Phylogenotyping of infectious bursal disease virus in Vietnam according to the newly unified genotypic classification scheme.

Since 1987, infectious bursal disease virus (IBDV) has circulated and evolved in Vietnam, but little is known about the genotypes present. IBDV samples were collected in 1987, 2001-2006, 2008, 2011, 2015-2019, and 2021 in 18 provinces. We conducted phylogenotyping analysis based on an alignment of 143 VP2-HVR (hypervariable region) sequences from 64 Vietnamese isolates (26 previous and 38 additional sequences and two vaccines, and alignment of 82 VP1 B-marker sequences, including one vaccine and four Vietnamese field strains. The analysis identified three A-genotypes, A1, A3, and A7, and two B-genotypes, B1 and B3, among the Vietnamese IBDV isolates. The lowest average evolutionary distance (8.6%) was seen between the A1 and A3 genotypes, and the highest (21.7%) was between A5 and A7, while there was a distance of 14% between B1 and B3 and 17% between B3 and B2. Unique signature residues were observed for genotypes A2, A3, A5, A6, and A8, which could be used for genotypic discrimination. A timeline statistical summary revealed that the A3-genotype predominated (79.8% presence) in Vietnam from 1987 to 2021 and that it remained the dominant IBDV genotype over the last five years (2016-2021). The current study contributes to a better understanding of the circulating genotypes and evolution of IBDV in Vietnam and worldwide.

Identification and Pathogenicity Evaluation of a Novel Reassortant Infectious Bursal Disease Virus (Genotype A2dB3).

Infectious bursal disease virus (IBDV) is a non-enveloped, bi-segmented double-stranded RNA virus and the causative agent of a poultry immunosuppressive disease known as infectious bursal disease (IBD). The novel variant IBDV (nVarIBDV) recently posed a great threat to the development of the poultry industry. In this study, we identified a novel segment-reassortant IBDV strain, IBDV-JS19-14701 (Genotype A2dB3). Phylogenic analysis showed that Segments A and B of IBDV-JS19-14701 were derived from emerging nVarIBDV (Genotype A2dB1) and long-prevalent HLJ0504-like strains (Genotype A3B3) in China, respectively. The pathogenicity of IBDV-JS19-14701 was further evaluated via animal experiments. IBDV-JS19-14701 exhibited a similar virulence to chickens with the nVarIBDV. The identification of this reassortment event is beneficial for understanding the epidemiology of nVarIBDV and will contribute to the efficient prevention and control of IBD.

The Novel Genetic Background of Infectious Bursal Disease Virus Strains Emerging from the Action of Positive Selection.

The circulation in Europe of novel reassortant strains of infectious bursal disease virus (IBDV), containing a unique genetic background composition, represents a serious problem for animal health. Since the emergence of this novel IBDV mosaic was first described in Poland, this scenario has become particularly attractive to uncover the evolutionary forces driving the genetic diversity of IBDV populations. This study additionally addressed the phenotypic characterization of these emergent strains, as well as the main features affecting the viral fitness during the competition process of IBDV lineages in the field. Our results showed how different evolutionary mechanisms modulate the genetic diversity of co-existent IBDV lineages, leading to the error catastrophe effect, Muller ratchet effect, or prevalence, depending on their genetic compositions. We also determined that the action of the positive selection pressure, depending on the genomic segment on which it is acting, can drive two main phenotypes for IBDV: immune-escaping strains from the selection on segment A or strains with functional advantages from the selection on segment B. This last group seems to possess an increased fitness landscape in the viral quasispecies composition, presenting better adaptability to dissimilar environmental conditions and likely becoming the dominant population. The reassortant strains also exhibited a lower mortality rate compared with the well-known vvIBDV strains, which can facilitate their spreading.

A unified genotypic classification of infectious bursal disease virus based on both genome segments.

Infectious bursal disease virus (IBDV) of chickens is a birnavirus with a bi-segmented double-stranded RNA genome, the segments designated as A and B. We performed phylogenetic analysis using a 366-bp fragment of segment A (nt 785-1150) and a 508-bp fragment of segment B (nt 328-835) of IBDV. A total of 463 segment A and 434 segment B sequences from GenBank, including the sequences of eight recent Bangladeshi isolates, were used in the analysis. The analysis revealed eight genogroups of segment A under serotype 1, designated as A1 (classical), A2 (US antigenic variant), A3 (very virulent), A4 (dIBDV), A5 (atypical Mexican), A6 (atypical Italian), A7 (early Australian) and A8 (Australian variant), and a single genogroup under serotype 2, designated as A0. On the other hand, segment B could be categorized into five genogroups irrespective of serotype, these being B1 (classical-like), B2 (very virulent-like), B3 (early Australian-like), B4 (Polish & Tanzanian) and B5 (Nigerian). Segment B of serotype 2 strains clustered within genogroup B1. With the bi-segmented genome of IBDV, these differences would allow for a total of 45 possible assortments. Based on the combinations of segment A and segment B genogroups observed in 463 IBDV strains, a total of 15 genotypes could be recognized. Recent Bangladeshi IBDV strains, isolated in 2016, appeared to be segment reassortants having segment A of genogroup A3 (very virulent) and segment B of genogroup B3 (early Australian-like). An extended system of nomenclature of IBDV strains is proposed.

A reassortment vaccine candidate of the novel variant infectious bursal disease virus.

Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is the most important immunosuppressive disease threatening the poultry industry worldwide. Recently, the novel variant IBDV has been emerging in large-scale in Asia including China and is becoming a new threat to the healthy development of the poultry industry, but no ideal vaccine is available. Therefore, it is necessary and urgent to develop a new vaccine against the novel variant IBDV. In this study, based on the skeleton of an attenuated vaccine strain Gt, a reassortment virus strain rGtVarVP2 was constructed for the first time, which could express the main protective antigen VP2 of the novel variant IBDV and replicate well in cell culture. Subsequently, the safety and effectiveness of rGtVarVP2 were further evaluated using animal experiments. The rGtVarVP2 is nonpathogenic to specific-pathogen-free (SPF) chicken. The immunization of rGtVarVP2 could induce the specific neutralizing antibodies against the novel variant IBDV. The challenge protection tests further confirmed the effectiveness of the IBDV reassortment virus rGtVarVP2. No atrophy and obvious lesions were observed in the immunization group while the bursae of non-immunization control group were severely destroyed after challenge, which showed that rGtVarVP2 could provide complete protection against the novel variant IBDV. These data indicate that the vaccine candidate (rGtVarVP2 strain) is safe and effective, which is of great significance for comprehensive control of IBD and healthy breeding.

Naturally occurring homologous recombination between novel variant infectious bursal disease virus and intermediate vaccine strain.

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease (IBD), an important immunosuppressive disease seriously threatening poultry farming worldwide. Since the identification of the classic strain in 1957, variant IBDV, very virulent IBDV, and novel variant IBDV have successively emerged brought severe challenges. Over the years, attenuated, intermediate, and intermediate-plus live vaccines have been developed to control the disease. The coexistence of various strains in flocks increases the probability of homologous recombination, and in this study, a naturally occurring homologous recombination between a novel variant strain and an intermediate vaccine strain of IBDV was first identified. Sequence analyses demonstrated that the IBD16HeN01 strain was a recombinant IBDV incorporating the skeleton of the novel variant IBDV (SHG19-like strain), where the 3' region of segment A (nt 1539-3260) was replaced by an intermediate vaccine strain (W2512-like strain). Pathogenicity experiments indicated that IBD16HeN01 could cause severe bursal lesions and the recombination increased viral pathogenicity to chick embryos compared with the novel variant IBDV. Homologous recombination in IBDV has increased the complexity of disease prevention and control and reminds us that we should use live vaccines more scientifically and cautiously.

Continuous circulation of an antigenically modified very virulent infectious bursal disease virus for fifteen years in Egypt.

Infectious bursal disease virus (IBDV), the agent of an immunosuppressive and sometimes lethal disease in chickens, is causing recurrent outbreaks in broiler chickens in Egypt. In particular, an antigenically modified isolate of very virulent IBDV (vvIBDV) called 99323 was detected in Egypt nearly twenty years ago; this isolate was shown to be experimentally controlled by an antigenically classical live vaccine. However, acute IBD is still reported, even in vaccinated flocks, and little is known about the genetic and antigenic properties of viruses currently circulating in Egypt. In the present study, ten samples collected in Egyptian broiler farms in 2015 as well as five samples collected in 2001 were analyzed. Genetic analyses of partial VP2 sequences revealed that 8 isolates clustered with vvIBDV strains, and 5 with tissue culture adapted and vaccine strains. Similar results were observed for partial VP1 sequences with the exception of isolate 160019, for which VP2 clustered with the vaccine strain Bursine while VP1 clustered with vvIBDV, suggesting reassortment. For isolates genetically related to vvIBDV, antigenic profiling revealed two patterns: while some isolates exhibited typical European vvIBDV reactivity with lack of binding of mAbs 5, other revealed extensive antigenic modifications, with lack of binding of mAbs 3, 5, 6, 8 and 9, similar to isolate 99323. These different patterns were associated with a single amino acid mutation at position 321 of VP2 that is located within peak PHI. Full genome sequencing was performed for three isolates, among which two were representative of the two antigenic patterns observed for vvIBDV as well as the reassortant isolate 160019. This study highlights the co-circulation of both antigenically typical and modified vvIBDV during the last fifteen years in Egypt.

Complete genome analysis and time scale evolution of very virulent infectious bursal disease viruses isolated from recent outbreaks in Morocco.

Emerging of very virulent infectious bursal disease virus (vvIBDV) genotype in poultry flocks in Morocco were characterized. VP2 sequence analysis showed that the strains of Moroccan vvIBDV genotypes clustered separately from classic and vaccine strains reference of IBDV. The full-length genome of four Moroccan vvIBDV strains was determined, in order to get a more exhaustive molecular characterization allowing to conduct the evolution time scale and speculations on their origin. In a phylogenetic tree, nucleotide sequences of segment A and B formed a common branch with those vvIBDV references strains published in GenBank, but they clearly grouped into a distinct subcluster. An alignment of deduced amino acid sequences segment B, confirmed the presence of the conserved TDN tripeptide found in all of the vvIBDV genotype and revealed the presence of 2 substitutions I472L and E688D specific for the vvIBDV Moroccan isolates. The deduced amino acid sequences of segment A genes showed the presence of the "signature" typical of the vvIBDV genotype and revealed the presence of 7 aa substitutions specific for the vvIBDV Moroccan strains. The evolution rate for IBDV VP2 gene was estimated at 5.875 × 10-4 substitutions/site/year. The estimation of the time to most common recent ancestor of Moroccan vvIBDV based on the VP2 sequences available was 31 years, corresponding to 3 years earlier than the first vvIBDV case detection in layers in the country.

Origin and global spreading of an ancestral lineage of the infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is an economically relevant and widespread pathogen that produces immunosuppression in young chickens. IBDV is genetically classified into seven genogroups (G1-G7), where the traditional classic, variant and very virulent strains correspond to G1, G2 and G3, respectively. The G4 strains, also known as 'distinct' (dIBDV), have recently acquired increased relevance because of their prevalence and notorious impair to the poultry industry in South America. Here, worldwide dIBDV strains were studied using phylogenetic and phylodynamic approaches. The phylogenetic analyses performed using partial and complete sequences of both viral segments (A and B) consistently clustered the dIBDV strains in a monophyletic group. The analyses of the VP5, polyprotein and VP1 coding regions identified amino acid residues that act as markers for the identification of the entire dIBDV group or different sub-populations. The phylodynamic analyses performed using the hypervariable region of VP2 indicated that the dIBDV strains emerged in the early 1930s in Eastern Europe, shortly after the emergence of classic strains (1927) and before variant (1949) and very virulent strains (1967). The analysis of the migration routes indicated that after its emergence, the dIBDV strains spread to Eastern Asia around 1959, to Brazil around 1963, and to Argentina around 1990. These inter-continental migrations resulted in three sub-populations that are currently represented by strains from (a) Brazil, (b) Eastern Asia and Canada, and (c) Eastern Europe, Argentina and Uruguay. Taken together, our results highlight the complex evolutionary history of IBDV and the importance of new phylodynamic data to unravel and nearly follow the different evolutionary pathways taken by this important poultry pathogen.

Infectious Bursal Disease Virus: Molecular Epidemiologic Perspectives and Impact on Vaccine Efficacy Against Avian Influenza and Newcastle Disease Viruses.

Infectious bursal disease (IBD) virus (IBDV) is the causative agent of a highly contagious and immunosuppressive disease of chickens with huge economic losses to the poultry industry despite extensive vaccination. Analysis of isolated IBDV field strains from vaccinated birds would greatly improve the current immunization regimens and support the development of vaccines that offer better immunity. The study investigated the genetic characteristics and pathologic features of IBDVs in commercial broiler chicken farms, as well as the effect of IBDV infection on the efficacy of vaccination against avian influenza virus (AIV) and Newcastle disease virus (NDV) under field conditions. A preliminary diagnosis of IBD was made on the basis of the flock history and the characteristic gross pathologic findings. Microscopically, lymphoid depletion in bursal follicles with infiltration of lymphomononuclear cells along with cystic cavitations reflected the IBDV infection. The molecular analysis confirmed the IBDV infection in (57.1%) of tested flocks. Upon phylogenetic analysis of the VP2 hypervariable region of 14 Egyptian IBDVs, most viruses (n = 12) were clustered within the genogroup 3, while two viruses were closely related to attenuated vaccine isolates in genogroup 1. The analysis of the amino acid (aa) sequences revealed that most of the strains possessed five consistent aas at the VP2 protein (222A, 242I, 256I, 294I, and 299S), which are characteristic for the very virulent IBDV (vvIBDV). Serology indicated the immunosuppressive effect of IBDV, which is represented by a decrease (1.6-2.6 and 1.4-2.6 mean log 2) in the hemagglutination inhibition titer of the low pathogenic AIV subtype H9N2 and NDV, respectively. The examined IBDVs showed a high mutation rate within the hypervariable domain of the VP2 peptide. The results highlighted the need for carrying out an inclusive surveillance of IBDV infections in chicken flocks in Egypt.

Virus de la enfermedad de la bolsa infecciosa: perspectivas epidemiológicas moleculares e impacto sobre la eficacia de la vacunación contra los virus de influenza aviar y de la enfermedad de Newcastle. El virus de la enfermedad de la bolsa infecciosa (IBD) es el agente causante de una enfermedad altamente contagiosa e inmunosupresora de los pollos con grandes pérdidas económicas para la industria avícola a pesar de la vacunación extensiva. El análisis de cepas de campo del virus de Gumboro aisladas de aves vacunadas mejoraría en gran medida los regímenes de inmunización actuales y respaldaría el desarrollo de vacunas que ofrezcan una mejor inmunidad. Este estudio investigó las características genéticas y patológicas de los virus de la enfermedad infecciosa de la bolsa en granjas comerciales de pollos de engorde, así como el efecto de la infección por el virus de Gumboro en la eficacia de la vacunación contra el virus de la influenza aviar (AIV) y el virus de la enfermedad de Newcastle (NDV) bajo condiciones de campo. Se realizó un diagnóstico preliminar de la enfermedad infecciosa de la bolsa con base en la historia de la parvada y de los hallazgos patológicos macroscópicos característicos. Microscópicamente, la despoblación linfoide en los folículos bursales con infiltración de células linfomononucleares junto con formaciones quísticas reflejó la infección por el virus de Gumboro. El análisis molecular confirmó la infección por este virus en 57.1% de las parvadas analizadas. Después del análisis filogenético de la región hipervariable del gene VP2 de 14 virus egipcios, la mayoría de los virus (n = 12) se agruparon dentro del genogrupo 3, mientras que dos virus estaban estrechamente relacionados con los aislamientos vacunales atenuados del genogrupo 1. El análisis de las secuencias de aminoácidos reveló que la mayoría de las cepas poseían consistentemente cuatro aminoácidos en la proteína VP2 (222A, 242I, 256I, 294I y 299S), que son características de cepas muy virulentas del virus de Gumboro (vvIBDV). La serología indicó el efecto inmunosupresor del virus de Gumboro, que está representado por una disminución (1.6­2.6 y 1.4­2.6 log2) en los títulos de anticuerpos por inhibición de la hemaglutinación contra el virus de influenza aviar de baja patogenicidad subtipo H9N2 y del virus de Newcastle, respectivamente. Los virus de Gumboro examinados mostraron una alta tasa de mutación dentro del dominio hipervariable del péptido VP2. Los resultados resaltaron la necesidad de llevar a cabo una vigilancia intensiva de las infecciones por el virus de Gumboro en parvadas de pollos en Egipto.

Serological study reveal different antigenic IBDV strains prevalent in southern China during the years 2000-2017 and also the antigenic differences between the field strains and the commonly used vaccine strains.

The aim of this study was to determine the antigenic relatedness of Infectious Bursal Disease Viruses (IBDVs) in the field in southern China during the period 2000-2017, as well as the antigenic relationship between the field strains and the most commonly used vaccine strains by using a virus neutralization (VN) test in vitro. The antigenic relatedness (R) value and the difference in VN titers were analyzed, and the antigenic index based on the sequences of the hypervariable region of VP2 (vVP2) of the strains was further evaluated. As a result, the R value of representative field strains showed that there were three subtypes present in the field strains examined, with 7 strains belonging to subtype 1, while strains BH11 and JS7 belonged to subtype 2 and subtype 3, respectively. The commonly used vaccine strains B87 and FW2512 belonged to subtype 1. The analysis of the VN titer differences revealed that all the 136 field strains were classified into subtype 1, except BH11 and JS7. All the field strains in subtype 1 have been divided into at least 5 subgroups, suggesting the antigenic diversity among these strains. The antigenic index based on IBDV-VP2 sequences further confirmed the antigenic differences between the three subtype strains and also the antigenic diversity among the subtype 1. The results demonstrated the antigenic diversity of field IBDVs in southern China during the years 2000-2017 and the antigenic differences between the field strains and the commonly used vaccine strains. This would indicate that the commonly used vaccines are only partially effective. These results enhance our understanding of IBDV genetic evolution and should help to develop more effective vaccines for the control of this disease in the future.

Identification and assessment of pathogenicity of a naturally reassorted infectious bursal disease virus from Henan, China.

Infectious bursal disease virus (IBDV) is still a vital etiological agent in poultry farms. IBDV outbreaks occasionally occur due to the presence of very virulent, reassortment or variant strains. Vaccine immunization has played crucial roles in IBD control for decades. However, survival pressure of IBDV from the vaccine immunization also increases the reassortments of circulating viruses. In this study, an IBDV strain was isolated from several broiler farms in Henan Province, central part of China, and named IBDV HN strain. Based on the results of RT-PCR, sequencing and phylogenic analyses of VP1 and VP2 genes, the IBDV HN strain is a novel reassortment strain in the Henan region. Segment A of this strain appears to originate from the very virulent IBDV strain, while segment B comes from the other field reassortment strains. This may be the result of natural reassortant of virus circulating in the field. About 60% (6/10) of experimentally infected specific pathogen-free chickens died after 3 to 5 d post-infection with typical symptom and pathological lesions. The IBDV HN strain was prone to horizontal transmission, which poses a serious threat to the chicken industry. Further investigation on the prevalence, virulence, and evolution of HN strain IBDV will provide a foundation for the prevention and control of the disease in this region.

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.

Molecular epidemiology of endemic and very virulent infectious bursal disease virus genogroups in backyard chickens in California, 2009-2017.

Pathogenic strains of infectious bursal disease virus (IBDV) are associated with increased morbidity, mortality, and immunosuppression in susceptible chickens. Backyard poultry is increasing in popularity in the United States, but very little is known about the prevalence and molecular epidemiology of IBDV within these flocks. We performed a retrospective study and phylogenetic analyses of IBDV detected in backyard chickens (BYCs) submitted to the California Animal Health and Food Safety (CAHFS) diagnostic laboratory system in 2009-2017. There were 17 CAHFS autopsy cases of very virulent IBDV (vvIBDV) segment A detected by RT-rtPCR in BYC flocks from 7 counties in California from 2009-2017. During this same time period, non-vvIBDV genotypes were detected by RT-rtPCR in 16 autopsy cases originating from BYC premises in 10 counties in California. Subsequent RT-PCR and phylogenetic analysis of a segment of the hvVP2 and VP1 gene identified vvIBDV, interserotypic reassortant IBDV (vvIBDV segment A and serotype 2 segment B), and non-vvIBDV (variant/subclinical IBDV and classic IBDV) strains in BYC flocks in California.

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.

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.

Genetic Characterization of Infectious Bursal Disease Viruses from Field Outbreaks of the North East Region of India.

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.

Differential activation of intraepithelial lymphocyte-natural killer cells in chickens infected with very virulent and vaccine strains of infectious bursal disease virus.

To gain insights into the role of CD3-/28.4+ intraepithelial lymphocytes-natural killer (CD3-/28.4+IEL-NK) cells during infectious bursal disease virus (IBDV) infection, characterisation of the cells was performed following infection with different strains of the virus. In vitro treatment with IL-18 or ionomycin/PMA successfully stimulated and activated the cells via a significant increase in the expression of CD69, B-Lec, CHIR-AB1 and NK-lysin. Similarly, chickens infected with the vaccine strain of IBDV also up-regulated the expression of CD69, B-Lec, CHIR-AB1 and NK-lysin in CD3-/28.4+ IEL-NK cells up to 3 days post infection (dpi) and down-regulated the expression of the inhibitory receptor B-NK at 3 dpi. On the contrary, infection with the very virulent IBDV (vvIBDV) strain lead to a reduced activation of the cells by down-regulating the expression of the CD69, CHIR-AB1 and NK-lysin especially at 1 dpi. These findings altogether demonstrate the differential activation of CD3-/28.4+IEL-NK cells in chicken following infection with the vaccine or very virulent strains of IBDV. The study therefore provides an important clue into the differential pathogenesis of IBDV infection in chicken. Further studies are however required to determine the functional importance of these findings during IBDV vaccination and infection.

Antigenicity characterization of four representative natural reassortment IBDVs isolated from commercial three-yellow chickens from Southern China reveals different subtypes co-prevalent in the field.

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.

Infectious bursal disease virus in Algeria: Detection of highly pathogenic reassortant viruses.

Infectious bursal disease (IBD) is an immunosuppressive viral disease, present worldwide, which causes mortality and immunosuppression in young chickens. The causative agent, the Avibirnavirus IBDV, is a non-enveloped virus whose genome consists of two segments (A and B) of double-stranded RNA. Different pathotypes of IBDV exist, ranging from attenuated vaccine strains to very virulent viruses (vvIBDV). In Algeria, despite the prophylactic measures implemented, cases of IBD are still often diagnosed clinically and the current molecular epidemiology of IBDV remains unknown. The presence of the virus and especially of strains genetically close to vvIBDV was confirmed in 2000 by an unpublished OIE report. In this study, nineteen IBDV isolates were collected in Algeria between September 2014 and September 2015 during clinical outbreaks. These isolates were analyzed at the genetic, antigenic and pathogenic levels. Our results reveal a broad genetic and phenotypic diversity of pathogenic IBDV strains in Algeria, with, i) the circulation of viruses with both genome segments related to European vvIBDV, which proved as pathogenic for specific pathogen-free chickens as vvIBDV reference strain, ii) the circulation of viruses closely related - yet with a specific segment B - to European vvIBDV, their pathogenicity being lower than reference vvIBDV, iii) the detection of reassortant viruses whose segment A was related to vvIBDV whereas their segment B did not appear closely related to any reference sequence. Interestingly, the pathogenicity of these potentially reassortant strains was comparable to that of reference vvIBDV. All strains characterized in this study exhibited an antigenicity similar to the cognate reference IBDV strains. These data reveal the continuous genetic evolution of IBDV strains in Algerian poultry through reassortment and acquisition of genetic material of unidentified origin. Continuous surveillance of the situation as well as good vaccination practice associated with appropriate biosecurity measures are necessary for disease control.