hepatitis syndrome (HHS) in chickens and ducks, which has caused huge economic losses for the Chinese poultry industry since 2015. In order to objectively determine the prevalence and co-infection status of the virus in Shandong province in China, we analyzed a total of 679 clinical cases of chickens and ducks from 36 farms in the province. The results showed that the FAdV-4 infection rate was 65.2% (443/679), and the rate in breeder ducks was almost two-fold higher than that in breeder chickens (68.57% vs. 34.30%). Notably, co-infection by H9N2 avian influenza virus, infectious bursal disease virus, and/or chicken infectious anemia virus was very common in the 443 FAdV-4-positive cases. Furthermore, phylogenetic analysis of the hexon genes of four Shandong FAdV-4 isolates revealed that these strains clustered into Indian reference strains, indicating that the Shandong FAdV-4 strains might have originated in India. These findings provide the first data on the prevalence and co-infection status of FAdV-4 in Shandong province, which may serve as a foundation for the prevention of FAdV-4 in the field.

Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious immunosuppressive disease affecting young chickens. The recently described "distinct IBDV" (dIBDV) genetic lineage encompasses a group of worldwide distributed strains that share conserved genetic characteristics in both genome segments making them unique within IBDV strains. Phenotypic characterization of these strains is scarce and limited to Asiatic and European strains collected more than 15 years ago. The present study aimed to assess the complete and comprehensive phenotypic characterization of a recently collected South American dIBDV strain (1/chicken/URY/1302/16). Genetic analyses of both partial genome segments confirmed that this strain belongs to the dIBDV genetic lineage and that it is not a reassortant. Antigenic analysis with monoclonal antibodies indicated that this strain has a particular antigenic profile, similar to that obtained in a dIBDV strain from Europe (80/GA), which differs from those previously found in the traditional classic, variant and very virulent strains. Chickens infected with the South American dIBDV strain showed subclinical infections but had a marked bursal atrophy. Further analysis using Newcastle disease virus-immunized chickens, previously infected with the South American and European dIBDV strains, demonstrated their severe immunosuppressive effect. These results indicate that dIBDV strains currently circulating in South America can severely impair the immune system of chickens, consequently affecting the local poultry industry. Our study provides new insights into the characteristics and variability of this global genetic lineage and is valuable to determine whether specific control measures are required for the dIBDV lineage. Research Highlights A South American strain of the dIBDV lineage was phenotypically characterized. The strain produced subclinical infections with a marked bursal atrophy. Infected chickens were severely immunosuppressed. The dIBDV strains are antigenically divergent from other IBDV lineages.

Background: Lates calcarifer, known as seabass in Asia and barramundi in Australia, is a widely farmed species internationally and in Southeast Asia and any disease outbreak will have a great economic impact on the aquaculture industry. Through disease investigation of Asian seabass from a coastal fish farm in 2015 in Singapore, a novel birnavirus named Lates calcarifer Birnavirus (LCBV) was detected and we sought to isolate and characterize the virus through molecular and biochemical methods., Methods: In order to propagate the novel birnavirus LCBV, the virus was inoculated into the Bluegill Fry (BF-2) cell line and similar clinical signs of disease were reproduced in an experimental fish challenge study using the virus isolate. Virus morphology was visualized using transmission electron microscopy (TEM). Biochemical analysis using chloroform and 5-Bromo-2'-deoxyuridine (BUDR) sensitivity assays were employed to characterize the virus. Next-Generation Sequencing (NGS) was also used to obtain the virus genome for genetic and phylogenetic analyses., Results: The LCBV-infected BF-2 cell line showed cytopathic effects such as rounding and granulation of cells, localized cell death and detachment of cells observed at 3 to 5 days' post-infection. The propagated virus, when injected intra-peritoneally into naïve Asian seabass under experimental conditions, induced lesions similar to fish naturally infected with LCBV. Morphology of LCBV, visualized under TEM, revealed icosahedral particles around 50 nm in diameter. Chloroform and BUDR sensitivity assays confirmed the virus to be a non-enveloped RNA virus. Further genome analysis using NGS identified the virus to be a birnavirus with two genome segments. Phylogenetic analyses revealed that LCBV is more closely related to the Blosnavirus genus than to the Aquabirnavirus genus within the Birnaviridae family., Conclusions: These findings revealed the presence of a novel birnavirus that could be linked to the disease observed in the Asian seabass from the coastal fish farms in Singapore. This calls for more studies on disease transmission and enhanced surveillance programs to be carried out to understand pathogenicity and epidemiology of this novel virus. The gene sequences data obtained from the study can also pave way to the development of PCR-based diagnostic test methods that will enable quick and specific identification of the virus in future disease investigations.

Background: Infectious bursal disease virus (IBDV) is the causative agent of Infectious Bursal Disease (IBD), the disease causes immunosuppression which leads to secondary infections among rearing poultry flocks. Characterization of the virus is important for its control and eradication. The circulating IBDVs are classified on the basis of their antigenic and pathogenic properties. The virus is categorised as classical, variant and very virulent IBDV (vvIBDV). IBDV is a non-envelop, icosahedral double stranded virus. Viral protein 2 (VP2) is the major structural protein of capsid that determines the host-pathogen relationship. The aim of this study was to characterise the IBD virus of Pak-Asian region., Methodology: IBDV suspected flocks were examined in Punjab, Pakistan from 2014-2018. Two hundred and fifty samples were collected with complete history of the disease. The suspected samples were collected from broiler, layer and rural poultry farms. RNA was extracted and hyper-variable region of VP2 gene was amplified using specific primers. Nucleotide sequence of the VP2 gene was determined and its Amino Acid sequence was deduced. Moreover, phylogenetic analysis was also performed., Results: The current classifications based in a hyper-variable region of the capsid protein VP2 (hvVP2), classification of IBDVs is split into newly proposed geno-groups according to Jackwood group. Among these prevailing, some IBDVs are limited geographically whereas, others are reported cosmopolitan. Genetic alterations are continuously playing role in evolution of new strains of the virus., Conclusion: During this study it was found that isolates of IBDV fall in first three geno-groups. Most of the geno-groups are prevalent around the world, whereas the mutated and re-assorted ones are confined in particular areas of the globe., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Fowl adenovirus (FAdV), which causes the high-impact diseases such as inclusion body hepatitis and hepatitis-hydropericardium syndrome, is of major concern to the poultry industry internationally. This study was carried out in direct response to mortality rates of up to 75% in commercial broiler flocks in Trinidad, West Indies. Symptoms in 3- to 8-week-old broilers and 13- to 18-week-old pullets pointed to infection with an immunosuppressive viral pathogen. The objectives of the study were to determine whether the infectious agent FAdV, along with other viral pathogens, was responsible for the clinical disease, and to obtain information on the serotypes of FAdV that were infecting the birds. Tissue samples from clinically affected birds from eight different farms were tested for chicken infectious anaemia virus (CIAV) and infectious bursal disease virus (IBDV) by real-time reverse transcription polymerase chain reaction (PCR) and for FAdV by conventional PCR. The birds tested positive for FAdV and CIAV, but negative for IBDV. The gene corresponding to the L1 loop of the hexon protein for FAdV was amplified and sequenced. Phylogenetic analysis of seven FAdV strains inferred that four serotypes were likely to be circulating in the chickens. Well supported genetic relatedness was observed for serotype 8a (97.8%), 8b (97.8%), 9 (95.8%) and 11 (98.8%-99.5%). This is the first published report from Trinidad and Tobago on the presence and circulation of pathogenic FAdV strains, in combination with CIAV, in poultry. The data demonstrate a possible need for the introduction of serotype-specific vaccines against FAdV, as well as vaccines against CIAV, in broilers in the region and emphasize the importance of maintaining high levels of biosecurity on farms to prevent the spread of these potentially devastating viruses between farms., (© 2019 Blackwell Verlag GmbH.)

Infectious bursal disease virus is a causative agent of one of the most important disease which causes frequent tragic disaster in the poultry industry all over the world. Therefore, in the present study a new fluorescence resonance energy transfer-based technique was developed to detect VP2 gene of infectious bursal disease virus using two oligonucleotide probes labeled with quantum dots and rhodamine- immobilized gold nanoparticles (AuNPs-Rh). Quantum dots labeled with an amino-modified first oligonucleotide, and AuNPs-Rh labeled with thiol-modified second oligonucleotides were added to the DNA targets upon which hybridization occurred. In the presence of target the AuNPs-Rh will be located in the vicinity of the quantum dots and leads to the fluorescence resonance energy transfer to be occurred and subsequently the fluorescence intensity of quantum dots was stimulated. The immobilization of rhodamine to the surface of AuNPs increased the fluorescence intensity of rhodamine. The maximum fluorescence resonance energy transfer efficiency for the developed sensor is monitored at a quantum dots-P A /AuNPs-Rh-P T molar ratio of 1:10. Moreover, the feasibility of the developed nanobiosensor was demonstrated by the detection of a synthetic 49-mer nucleotide derived from infectious bursal disease virus and the limit of detection was estimated as 3 × 10 -8  M. The developed DNA detection scheme is a simple, rapid and efficient technique which does not need excessive washing and separation steps., (Copyright © 2018. Published by Elsevier B.V.)

The infectious bursal disease (IBD) causes immunosuppression in chicken of all ages and high mortality in young chicken, posing serious threat to poultry industry worldwide. One promising strategy for preventing this highly contagious disease is using recombinant subunit vaccine, employing VP2 subviral particles (SVP) as epitomic antigen. Analytical techniques of viral-like particles such as SDS-PAGE, western blot, or high-performance size-exclusion chromatography have been widely applied, but mostly unsatisfactory. In the present study, a simple, fast and cost-effective capillary zone electrophoresis (CZE) method with UV-detection was developed to analyze purified IBDV-SVP (expressed by Escherichia coli system) using commercial monoclonal antibody (mAb) against VP2. To find satisfying CZE conditions, injection mode, separation voltage, and separation buffer were explored. Through the modified CZE, mAb and SVP could be well separated and shown distinct peaks in the electropherogram. Furthermore, to determine the stoichiometry, the area of the mAb peak versus SVP/mAb binding ratio was plotted and indicated that 2 or 3 receptor molecules were bound per SVP. The purity and integrity of SVP and the interactions between SVP and mAb could be analyzed by the developed simple CZE-UV method in less than half hour. This CZE-UV method proved to be a valuable and useful tool in detection, characterization, and quantification of IBDV-SVP and the mAb, offering potential applications of in-process quality control of vaccine production, surveillance of serum antibody produced against IBDV infection, or vaccine immunization., (© 2018 Poultry Science Association Inc.)

Infectious bursal disease (IBD) is one of the most important immunosuppressive diseases that seriously threaten poultry farming and food safety worldwide. The variant strain of infectious bursal disease virus (IBDV) has been greatly neglected for more than 30 years. Recently, the subclinical infection of suspected IBD, causing considerable economic losses, occurred in the main chicken-farming regions of China. Through RT-PCR, sequencing, and phylogenic analyses, novel variant IBDVs were first identified in six provinces of eastern China. Immunological detection further confirmed the antigenic variation of the Chinese variant IBDVs. The Chinese IBDV variants were obviously different from the American IBDV variants, with less than a 97.7% (VP1) or 98.7% (VP2) amino acid sequence identity. Animal experiments further confirmed the serious threat of the variant IBDVs to chickens, demonstrating irreversible damage to the central immune organ, obvious immunosuppression, and growth retardation. This study not only identified the pandemic nature of the novel variant IBDVs for the first time but also discovered the distinct molecular epidemiological characteristics of these viruses, which will contribute more to the control of the disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Very virulent infectious bursal disease virus (vvIBDV), the cause of significant economic losses in many poultry-producing areas, has been present in Morocco since 1991. In spite of the introduction of vaccination, disease outbreaks are frequently observed. To ascertain if vaccines failure may be due to the emergence of new strains, the aim of this study was to perform for the first time the molecular characterization of vvIBDV strains circulating in Morocco by focusing on the hypervariable region (HVR) of the VP2 protein, which is frequently used for molecular epidemiology and phylogenetic studies. Field samples of haemorrhagic bursae of Fabricius were collected for molecular characterization in different parts of the country during 2016-2017 from 48 chicken flocks showing symptoms of disease. In a phylogenetic tree, nucleotide sequences containing the VP2 HVR of 13 samples that were positive for vvIBDV formed a common branch with those of vvIBDV references strains published in GenBank, but they clearly grouped into a distinct subcluster. An alignment of the deduced amino acid sequences, in addition to confirming the presence of the "signature" typical of the vvIBDV HVR, also revealed the presence of substitutions in hydrophilic loops that are known to be involved in the elicitation of neutralizing antibodies. One of these substitutions is unique to the Moroccan isolates. These results represent the first molecular characterization of vvIBDV isolates in Morocco and may indicate that one of the causes of vaccine ineffectiveness is antigenic drift.

Infectious bursal disease virus (IBDV) and chicken anemia virus (CAV) cause relevant immunosuppressive diseases in poultry. Clinical diagnosis of these viruses is challenging given the different disease presentations and the frequent occurrence of co-infections with other pathogens. Here, we standardized and validated simplex and duplex RT-qPCR assays for the straightforward detection of IBDV and CAV. The qPCR assays are based on primers and hydrolysis probes that target highly conserved regions of IBDV and CAV genomes. Analytical sensitivity tests on 10-fold serial dilutions containing 10 0 -10 8 viral genomes indicated that the simplex assays have good determination coefficients and efficiency and detect a wide range of virus doses (10 2 to 10 8 molecules copies/reactions). The relatively small values of intra- and inter-assay variability ensure the repeatability and support its reproducibility in different diagnostic and research facilities. The assays are also efficient tools for absolute quantification as indicated by the analytical performance analysis. The assays have an excellent specificity and absence of cross-reactivity with negative samples, or with other common avian viruses. The simplex IBDV and CAV assays use probes labelled with different dyes (FAM and HEX) and can be multiplexed for the simultaneous detection of both viruses. The determination coefficients, PCR efficiencies, and relatively small intra- and inter-assay variability were comparable to the simplex assays. This duplex assay is the first to simultaneously detect IBDV and CAV using the same RNA extraction from the bursa of Fabricius in a single and straightforward step. Therefore, this method is time saving, provides quantitative results for both targets without any cross-reaction, and reduces the risk of carrying-over contaminations. The qPCR assays here developed can be used in simplex and duplex formats for detection and quantification of large number of samples with reliable sensitivity and specificity. These tools are expected to improve surveillance and control of these ubiquitous viruses., (Copyright © 2018. Published by Elsevier Ltd.)

Infectious bursal disease virus (IBDV) was initially identified in the USA. For decades, these viruses were not categorized using a typing system because they were considered to be antigenically and pathogenically similar. In the 1980s, a second major serotype, serotype 2, was found in turkeys. Classification of IBDV became more complex with the discovery of antigenic variant strains called "variants" in the United States and a highly virulent strain known as "very virulent" or vvIBDV identified in Europe. To distinguish the IBDV strains identified prior to this time from the antigenic variant viruses, the term "classic viruses" was adopted. Studies over the next three decades produced a wealth of information on the antigenicity, pathogenicity and molecular structure of IBDV isolates. These data made it clear that the descriptive nomenclature used for IBDV was inadequate. For example, not all viruses identified as vvIBDV by genotyping are highly pathogenic; some have reassorted genome segments that result in lower virulence. Furthermore, variant viruses are not an antigenically homogeneous group and the term "classic virus" has been used interchangeably to describe antigenic and pathogenic types of IBDV. These and other issues make the current naming system for strains of IBDV archaic. The lack of uniform testing and standards for antigenicity and pathogenicity makes it difficult to categorize IBDV strains on a global basis. A new nomenclature that includes a genotyping system that can easily be applied worldwide is proposed and serves as a platform to begin discussions on its value to the scientific community.

In recent years, acute severe outbreaks of infectious bursal disease (IBD) are frequently observed in commercial chicken populations of the North East Region (NER) of India, resulting in huge economic loses to poultry farmers. Field outbreaks of IBD in 30 different poultry farms in the NER were confirmed by clinicopathologic examination and reverse transcriptase PCR. A total of 10 isolates of IBD virus (IBDV) from these outbreaks were characterized by the genetic analysis of VP1 and the hypervariable region of the VP2 gene. Nucleotide sequences, deduced amino acid sequences, and phylogenetic analysis of both VP2 and VP1 genes revealed two genetically diverse strains of very virulent IBDV (vvIBDV) and one intermediate strain circulating in the NER. These isolates differ at nucleotide and amino acid levels from vvIBDV isolates of mainland India and are clustered in distinctly separate groups in the phylogenetic tree. Six of the isolates revealed a unique combination of vvIBDV amino acid signatures in the VP2 gene (A222, I256, I294), while bearing the non-vvIBDV amino acid signatures of the VP1 gene (146E, 147G, 242D), but they are clearly classified as vvIBDV in a phylogenetic analysis of both genes. Interestingly, one of the isolates showed 99% sequence homology with attenuated vaccine strains in the VP2 gene and clustered together. This study demonstrates the diversity of IBDVs in India and document for the first time the possible involvement of attenuated vaccine strains in the epidemiology of IBD in India.

Infectious bursal disease (IBD) is a highly contagious, immunosuppressive disease of chickens and causes substantial economic losses to the poultry industry globally. This study investigated the genetic characteristics and pathological lesions induced by IBD viruses (IBDVs) that were associated with 60 suspected outbreaks in chickens during 2015-2016 in Lusaka Province, Zambia. Nucleotide sequences of VP2 hypervariable region (VP2-HVR) (n = 38) and part of VP1 (n = 37) of Zambian IBDVs were phylogenetically analysed. Phylogenetic analysis of the VP2-HVR and VP1 revealed that most viruses (n = 31 of each genome segment) clustered with the very virulent (vv) strains. The rest of the viruses clustered with the classical strains, with two of the viruses being closely related to attenuated vaccine isolates. Two of the viruses that belonged to the vv genotype had a unique amino acid (aa) substitution Q324L whereas one virus had two unique changes, N280S and E300A in the VP2-HVR aa sequence. Although Zambian strains with a vv genotype possessed virulence marker aa within VP1 at 145T, 146D and 147N, two viruses showed unique substitutions, with one virus having 147T while the other had 147H. Pathologically, it was noted that only viruses with a vv genotype appeared to be associated with inducing pathological lesions in non-lymphoid organs (proventriculus and gizzard). Whilst documenting for the first time the presence of classical virulent IBDVs, this study demonstrates the involvement of multiple genotypes, with predominance of vvIBDVs in the epidemiology of IBD in Zambia.

The antigenic relationships between the natural reassortment field strains of infectious bursal disease virus (IBDV), and between the field strains and the vaccine strains are poorly understood. In the present study, the antigenicity of four representative natural reassortment IBDV isolates designated JS7, GD10111, NN1005 and NN1172 from southern China during the years 2005-2011 and their antigenic relationship with the most commonly used vaccine strain B87 were investigated in vivo. For this purpose, cross-challenge studies were performed on 28-day-old birds, which were 2 weeks post-vaccination by oil-emulsion vaccines (OEVs) prepared from the four field viruses and B87, respectively. The protection related values (PRV) were evaluated based on the protection rate measured by clinical signs and mortality, bursa/body weight (B/BW) ratio and the viral load in the bursal samples at 3 and 7 days post challenge. As a result, the PRV showed that the isolates NN1172 and GD10111 belonged to the same antigenic subtype, while the isolates NN1005 and JS7 belong to another subtype. The vaccine strain B87 was grouped with the isolates NN1005 and JS7 but actually belongs to another small subgroup and provided only 60-80% protection against the challenge of the four field strains. The results demonstrated that different antigenic subtypes co-existed among the field natural reassortment IBDV strains and the commonly used vaccine strain B87 was antigenically different from the prevalent IBDVs in southern China., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: To compare the effects of infectious bursal disease virus (IBDV) infection of commercial meat chickens at 0 and 16 days old (d.o.) and determine if IBDV vRNA is quantifiable in litter and dust samples., Methods: Ross meat chickens (n = 60) were orally infected or not with IBDV at 0 or 16 d.o. Blood and faecal samples were collected longitudinally to 28 days post infection (dpi) from six chickens and tissues collected weekly from three euthanased chickens. Relative bursal weight was recorded postmortem. IBDV antibody titres in sera were measured using ELISA and VCN was determined in tissues, faeces, litter and dust using qRT-PCR., Results: Chickens infected at 16 d.o. had earlier and more severe bursal atrophy, earlier and higher IBDV vRNA load in lymphoid organs and an earlier and greater antibody response to infection than those infected at 0 d.o. Faecal shedding of IBDV between 2 and 6 dpi was observed in both groups followed by cessation with the 0 d.o. group and re-initiation of shedding at 28 dpi. IBDV was readily detected and quantified in litter and dust samples., Conclusions: The presence of significant maternal antibody (MAb) titres in 0 d.o. chickens provided protection against IBDV replication and bursal atrophy at 7 and 14 days post infection. The reduced titres of MAb present at 16 d.o. did not prevent rapid IBDV replication and early marked bursal atrophy. The observed resistance of 0 d.o. chickens is likely to be a combination of MAb inhibition of IBDV and true age resistance of neonatal chicks. Measurement of IBDV in litter and dust may have research or diagnostic application., (© 2018 Australian Veterinary Association.)

Infectious Bursal Disease (IBD) is an acute, highly contagious and immunosuppressive disease of young chicken. The causative virus (IBDV) is a bi-segmented, double-stranded RNA virus. The virus encodes five major proteins, viral protein (VP) 1-5. VPs 1-3 have been characterized crystallographically. Albeit a rise in the number of studies reporting successful heterologous expression of VP5 in recent times, challenging the notion that rapid death of host cells overexpressing VP5 disallows obtaining sufficiently pure preparations of the protein for crystallographic studies, the structure of VP5 remains unknown and its function controversial. Our study describes the first 3D model of IBD VP5 obtained through an elaborate computational workflow. Based on the results of the study, IBD VP5 can be predicted to be a structural analog of the leucine-rich repeat (LRR) family of proteins. Functional implications arising from structural similarity of VP5 with host Toll-like receptor (Tlr) 3 also satisfy the previously reported opposing roles of the protein in first abolishing and later inducing host-cell apoptosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the present study, four very virulent infectious bursal disease virus (vvIBDV) isolates from flocks of chickens with vaccination failure in Egypt in 2003, 2007, 2010 and 2014 were characterized. The four viruses, designated USC2003, USC2007, USC2010 and USC2014, were detected by reverse transcription PCR, subjected to sequencing of both genomic segments (A and B) and compared with geographically and phylogenetically diverse IBDV strains. Phylogenetic analysis of segment A (complete) and B (partial) revealed a close relationship between Egyptian and vvIBDV reference strains of European and Asian origin. The sequences of segments of A and B the current Egyptian isolates were 96.1-98.2% and 96.5-98.7% identical, respectively, to those of other known vvIBDV isolates. The deduced amino acid sequences of VP1, polyprotein (pVP2-VP4-VP3) and VP5 revealed the presence of putative virulence determinants of Egyptian isolates compared with vvIBDV and less virulent (classical and variant) strains. The Egyptian isolates also possess unique amino acids substitutions within the hypervariable region of VP2 that differ from those of other reference IBDV strains. Further studies may be necessary to determine the pathogenic significance of these amino acid substitutions to fully understand the molecular epidemiology and evolution of IBDV.

The study was conducted with the objective of isolation and molecular characterization of infectious bursal disease virus (IBDV) circulating in Ethiopia and to assess the immunogenicity of different commercially available live attenuated IBD vaccines and finally to select the appropriate vaccine strain for the existing IBDV. Outbreak samples collected from different poultry farms with IBD infection between 2013 and 2015 were used for the virus isolation and molecular characterization. IBD vaccine immunogenicity test was conducted using four different commercially available live attenuated IBD vaccine strains: namely D78, B2K, LC75, and EXTREM. Day-old Bowman brown chickens purchased from commercial farm in Debre Zeit were used for the experiment. Serum samples were collected at days 14 and 21 and screened for the presence of maternal IBDv antibodies. The screening test result revealed that most of the chickens from vaccinated progeny were positive at the age of day 14 with mean antibody titer of .42, but declined at day 21 to 0.049 below cut-off point (S/P < 0.3). Chickens were divided into five different groups (four vaccinal and one control) and vaccinated at the age of day 21 and boosted after 14 days. Serum samples were collected and all of them were challenged at their 42 days of age with locally isolated very virulent infectious bursal disease virus (vvIBDV). From four of the vaccine strains used for immunogenicity study, the intermediate plus strains (LC75 and EXTREM) found to be superior and efficiently cross protect against the challenge with locally isolated vvIBDV. The development of clinical signs was studied and post-mortem examinations were conducted both on dead and sacrificed birds. From a total of 25 tissue samples processed for virus isolation on chicken fibroblast cell culture, 95% (18/20) of bursa and 80% (4/5) of the spleen samples showed visible cytopathic effect (CPE). The positive samples were tested by PCR and 19 of them had the expected band (643 bp). Further 11 representative samples were sequenced and confirmed that the circulating virus among poultry population in the country is vvIBDV. The study has recommended to produce vaccine using intermediate plus strains to prevent and control currently circulating vvIBDV.

The infectious bursal disease virus (IBDV) is a major health threat to the world's poultry industry despite intensive controls including proper biosafety practices and vaccination. IBDV (Avibirnavirus, Birnaviridae) is a non-enveloped virus with a bisegmented double-stranded RNA genome. The virus is traditionally classified into classic, variant and very virulent strains, each with different epidemiological relevance and clinical implications. Recently, a novel worldwide spread genetic lineage was described and denoted as distinct (d) IBDV. Here, we report the development and validation of a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay for the specific detection of dIBDVs in the global poultry industry. The assay employs a TaqMan-MGB probe that hybridizes with a unique molecular signature of dIBDV. The assay successfully detected all the assessed strains belonging to the dIBDV genetic lineage, showing high specificity and absence of cross-reactivity with non-dIBDVs, IBDV-negative samples and other common avian viruses. Using serial dilutions of in vitro-transcribed RNA we obtained acceptable PCR efficiencies and determination coefficients, and relatively small intra- and inter-assay variability. The assay demonstrated a wide dynamic range between 10 3 and 10 8 RNA copies/reaction. This rapid, specific and quantitative assay is expected to improve IBDV surveillance and control worldwide and to increase our understanding of the molecular epidemiology of this economically detrimental poultry pathogen.

Infectious bursal disease virus (IBDV, family Birnaviridae) is a bi-segmented double-stranded RNA virus for which two serotypes are described. Serotype 1 replicates in the bursa of Fabricius and causes an immunosuppressive and potentially fatal disease in young chickens. Serotype 2 is apathogenic in poultry species. Up to now, only one natural event of interserotypic reassortment has been described after the introduction of very virulent IBDV (vvIBDV) in the USA in 2009, resulting in an IBDV strain with its segment A related to vvIBDV and its segment B related to US serotype 2 strain OH. Here, we present the first European isolate illustrative of interserotypic reassortment. The reassorting isolate, named 100056, exhibits a genomic segment A typical of current European vvIBDV but a segment B close to European serotype 2 viruses, supporting an origin distinct from US strains. When inoculated into SPF chickens, isolate 100056 induced mild clinical signs in the absence of mortality but caused a severe bursal atrophy, which strongly suggests an immunosuppressive potential. These results illustrate that interserotypic reassortment is another mechanism that can create IBDV strains with a modified acute pathogenicity. As a consequence, and for a more precise inference of the possible phenotype, care should be taken that the molecular identification of IBDV strains is targeted to both genome segments.

We attempted the isolation of variant infectious bursal disease (IBD) viruses by using sentinel chickens immunized with inactivated classical-type IBD vaccine. Immunized sentinel chickens with high levels of neutralizing antibodies and non-immunized sentinel chickens were raised together with broiler chickens in a commercial farm. Severe atrophy of the bursa of Fabricius was observed from the second week after cohabitation in non-immunized sentinel chickens. However, in immunized sentinel chickens and broiler chickens, atrophy was observed from the third week after cohabitation. The IBD virus (IBDV) isolated from the bursa of Fabricius of immunized sentinel chickens, designated as strain IBDV TY2, showed severe atrophy of the bursa in infected SPF chickens. Antiserum to the IBDV TY2 strain showed higher neutralizing activity to heterologous IBDV strains than did antiserum to the K strain vaccine virus. Phylogenetic analysis revealed that the nucleotide sequences encoding the hypervariable region of virus protein 2 of the IBDV TY2 strain did not cluster with the classical, variant or very virulent IBDV groups. Based on these results, we suggest that the IBDV TY2 strain may constitute a novel variant type of IBDV.

Infectious bursal disease virus (IBDV) causes important economic losses and negatively affects global trade in poultry and poultry products. This study determined the presence of IBDV in primary lymphoid tissues and muscle tissue of infected broilers and the role of vaccination as a mitigation strategy. In the first study, specific-pathogen-free (SPF) broiler chickens were challenged with STC (classical [cIBDV]), Indiana (variant [varIBDV]), rA (very virulent [vvIBDV]), or Ohio (serotype 2, avirulent) IBDV. Infection was confirmed in all groups, but only the cIBDV group experienced morbidity or mortality. Virus was only isolated in low titers from a few breast and/or thigh muscle tissue samples from cIBDV and vvIBDV-infected chickens. For the second study, SPF broilers from three different treatment groups were challenged with IBDV viruses that currently circulate in the United States, varIBDV or vvIBDV: 1) maternal antibody-positive (MAb+), vaccinated with recombinant HVT-IBDV vaccine (Vaxxitek ® , Merial; MAb+/Vax); 2) MAb+, not-vaccinated (MAb+/Unvax); and 3) maternal antibody-negative, not-vaccinated chickens (MAb-/Unvax). MAb+/Vax and MAb+/Unvax chickens had significantly lower virus titers in primary lymphoid tissues compared to MAb-/Unvax chickens. No virus was detected in muscle tissues from any of the groups challenged with varIBDV, confirming the results of the first experiment. Only 1 of 36 (MAb+/Vax) and 2 of 36 (MAb+/Unvax) muscle samples were positive at minimal amounts (10 1.97 EID 50 /ml) in vvIBDV challenge, compared to the 9 of 36 muscle samples that were positive in the MAb-/Unvax group. This study indicates that only cIBDV and vvIBDV strains can be found in muscle at low titers of SPF meat chickens and that the breeder vaccination with MAb transfer to progeny with or without accompanying progeny vaccination, as practiced in the United States, was an effective mitigation strategy for vvIBDV-challenged birds.

A distinctive infectious bursal disease (IBD) virus genotype (ITA) was detected in IBD-live vaccinated broilers in Italy without clinical signs of IBD. It was isolated in specific-pathogen-free eggs and molecularly characterized in the hypervariable region of the virus protein (VP) 2. Phylogenetic analysis showed that ITA strains clustered separately from other homologous reference sequences of IBDVs, either classical or very virulent, retrieved from GenBank or previously reported in Italy, and from vaccine strains. The new genotype shows peculiar molecular characteristics in key positions of the VP2 hypervariable region, which affect charged or potentially glycosylated amino acids virtually associated with important changes in virus properties. Characterization of 41 IBDV strains detected in Italy between 2013 and 2014 showed that ITA is emergent in densely populated poultry areas of Italy, being 68% of the IBDV detections made during routine diagnostic activity over a two-year period, in spite of the immunity induced by large-scale vaccination. Four very virulent strains (DV86) and one classical strain (HPR2), together with eight vaccine strains, were also detected. The currently available epidemiological and clinical data do not allow the degree of pathogenicity of the ITA genotype to be defined. Only in vivo experimental pathogenicity studies conducted in secure isolation conditions, through the evaluation of clinical signs and macro/microscopic lesions, will clarify conclusively the virulence of the new Italian genotype.

Since the first report of infectious bursal disease in Pakistan in 1987, outbreaks have been common even in vaccinated flocks. Despite appropriate administration of vaccines, concerns arise if the circulating strains are different from the ones used in the vaccine. Here, we sequenced the hypervariable region (HVR) of the VP2 gene of circulating strains of infectious bursal disease virus (IBDV) originating from outbreaks (n = 4) in broiler flocks in Pakistan. Nucleotide sequencing followed by phylogeny and deduced amino acid sequence analysis showed the circulating strains to be very virulent (vv) and identified characteristic residues at position 222 (A), 242 (I), 256 (I), 294 (I) and 299 (S). In addition, a substitution at positions 221 (Q→H) was found to be exclusive to Pakistani strains in our analysis, although a larger dataset is required to confirm this finding. Compared to vaccine strains that are commonly used in Pakistan, substitution mutations were found at key amino acid positions in VP2 that may be responsible for potential changes in neutralization epitopes and vaccine failure.

Infectious bursal disease virus (IBDV) is an important representative of Birnaviridae, which causes infectious bursal disease (IBD), one important immuno-suppressive and fatal disease threatening the poultry husbandry. The naturally occurring reassortants of IBDV induced new risks to disease prevention and control. Here, we reviewed the main types of the genome segments reassortants and intragenic recombination, the inherent mechanism and the biological significances were analyzed, which would give us new insights into the virus genetic evolution research and the disease control strategy.

Infectious bursal disease virus (IBDV) is a highly infectious disease of young chicken that predominantly affects the immune system. In the present study, we are reporting first comprehensive study of IBDV outbreaks from the Northeastern part of India. Northeast India shares a porous border with four different countries; and as a rule any outbreak in the neighboring countries substantially affects the poultry population in the adjoining states. Nucleotide sequence analysis of the VP2 gene of the IBDV isolates from the Northeastern part of India suggested the extreme virulent nature of the virus. The virulent marker amino acids (A222, I242, Q253, I256 and S299) in the hypervariable region of the Northeastern isolates were found identical with the reported very virulent strains of IBDV. A unique insertion of I/L294V was recorded in all the isolates of the Northeastern India. The study will be useful in understanding the circulating pathotypes of IBDV in India., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The reverse transcription PCR (RT-PCR) combined with restriction fragment length polymorphism (RFLP) is used for the differentiation of classical virulent (cv), virulent (v) and very virulent (vv) strains of infectious bursal disease virus (IBDV) isolates from chicken bursal tissues in southern states of India. In the present study, six different isolates (MB11, HY12, PY12, BGE14, VCN14 and NKL14) of IBDV strains were subjected for genotyping along with vaccine virus (Georgia, intermediate strain) using RT-PCR for amplification of a 743 bp sequence in the hypervariable region of VP2 gene followed by restriction enzyme digestion with 5 different restriction enzymes (BspMI, SacI, HhaI, StuI and SspI). The RT-PCR products obtained from vvIBDV strains were digested by SspI enzyme except PY12, BGE14 and MB11 isolates. The SacI digested the isolate MB11, PY12 and the vaccine strain, but it did not cleave the very virulent isolates of IBDV. HhaI cleaved all the isolates with different restriction profile patterns. StuI digested all the vvIBDV isolates and BspMI was not able to differentiate field isolates from vaccine strain. Though RT-PCR combined with RFLP is a genotypic method, further confirmation of serotypes to distinguish the vvIBDV from cvIBDV has to be carried out using pathogenicity studies.

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive viral disease of young chickens and remains one of the economically most important diseases threatening the poultry industry worldwide. In this study, 16 and 11 nucleotide sequences of the VP2 hypervariable region (VP2-HVR) and part of VP1, respectively, of IBD virus (IBDV) detected in vaccinated broiler chickens in Lusaka in 2012 were determined. Phylogenetic analysis revealed that these Zambian IBDVs separated into three genotypes of very virulent (VV) IBDVs. Although the majority of these viruses belonged to the African VV type (VV1), which consisted of viruses from West Africa, South Africa and Zambia, one virus belonged to the East African VV type (VV2). Interestingly, a Zambian IBDV belonging to the VV3 genotype (composed of viruses from several continents) clustered with attenuated vaccine strains. Although sequence analysis of VP2-HVR showed that all detected Zambian IBDVs had conserved putative virulence marker amino acids (i.e., 222A, 242I, 256I, 294I and 299S), one virus had two unique amino acid substitutions, N280S and E300A. This study demonstrates the diversity of Zambian IBDVs and documents for the first time the possible involvement of attenuated vaccine strains in the epidemiology of IBD in Zambia. Strict biosecurity of poultry farms, monitoring of live vaccine use in the field, surveillance and characterization of IBDV in poultry and development of a vaccine from local or regional IBDV field strains are recommended for improved IBD control in Zambia.

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.

In Argentina, classical vaccines are used to control infectious bursal disease virus (IBDV); however, outbreaks of IBDV are frequently observed. This could be due to failures in the vaccination programs or to the emergence of new strains, which would be able to break through the protection given by vaccines. Hence, genetic characterization of the viruses responsible for the outbreaks that occurred in recent years is crucial for the evaluation of the control programs and the understanding of the epidemiology and evolution of IBDV. In this study, we characterized 51 field samples collected in Argentina (previously identified as IBDV positive) through the analysis of previously identified apomorphic sequences. Phylogenetic analysis of regVP2 showed that 42 samples formed a unique cluster (Argentinean lineage), seven samples were typical classical strains (one of them was a vaccine strain), and two belonged to the very virulent lineage (vvIBDV). Interestingly, when the analysis was performed on the regVP1 sequences, the field samples segregated similarly to regVP2; thus, we observed no evidence of a reassortment event in the Argentinean samples. Amino acid sequence analysis of regVP2 showed a particular pattern of residues in the Argentinean lineage, particularly the presence of T272, P289 and F296, which had not been reported before as signature sequences for any IBDV phenotype. Notably, the residue S254, characteristic of the antigenic variant, was not present in any of the Argentinean samples.

Infectious bursal disease virus (IBDV) enters the host cells via endocytic pathway to achieve viral replication in the cytoplasm. Here, we performed LC-MS/MS coupled with isobaric tags for relative and absolute quantification labeling of differentially abundant proteins of IBDV-infected cells using a subcellular fractionation strategy. We show that the viral infection regulates the abundance and/or subcellular localization of 3211 proteins during early infection. In total, 23 cellular proteins in the cytoplasmic proteome and 34 in the nuclear proteome were significantly altered after virus infection. These differentially abundant proteins are involved in such biological processes as immune response, signal transduction, RNA processing, macromolecular biosynthesis, energy metabolism, virus binding, and cellular apoptosis. Moreover, transcriptional profiles of the 25 genes corresponding to the identified proteins were analyzed by quantitative real-time RT-PCR. Ingenuity Pathway Analysis clustered the differentially abundant proteins primarily into the mTOR pathway, PI3K/Akt pathway, and interferon-β signaling cascades. Confocal microscopy showed colocalization of the viral protein VP3 with host proteins heterogeneous nuclear ribonucleoprotein H1, nuclear factor 45, apoptosis inhibitor 5, nuclear protein localization protein 4 and DEAD-box RNA helicase 42 during the virus infection. Together, these identified subcellular constituents provide important information for understanding host-IBDV interactions and underlying mechanisms of IBDV infection and pathogenesis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Infectious bursal disease virus (IBDV) is a bi-segmented, double-stranded RNA virus that belongs to the genus Avibirnavirus of the family of Birnavirideae. The co-evolution of genome segments is a major evolutionary feature for IBDV. However, in recent years, some strains exhibited markedly different genetic relationships for segments A and B. In this study, we firstly isolated a new type of reassortment IBDV strain named IBD13HeB01 from northern China. The full-length genomes of segments A and B were cloned and identified. Sequence analysis revealed that IBD13HeB01 was a segment-reassortment strain, whose segment A was derived from very virulent strain and segment B from attenuated IBDV. In addition, the virulence of IBD13HeB01 strain was evaluated using SPF chickens. This study is not only beneficial for further understanding of the viral evolution but also suggests the potential risk of application of the live vaccines of IBDV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.

Two experiments were conducted to determine the persistence and tissue distribution of serotypes 1 and 2 of infectious bursal disease virus (IBDV) in specific-pathogen-free and vaccinated turkeys. In Experiment 1, three groups of 2-wk-old turkey poults, including a negative control group, were used. In groups 1 and 2, 13 poults in each group were challenged with either serotype 1 (STC) or serotype 2 (OH) strains using an inoculum of 10(4) 50% embryo infectious dose (EID50)/0.2 ml/bird. Thymus, bursa, spleen, kidney, lungs, liver, pancreas, caecum, and breast and thigh muscles were sampled at predetermined intervals. The bursal tissues from birds inoculated with either serotype were reverse transcriptase-PCR (RT-PCR) positive up to 21 days postinoculation (DPI). In both groups virus isolation from bursas was possible up to 14 DPI. Except for the bursas and spleens in birds inoculated with serotype 1 and bursas in birds inoculated with serotype 2, all other tissues were RT-PCR negative. In Experiment 2, five groups of turkey poults were used. At 4 wk of age, group 1 was challenged with a serotype 1 STC strain and group 2 with serotype 2 OH strain using an inoculum size of 10(2) EID50/0.2 ml for both serotypes. Groups 3 and 4 were vaccinated at 2 wk of age using an inactivated serotype 1 IBDV vaccine. At 2 wk postvaccination, groups 3 and 4 were challenged with STC and OH strains respectively. From group 1, bursal, spleen, and liver tissues were RT-PCR positive up to 14 DPI; breast muscle and kidney tissues were positive up to 7 DPI; and lungs and pancreatic tissues were positive up to 3 DPI. From group 2, bursal tissues were RT-PCR positive up to 14 DPI and lung tissues up to 3 DPI. All of the tissue samples collected from groups 3, 4, and 5 were RT-PCR negative. Virus could not be isolated from RT-PCR positive bursal homogenate. In this work, it was confirmed that the virus persisted in the bursa longer than in any other tissues. The difference in the results between Experiments 1 and 2 could be due to the age of poults at vaccination and the higher inoculum size used in Experiment 1. This study indicates that turkeys are more resistant to IBDV as compared to chickens. Viruses of serotypes 1 and 2 infect turkeys and persist in bursal tissue for 14 days and RNA was detected up to 21 days.

A molecular epidemiology study of infectious bursal disease viruses (IBDVs) isolated from seven provinces in southern China during the years 2000-2012 was performed based on partial sequences of genome segments A and B, namely the hypervariable region of the A-VP2 gene (A-vVP2) and the b fragment of VP1 gene (B-VP1b) from a total of 91 field isolates. Sequence analysis based on vVP2 revealed that 72 out of 91 isolates had the same characteristic amino acid (aa) sequences as vvIBDV. The mutation of D212N in A-vVP2 has become prevalent in the recent isolates. The origin of the field isolates with vvIBDV characteristic amino acid residues was complex, evidenced by the findings that more than one subgroup of strains prevailed in each province. When B-VP1b was analyzed, there were three lineages among the field isolates, and none of the isolates had a relationship to vvIBDV-related segment B. Phylogenetic analysis of both segments revealed that only a few isolates (13/91) had the same genetic relatives in consensus trees based on segments A and B, whereas the majority of the isolates (85.71%, 78/91) were identified to be naturally reassorted strains. Based on the origin of each segment, at least six types of reassortant IBDVs prevailed in southern China, three of which were shown to be dominant: segment A from vvIBDV and B from attenuated IBDV, segment A of vvIBDV and B from 002-73-like IBDV, and segment A of vvIBDV and B from HLJ0504 or a similar strain. Our findings suggest that both genomic segments of field IBDVs has been evolving, and continuous monitoring of the evolution of field IBDV genome is therefore urgently needed in the control of IBDV.

Samples from 231 randomly selected commercial broiler chicken flocks in Ontario were tested at slaughter for exposure to chicken anemia virus (CAV), fowl adenovirus (FAdV), and infectious bursal disease virus (IBDV). Fifteen blood samples per flock were collected and analyzed for the presence of antibodies against CAV, FAdV, and IBDV by ELISA or agar gel immunodiffusion test. Fifteen cecal tonsils and cloacal swabs per flock were analyzed for the presence of CAV, FAdV, and IBDV by PCR. The prevalence of exposure to avian adeno-associated virus (AAAV) was estimated by a PCR test on a subset of FAdV-PCR-positive samples from 178 flocks. Genotypes of FAdV and IBDV were identified on a subset of isolates (n = 353 and 45, respectively). The flock-level period prevalence of exposure to AAAV, CAV, FAdV, and IBDV during grow-out were 88.76% (95% CI: 84.08-93.45%), 77.06% (95% CI: 71.59-82.52%), 96.54% (95% CI: 94.16-98.91%), and 48.92% (95% CI: 42.42-55.41%), respectively. Results of a multivariable logistic regression model showed a significant association of exposure to FAdV with exposure to AAAV (OR = 18.57, 95% CI: 3.67-93.86, P = 0.004) but not with exposure to CAV (P = 0.7752) or exposure to IBDV (P = 0.2274). Pathogenic FAdV genotypes (FAdV-02, FAdV-08, and FAdV-11) constituted 39.38% of the isolates. The most-common IBDV genotypes identified were IBDV NC171 (60%) and IBDV 05SA8 (28.89%). This is the first large-scale study to estimate the baseline flock prevalence of exposure to AAAV, CAV, FAdV, and IBDV in commercial broiler flocks in Canada. Potentially pathogenic genotypes of FAdV and IBDV that can guide vaccine development and disease control efforts in Ontario were identified.

Introduction: The airborne transmission of infectious diseases in livestock production is increasingly receiving research attention. Reliable techniques of air sampling are crucial to underpin the findings of such studies. This study evaluated the physical and biological efficiencies and detection limits of four samplers (Andersen 6-stage impactor, all-glass impinger "AGI-30", OMNI-3000 and MD8 with gelatin filter) for collecting aerosols of infectious bursal disease virus (IBDV)., Materials and Method: IBDV aerosols mixed with a physical tracer (uranine) were generated in an isolator, and then collected by the bioaerosol samplers. Samplers' physical and biological efficiencies were derived based on the tracer concentration and the virus/tracer ratio, respectively. Detection limits for the samplers were estimated with the obtained efficiency data., Results: Physical efficiencies of the AGI-30 (96%) and the MD8 (100%) were significantly higher than that of the OMNI-3000 (60%). Biological efficiency of the OMNI-3000 (23%) was significantly lower than 100% (P < 0.01), indicating inactivation of airborne virus during sampling. The AGI-30, the Andersen impactor and the MD8 did not significantly inactivate virus during sampling. The 2-min detection limits of the samplers on airborne IBDV were 4.1 log10 50% egg infective dose (EID50) m (-3) for the Andersen impactor, 3.3 log10 EID50 m (-3) for the AGI-30, 2.5 log10 EID50 m (-3) for the OMNI-3000, and 2.9 log10 EID50 m (-3) for the MD8. The mean half-life of IBDV aerosolized at 20 °C and 70% was 11.9 min., Conclusion: Efficiencies of different samplers vary. Despite its relatively low sampling efficiency, the OMNI-3000 is suitable for use in environments with low viral concentrations because its high flow rate gives a low detection limit. With the 4 samplers investigated, negative air samples cannot guarantee virus-free aerial environments, which means that transmission of infectious agents between farms may still occur even when no virus has been detected.