Infectious bursal disease virus: predicting viral pathotype using machine learning models focused on early changes in total blood cell counts.

Infectious bursal disease (IBD) is an avian viral disease caused in chickens by infectious bursal disease virus (IBDV). IBDV strains (Avibirnavirus genus, Birnaviridae family) exhibit different pathotypes, for which no molecular marker is available yet. The different pathotypes, ranging from sub-clinical to inducing immunosuppression and high mortality, are currently determined through a 10-day-long animal experiment designed to compare mortality and clinical score of the uncharacterized strain with references strains. Limits of this protocol lie within standardization and the extensive use of animal experimentation. The aim of this study was to establish a predictive model of viral pathotype based on a minimum number of early parameters measured during infection, allowing faster pathotyping of IBDV strains with improved ethics. We thus measured, at 2 and 4 days post-infection (dpi), the blood concentrations of various immune and coagulation related cells, the uricemia and the infectious viral load in the bursa of Fabricius of chicken infected under standardized conditions with a panel of viruses encompassing the different pathotypes of IBDV. Machine learning algorithms allowed establishing a predictive model of the pathotype based on early changes of the blood cell formula, whose accuracy reached 84.1%. Its accuracy to predict the attenuated and strictly immunosuppressive pathotypes was above 90%. The key parameters for this model were the blood concentrations of B cells, T cells, monocytes, granulocytes, thrombocytes and erythrocytes of infected chickens at 4 dpi. This predictive model could be a second option to traditional IBDV pathotyping that is faster, and more ethical.

Folic acid enhances proinflammatory and antiviral molecular pathways in chicken B-lymphocytes infected with a mild infectious bursal disease virus.

1. This study evaluated the effect of folic acid (FA) supplementation on the proinflammatory and antiviral molecular pathways of B-lymphocytes infected with a modified live IBDV (ST-12) mild vaccine strain during a timed post-infection analysis.2. A chicken B-lymphocytes (DT-40) cell line was cultured in triplicate at a concentration of 5 × 105 cells per well in 24-well plates; and was divided into three groups: 1: No virus, FA; 2: Virus, no FA; 3: Virus + FA at a concentration of 3.96 mM. The experiment was repeated three times.3. Cells in groups 2 and 3 were infected with a modified live IBDV (ST-12) mild vaccine strain at one multiplicity of infection (MOI: 1). After 1 hour of virus adsorption, samples were collected at 0, 3, 6, 12, 24 and 36 hours post-infection (hpi).4. The modified live IBDV (ST-12) mild vaccine strain triggered a B-lymphocyte specific immune response associated with the upregulation of genes involved in virus recognition (Igß), virus sensing (TLR-2, TLR-3, TLR-4 and MDA5), signal transduction and regulation (TRIF, MyD88 and IRF7), and the antiviral effector molecules (IFN-α, OAS, PKR, and viperin).5. FA supplementation modulated IBDV replication and regulated the proinflammatory and antiviral downstream molecular pathways.6. In conclusion, the low virulent pathotype serotype I modified live IBDV (ST-12) mild vaccine strain was able to trigger and mount an immune response in chicken B-lymphocytes without affecting B-cell viability. FA supplementation modulated B lymphocytes response and improved their innate immune proinflammatory and antiviral response molecular pathways.

Assessing the antigenicity of different VP3 regions of infectious bursal disease virus in chickens from South Brazil.

BACKGROUND: Infectious bursal disease (IBD), also known as Gumboro disease, is a viral infection that causes mortality and immunosuppression in chickens (Gallus gallus). VP2 and VP3 are the major structural viral capsid components and are the most immunogenic proteins of IBD virus (IBDV). Reliable diagnostic tests using VP2 and VP3 produced in heterologous systems are important tools to control this infection. One advantage of an IBD diagnostic based on VP3, over those that use VP2, is that VP3 has linear epitopes, enabling its production in bacteria. RESULTS: We tested the suitability of recombinant VP3 (rVP3) as a diagnostic reagent in an enzyme-linked immunosorbent assay (ELISA). Compared with a commercial test, rVP3 ELISA showed high sensitivity and specificity as a diagnostic tool for vaccinated animals. In addition, rVP3, but not the commercial ELISA, was able to detect antibodies in nonvaccinated chickens, probably developed against circulating IBDV strains. It was possible the assessment of VP3 regions antigenicity using chicken antisera. CONCLUSIONS: The full-length recombinant VP3 can be used to assess post vaccination immunological status of chickens and its production is feasible and inexpensive. The evaluation of VP3 regions as candidates for general use in the diagnosis of IBD in chickens should be conducted with caution. Our work was the first to identify several regions of VP3 recognized by chicken antibodies.

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication.

Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.

Propagation and titration of infectious bursal disease virus, including non-cell-culture-adapted strains, using ex vivo-stimulated chicken bursal cells.

Infectious bursal disease virus (IBDV) is a Birnaviridae family member of economic importance for poultry. This virus infects and destroys developing B lymphocytes in the cloacal bursa, resulting in a potentially fatal or immunosuppressive disease in chickens. Naturally occurring viruses and many vaccine strains are not able to grow in in vitro systems without prior adaptation, which often affects viral properties such as virulence. Primary bursal cells, which are the main target cells of lymphotropic IBDV in vivo, may represent an attractive system for the study of IBDV. Unfortunately, bursal cells isolated from bursal follicles undergo apoptosis within hours following their isolation. Here, we demonstrate that ex vivo stimulation of bursal cells with phorbol 12-myristate 13-acetate maintains their viability long enough to allow IBDV replication to high titres. A wide range of field-derived or vaccine serotype 1 IBDV strains could be titrated in these phorbol 12-myristate 13-acetate -stimulated bursal cells and furthermore were permissive for replication of non-cell-culture-adapted viruses. These cells also supported multistep replication experiments and flow cytometry analysis of infection. Ex vivo-stimulated bursal cells therefore offer a promising tool in the study of IBDV.

Circulation of very virulent avian infectious bursal disease virus in Finland.

In the spring of 2014 infectious bursal disease (IBD) was confirmed in a Finnish layer flock exhibiting clinical signs and increased mortality. Organ and blood samples were sent for diagnosis to the Finnish Food Safety Authority Evira. IBD virus (IBDV) was detected in RT-PCR studies. Altogether hens from six layer farms associated with increased mortality (7-10%, worst case 30%) were diagnosed with IBD during 2014. Antibodies were also detected with IBD-ELISA tests in hens on two farms. Phylogenetic analysis showed that the causative agent of the 2014 IBD outbreak was a non-reassortant very virulent type IBDV. The representative virus strains from previous IBD outbreaks in 1978, 1987 and 1993 were also included in the analysis. The strains isolated in 2014 and 1993 were very similar indicating circulation of a very virulent IBDV for over 20 years in the country. In spite of the comprehensive phylogenetic analysis, the definitive origin of the viruses from 2014 and previous outbreaks remains unclear.

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.

Induction of apoptosis in the bursa of Fabricius by vaccination against Gumboro disease.

Infectious bursal disease is a severe acute viral disease of young chickens, affecting mainly the B-lymphocytes in the bursa of Fabricius, leading to severe immunosuppression as a result of the death of lymphoid cells. In the bursa infected with infectious bursal disease virus, viral replication is associated with apoptosis of lymphoid cells, inflammatory change and atrophy. Vaccination has appeared to be a crucial factor for control, with live attenuated vaccines being the most used. However, the apoptotic effect of these vaccines on the bursa has not been tested. We determined the apoptotic effect caused by the most used vaccines in local production on the bursa of Fabricius cells and the correlation with histological changes. In this study, it was demonstrated that apoptosis levels in the vaccinated groups were higher than those observed in the non-vaccinated birds leading to the conclusion that the action of the live virus vaccine strains modifies the boundary of the bursa and shapes processes of cell death by apoptosis. In contrast to other studies, the vaccine strains used did not show the phenomenon of bursal atrophy during the experimental period.

A molecular epidemiology study based on VP2 gene sequences reveals that a new genotype of infectious bursal disease virus is dominantly prevalent in Italy.

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.

Virus mutations and their impact on vaccination against infectious bursal disease (Gumboro disease). / Les mutations virales et leur impact sur la vaccination contre la bursite infectieuse (maladie de Gumboro).

Infectious bursal disease (also known as Gumboro disease) is an immunosuppressive viral disease specific to chickens. In spite of all the information amassed on the antigenic and immunological characteristics of the virus, the disease has not yet been brought fully under control. It is still prevalent in properly vaccinated flocks carrying specific antibodies at levels normally high enough to prevent the disease. Common causes apart, failure of vaccination against infectious bursal disease is associated mainly with early vaccination in flocks of unknown immune status and with the evolution of viruses circulating in the field, leading to antigenic drift and a sharp rise in pathogenicity. Various highly sensitive molecular techniques have clarified the viral determinants of antigenicity and pathogenicity of the infectious bursal disease virus. However, these markers are not universally recognised and tend to be considered as evolutionary markers. Antigenic variants of the infectious bursal disease virus possess modified neutralising epitopes that allow them to evade the action of maternally-derived or vaccine-induced antibodies. Autogenous or multivalent vaccines are required to control antigenic variants in areas where classical and variant virus strains coexist. Pathotypic variants (very virulent viruses) remain antigenically related to classical viruses. The difficulty in controlling pathotypic variants is linked to the difficulty of eliciting an early immune response, because of the risk of the vaccine virus being neutralised by maternal antibodies. Mathematical calculation of the optimal vaccination time and the use of vaccines resistant to maternally-derived antibodies have improved the control of very virulent viruses.

La bursite infectieuse (maladie de Gumboro) est une pathologie virale immunodépressive spécifique du poulet. En dépit des informations accumulées sur les caractères antigéniques et immunologiques du virus, la maladie reste imparfaitement contrôlée. Elle sévit aujourd'hui dans des cheptels correctement vaccinés et porteurs d'anticorps spécifiques à des niveaux habituellement suffisants pour prévenir la maladie. Au-delà des causes triviales, les échecs de la vaccination contre la maladie de Gumboro sont essentiellement liés aux vaccinations précoces de cheptels au statut immunitaire inconnu et à l'évolution des virus qui circulent sur le terrain, se traduisant par une dérive antigénique et une hausse sensible de la pathogénicité. Diverses techniques moléculaires hautement sensibles ont permis d'identifier les déterminants viraux d'antigénicité et de pathogénicité du virus. Ces marqueurs ne sont cependant pas unanimement reconnus et sont pour la plupart considérés comme des marqueurs évolutionnaires. Les virus variants antigéniques possèdent des épitopes neutralisants modifiés qui leur permettent de se soustraire à l'action des anticorps résiduels ou vaccinaux. Leur contrôle passe par l'utilisation d'autovaccins ou de vaccins multivalents dans les régions où coexistent virus classiques et variants. Les variants pathotypiques (virus hypervirulents) restent antigéniquement apparentés aux virus classiques. La difficulté de contrôler ce type de variant est liée à celle d'obtenir une réponse immune précoce, en raison du risque de neutralisation du virus vaccinal par les anticorps d'origine maternelle. Le calcul mathématique de la date optimale de vaccination et l'utilisation de vaccins insensibles aux anticorps résiduels ont permis un meilleur contrôle des virus hypervirulents.

La bursitis infecciosa (enfermedad de Gumboro) es una patología viral específica del pollo, en el que provoca inmunodeficiencia. Pese al acervo de datos existentes sobre los caracteres antigénicos e inmunológicos del virus, la enfermedad no está aún del todo controlada. Hoy en día afecta a bandadas que están correctamente vacunadas y presentan niveles de anticuerpos específicos que en general son suficientes para prevenir la enfermedad. Más allá de las causas corrientes, el fracaso de las vacunaciones contra la enfermedad de Gumboro tiene que ver esencialmente con la vacunación precoz de bandadas cuyo estado inmunológico se desconoce y con la evolución de los virus circulantes sobre el terreno, que se traduce en una deriva antigénica y un sensible aumento de la patogenicidad. Gracias a diversas técnicas moleculares de gran sensibilidad se han podido discernir los determinantes de antigenicidad y patogenicidad del virus, aunque se trata de marcadores no reconocidos unánimemente y considerados en su mayor parte marcadores evolutivos. Los virus que representan variantes antigénicas poseen epitopos neutralizantes modificados que les permiten escapar a la acción de los anticuerpos residuales o inducidos por vacunación. Para combatirlos es preciso utilizar autovacunas o vacunas polivalentes allí donde coexisten los virus clásicos y las variantes. Las variantes patotípicas (virus hipervirulentos) no dejan de estar emparentadas antigénicamente con los virus clásicos. La dificultad de luchar contra este tipo de variante está ligada a la de obtener una respuesta inmunitaria precoz, en razón del riesgo de neutralización del virus vacunal por acción de los anticuerpos de origen materno. El cálculo matemático de la fecha idónea de vacunación y el uso de vacunas insensibles a los anticuerpos residuales han permitido luchar más eficazmente contra los virus hipervirulentos.

Research Note: The infectious bursal disease (Gumboro disease) vaccination scheme affects the quantitative and qualitative carcass characteristics and the immune response of Ross 308 broiler chickens.

After different vaccinations, the study aimed to assess carcass composition, meat quality, and the level of the infectious bursal disease antibody titers in broiler chickens. In the experiment, 4 (30 chicks per group, 120 total) groups of Ross 308 broiler chickens (males and females) were included. Then, they were vaccinated at the hatchery. Group I was immunized against infectious avian bronchitis (IB) and Newcastle disease (ND) using vaccine A (20 mL/100 birds) and vaccine B (20 mL/100 birds), respectively, with injectable vaccine C (0.2 mL/each) targeting Infectious bursal' and Marek's diseases (MD). Group II received the same vaccinations for IB and ND, with separate injectable D vaccines for MD (0.2 mL/unit) and E for infectious bursal' disease (0.2 mL/each). Group III was similarly vaccinated for IB and ND; however, they received only vaccine D (0.2 mL/each) for MD, and on d 23, vaccine F was administered via drinking water against Gumboro disease (0.05 mL/each). Group IV served as the control and was not vaccinated. Ten carcasses from each group on d 42 were taken for carcass composition analysis and meat quality, including pH, color, and water-holding capacity (WHC). During rearing, on d 14, 28, and 42, blood was collected to determine the level of antibodies against infectious bursal' disease. Additionally, hematocrit (HCT), hemoglobin (HGB), and red blood cells were analyzed. In group I, a higher bursa of Fabricius weight than in the other groups was found (P < 0.001), and the liver weight was lower compared to group II (P = 0.020). In breast muscle, the pH24 was higher in the control group than in group I (P = 0.036). Lightness (L*) in breast and leg muscles from group I was higher than in group II (P < 0.05). WHC was higher in group I than in group II (P = 0.025). The antibody titers of infectious bursal' disease was higher in group I on d 28 than in other groups (P < 0.05). Vaccine C (1 dose 0.2 mL/1 bird) of the reconstituted vaccine with the live recombinant virus strain vHVT013-69, not less than 3.6 - 5.0 log10 PFU) had a beneficial effect.

Continuous clinicopathological and molecular recognition of very virulent infectious bursal disease virus in commercial broiler chickens.

Gumboro virus is one of the most dangerous immunosuppressant viruses that infect chickens and causes massive financial losses worldwide. The current study aims to conduct a molecular characterization of chicken farms for the infectious bursal disease virus (IBDV). Based on postmortem (PM) lesions, 125 bursal samples from 25 farms were collected from clinically diseased commercial chicken farms with increased mortality and suspected Gumboro virus infection. Pooled bursal samples from suspected IBD-vaccinated flocks were tested for IBDV by reverse transcriptase polymerase chain reaction (RT-PCR). Fifteen out of 25 pooled specimens were found positive for IBDV, with a 60% detection rate, and confirmed positive for very virulent IBDV (vvIBDV) by sequence analysis. Nucleotide phylogenetic analysis of VP1 and VP2 genes was employed to compare the 5 chosen isolates with strains representing different governorates in Egypt during 2022. All strains were clustered with vvIBDV with no evidence of reassortment in the VP1 gene. The VP1 and VP2 genes are divided into groups (I, II). The strains in our study were related to group II, and it acquired a new mutation in the VP2 gene that clustered it into new subgroup B. By mutation analysis, the VP2 gene of all strains had a characteristic mutation to vvIBDV. It acquired new mutations in HVRs compared with HK46 in Y220F, A222T/V in all strains in our study, and Q221K that was found in IBD-EGY-AH5 and AH2 in the loop PBC in addition to G254S in all strains in our study and Q249k that found in IBD-EGY-AH1 and AH3 in the loop PDE. These mutations are important in the virulency and antigenicity of the virus. The VP1 had 242E, 390M, and 393D which were characteristic of vvIBDV and KpnI restriction enzyme (777GGTAC/C782) in addition to a new mutation (F243Y and N383H) in IBD-EGY-AH1 and AH4 strains. According to the current study, the strains were distinct from the vaccinal strain; they could be responsible for the most recent IBDV outbreaks observed in flocks instead of received vaccinations. The current study highlighted the importance of molecular monitoring to keep up to date on the circulating IBDV for regular evaluation of commercial vaccination programs against circulating field viruses.

A rapid and affordable amplicon-based method for next-generation genome sequencing of the infectious bursal disease virus.

The infectious bursal disease virus (IBDV) causes a severe immunosuppressive disorder in young chickens. IBDV evolution resulted in the emergence of strains with divergent genetic, antigenic, and pathogenic characteristics. Genetic classification is typically performed by sequencing the coding region of the most immunogenic region of the viral protein 2 (VP2). Sequencing both double-stranded RNA genome segments is essential to achieve a more comprehensive IBDV classification that can detect recombinants and reassortments. Here, we report the development and standardization of a tiled PCR amplicon protocol for the direct and cost-effective genome sequencing of global IBDV strains using next-generation technology. Primers for tiled PCR were designed with adapters to bypass expensive and time-consuming library preparation steps. Sequencing was performed on Illumina MiniSeq equipment, and fourteen complete genomes of field strains were assembled using reference sequences. The PCR-enrichment step was used to obtain genomes from low-titer biological samples that were difficult to amplify using traditional sequencing. Phylogenetic analyses of the obtained genomes confirmed previous strain classification. By combining the enrichment methodology with massive sequencing, it is possible to obtain IBDV genomic sequences in a fast and affordable manner. This procedure can be a valuable tool to better understand virus epidemiology.

Immunization and Host Responses to MB-1, a Live Hatchery Vaccine against Infectious Bursal Disease.

MB-1 is an attenuated infectious bursal disease virus vaccine. Previously, we observed a temporal delay of vaccine virus replication in the bursae of chicks due to maternally derived antibodies (MDAs). The mechanism that allowed its survival despite MDA neutralization remained unclear. We hypothesized that after vaccination at 1 day of age (DOA), the MB-1 virus penetrates and resides in local macrophages that are then distributed to lymphoid organs. Furthermore, MB-1's ability to survive within macrophages ensures its survival during effective MDA protection. PCR analysis of lymphoid organs from chicks with MDA, vaccinated on 1 DOA, demonstrated that the MB-1 virus was identified at low levels solely in the spleen pre-14 days of age. Fourteen days after vaccination, the virus was identified using PCR in the bursa, with viral levels increasing with time. The possible delay in viral colonization of the bursa was attributed to the presence of anti-IBDV capsid VP2 maternal IgA and IgY in the bursa interstitium. These indicate that during the period of high MDA levels, a small but viable MB-1 viral reservoir was maintained in the spleen, which might have served to colonize the bursa after MDA levels declined. Thereafter, individual immunization of chicks against Gumboro disease was achieved.

First Detection and Molecular Characterization of Novel Variant Infectious Bursal Disease Virus (Genotype A2dB1b) in Egypt.

Infectious bursal disease (IBD) is an immunosuppressive disease causing significant damage to the poultry industry worldwide. Its etiological agent is infectious bursal disease virus (IBDV), a highly resistant RNA virus whose genetic variability considerably affects disease manifestation, diagnosis and control, primarily pursued by vaccination. In Egypt, very virulent strains (genotype A3B2), responsible for typical IBD signs and lesions and high mortality, have historically prevailed. The present molecular survey, however, suggests that a major epidemiological shift might be occurring in the country. Out of twenty-four samples collected in twelve governorates in 2022-2023, seven tested positive for IBDV. Two of them were A3B2 strains related to other very virulent Egyptian isolates, whereas the remaining five were novel variant IBDVs (A2dB1b), reported for the first time outside of Eastern and Southern Asia. This emerging genotype spawned a large-scale epidemic in China during the 2010s, characterized by subclinical IBD with severe bursal atrophy and immunosuppression. Its spread to Egypt is even more alarming considering that, contrary to circulating IBDVs, the protection conferred by available commercial vaccines appears suboptimal. These findings are therefore crucial for guiding monitoring and control efforts and helping to track the spread of novel variant IBDVs, possibly limiting their impact.

Sequence diversity and evolution of infectious bursal disease virus in Iraq.

Background: Infectious Bursal Disease (IBD) is a highly infectious disease which causes huge economic losses to the poultry industry due to the direct impact of the illness and indirect consequences such as decreasing the general immunity of the flock, leaving it naive to other diseases. In Iraq, IBD is highly prevalent despite vaccination programs, yet studies on sequence diversity of the causative virus are still rare.  Methods: A sample from Bursa of Fabricius from an IBD outbreak in a flock in the city of Najaf in Iraq was smeared on an FTA card. Amplicons of targeted regions in VP1 and VP2 genes were generated and sequenced. Sequences were then compared with other local and global sequences downloaded from GenBank repositories. Sequence alignment and DNA sequence analyses were achieved using MUSCLE, UGENE and MEGAx software. The molecular clock and sequence evolutionary analyses were applied using MEGAx tools.  Results: The strain sequenced in this study belongs to a very virulent Infectious Bursal Disease Virus (vvIBDV) as the DNA and phylogenetic analysis of VP1 and VP2 gene sequences showed a mutual clustering with similar sequences belonging to vvIBDV genogroup 3. Analyses of the hyper variable region of VP2 gene (hvVP2) of IBDV isolates from Iraq indicates a presence of sequence diversity. Interestingly, the two vaccine strains Ventri IBDV Plus and ABIC MB71 that showed the highest sequence similarity to the local isolates in the hvVP2 region are not used in vaccination routine against IBDV in Iraq.  Conclusion: Sequences of vvIBDV in Iraq are diverse. Remarkably, some of the available vaccine strains show high sequence similarity with local strains in Iraq; however, they are not included in the routine vaccination programs. Analysis of more samples involving more geographical regions is needed to draw a detailed map of antigenic diversity of IBDV in Iraq.

Safety and Efficacy Profile of Live, Intermediate Plus Vaccine Against Infectious Bursal Disease Based on Strain G6.

Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive disease of young chickens that causes considerable economic loss in the poultry industry worldwide. Vaccination with live attenuated vaccines is still the most important method used for the control and prevention of IBD in chickens. Here we present the results of in vitro characterization, as well as efficacy and safety testing of a live, intermediate plus vaccine against IBD based on strain G6. Strain characterization confirmed that G6 strain is an intermediate plus strain, showing a high degree of homology with the existing vaccine strains of the same virulence. Safety studies showed that chickens can be vaccinated from 10 days of age. Onset and duration of immunity in specific pathogen free and maternally derived antibodies (MDA) chickens was proven to be 14 and 35 days after vaccination, respectively. When immunizing MDA-positive chickens, vaccine is capable of breakthrough at a titer of ≤500 ELISA units. The field trial conducted on commercial broilers showed a 95% protection against vvIBDV challenge. Stability of the freeze-dried vaccine after reconstitution was confirmed over a period of 3 h. Overall, IBD G6 vaccine has shown good safety and efficacy profile in accordance with European Pharmacopoeia requirements.

Comparative in vivo pathogenicity study of an ITA genotype isolate (G6) of infectious bursal disease virus.

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.

Effect of the polysaccharide extract from the edible mushroom Pleurotus ostreatus against infectious bursal disease virus.

The polysaccharide-containing extracellular fractions (EFs) of the edible mushroom Pleurotus ostreatus have immunomodulating effects. Being aware of these therapeutic effects of mushroom extracts, we have investigated the synergistic relations between these extracts and BIAVAC and BIAROMVAC vaccines. These vaccines target the stimulation of the immune system in commercial poultry, which are extremely vulnerable in the first days of their lives. By administrating EF with polysaccharides from P. ostreatus to unvaccinated broilers we have noticed slow stimulation of maternal antibodies against infectious bursal disease (IBD) starting from four weeks post hatching. For the broilers vaccinated with BIAVAC and BIAROMVAC vaccines a low to almost complete lack of IBD maternal antibodies has been recorded. By adding 5% and 15% EF in the water intake, as compared to the reaction of the immune system in the previous experiment, the level of IBD antibodies was increased. This has led us to believe that by using this combination of BIAVAC and BIAROMVAC vaccine and EF from P. ostreatus we can obtain good results in stimulating the production of IBD antibodies in the period of the chicken first days of life, which are critical to broilers' survival. This can be rationalized by the newly proposed reactivity biological activity (ReBiAc) principles by examining the parabolic relationship between EF administration and recorded biological activity.

Phylogeny and evolution of infectious bursal disease virus circulating in Turkish broiler flocks.

The emergence of new infectious bursal disease virus (IBDV) variants can threaten poultry health and production all over the world causing significant economic losses. Therefore, this study was performed to determine IBDV molecular epidemilogy, VP2 gene variation, and corresponding pathological lesions in IBDV infected chickens in Turkey. For this, 1855 bursa of Fabricius samples were collected from 371 vaccinated broiler flocks. Atrophia and haemorrhages were seen in the bursa Fabricius of very virulent IBDV (vvIBDV) infected chickens. Partial VP2 gene was sequenced and phylogenetic, recombination, and evolutionary analyses were performed. 1548 (83.5%) out of 1855 of bursa of Fabricius samples were IBDV positive and 1525 of those could be sequenced. The recombination analysis did not detect occurrence of any recombination event among the Turkish strains. Among 1525 sequenced samples, 1380 of them were found to be classical strains. Among 1380 classical strains, 1317 were similar to IBDV 2512, 11 to Faragher 52/70, 40 to 228 E, and 12 to Lukert strain. Out of 1525 reverse transcriptase ploymerase chain reaction positive samples, 144 of them were found to be similar to vvIBDV-VP2 gene reported to GenBank previously. The phylogenetic tree performed on a broad sequence dataset demonstrated grouping of vvIBDV Turkish strains in three different clusters, including sequences collected also from Iraq and Kuwait (Cluster 1), Indian (Cluster 2), and a distinct Turkish-only cluster (Cluster 3). The evolutionary rate estimation on branches/clades including Turkish strain mirrored the expected one for RNA viruses and no significant differences were found among different considered branches. In conclusion, results of this study indicate that vvIBDV strains similar to those circulating in various countries in the Middle East are present and undergoing evolution in chickens from Turkish broiler flocks. This point needs to be taken into account in planning adequate control strategies.