Emergence of the novel infectious bursal disease virus variant in vaccinated poultry flocks in Egypt.

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.

Characterization of antigenic variant infectious bursal disease virus strains identified in South Korea.

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.

Generation of recombinant VP3 protein of infectious bursal disease virus in three different expression systems, antigenic analysis of the obtained polypeptides and development of an ELISA test.

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.

Potential reverse spillover of infectious bursal disease virus at the interface of commercial poultry and wild birds.

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.

Detecting Infectious Bursal Disease Using a VP1 Gene-Based RT-qPCR Assay Compared to Standard Methods of Virus Isolation, ELISA, and Histopathology.

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.

Detection and characterization of a novel marine birnavirus isolated from Asian seabass in Singapore.

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.

Development of real-time PCR assays for single and simultaneous detection of infectious bursal disease virus and chicken anemia virus.

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 100-108 viral genomes indicated that the simplex assays have good determination coefficients and efficiency and detect a wide range of virus doses (102 to 108 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.

Molecular characterization of field isolates of infectious bursal disease virus from three decades, 1987-2018, reveals a distinct genotypic subgroup in Vietnam.

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 PAB 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.

Molecular characterization and phylogenetic analysis of very virulent infectious bursal disease virus circulating in Morocco during 2016-2017.

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.

Antigenicity, pathogenicity and immunosuppressive effect caused by a South American isolate of infectious bursal disease virus belonging to the "distinct" genetic lineage.

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.

New introduction of a very virulent infectious bursal disease virus in New York, USA.

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.

A proposed nomenclature for infectious bursal disease virus isolates.

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.

Characterization of field infectious bursal disease viruses in Zambia: evidence of co-circulation of multiple genotypes with predominance of very virulent strains.

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.

Molecular characterization of very virulent infectious bursal disease virus strains circulating in Egypt from 2003 to 2014.

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.

Development of an RT-qPCR assay for the specific detection of a distinct genetic lineage of the infectious bursal disease virus.

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 103 and 108 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.

Identification of a European interserotypic reassortant strain of infectious bursal disease virus.

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.

Infectious bursal disease: outbreak investigation, molecular characterization, and vaccine immunogenicity trial in Ethiopia.

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.

Isolation of novel variants of infectious bursal disease virus from different outbreaks in Northeast India.

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.

Molecular characterization of infectious bursal disease viruses detected in vaccinated commercial broiler flocks in Lusaka, Zambia.

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.

Molecular characterization of infectious bursal disease viruses from Pakistan.

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.