Infectious Bursal Disease Virus Strains Research Articles

Genetic variability in VP1 gene of infectious bursal disease virus from the field outbreaks of Kerala, India.

Infectious bursal disease (IBD) is considered as menace as it affects poultry industry globally causing immunosuppression, high mortality and heavy economic loss. Outbreaks of IBD were reported in many states of India including Kerala. VP1 gene acts as an important factor in the process of virus encapsidation and its involvement in viral virulence and viral replication indicates its importance in infectious bursal disease virus (IBDV). The present study was conducted to carry out the molecular characterization of VP1 gene of virulent IBDV in Kerala. A total of 42 samples were processed for the detection and analysis of VP1 gene of IBDV. Out of 42 samples, 21 samples were positive for VP1 gene of IBD. The phylogenetic analysis of the partial VP1 gene sequences reveals the clustering of IBDV isolates into very virulent IBDV (vvIBDV) and non-virulent IBDV (vIBDV). Eighteen isolates (11 isolates from vaccinated flock and 7 from non-vaccinated flocks) clustered with very virulent strains. Three isolates (2 isolates were from vaccinated flock and 1 from non-vaccinated flock) clustered with non-virulent IBDV strains, showing more evolutionarily similarity to south Indian strain VCN14/ABT/MVC/India. It is observed that vvIBDV isolates from this study have common ancestor with the south Indian strain PY12 but showed 9-10% divergence from this strains. The amino acid analysis of these 21 isolates revealed that 17 isolates possessed the characteristic vvIBDV TDN amino acid triplet, while the three isolates had non-vIBDV NEG amino acid triplet at 145/146/147 position. The remaining isolate 1/CVASP/IBDV/VP1 shows unique PDN triplet instead of TDN. Two vvIBDV isolates (15/CVASP/IBDV/VP1 and 18/CVASP/IBDV/VP1) showed 100% nucleotide and amino acid similarity with intermediate plus vaccine strain. Four vvIBDV isolates showed neutral amino acid substitution K251R which was earlier reported in Indian strains but first time in south Indian isolates. The most common unique amino acid substitution observed in our study was neutral E269D amino acid substitution in 12 isolates, neutral amino acid substitution T329S in five isolates, neutral T174N and non-polar to polar amino acid substitution A178T in isolate 10/CVASP/IBDV/VP1, non-polar to polar amino acid substitution P360R in isolate 17/CVASP/IBDV/VP1 and non-polar to polar amino acid substitution P188S in isolate 1/CVASP/IBDV/VP1. These novel mutations in our study reveal the role of genetic drift in the evolution of vvIBDV strains. The isolate 2/CVASP/IBDV/VP1 from non-vaccinated flock shows VP1 gene of non-vIBDV, but possessing VP2 of vvIBDV type indicates this is evolved by genetic shift of segments A and B. This is the first genetic characterization study of field VP1 gene of IBDV isolates in Kerala, India.

Genome Evolution of Two Genetically Homogeneous Infectious Bursal Disease Virus Strains During Passages in vitro and ex vivo in the Presence of a Mutagenic Nucleoside Analog.

The avibirnavirus infectious bursal disease virus (IBDV) is responsible for a highly contagious and sometimes lethal disease of chickens (Gallus gallus). IBDV genetic variation is well-described for both field and live-attenuated vaccine strains, however, the dynamics and selection pressures behind this genetic evolution remain poorly documented. Here, genetically homogeneous virus stocks were generated using reverse genetics for a very virulent strain, rvv, and a vaccine-related strain, rCu-1. These viruses were serially passaged at controlled multiplicities of infection in several biological systems, including primary chickens B cells, the main cell type targeted by IBDV in vivo. Passages were also performed in the absence or presence of a strong selective pressure using the antiviral nucleoside analog 7-deaza-2′-C-methyladenosine (7DMA). Next Generation Sequencing (NGS) of viral genomes after the last passage in each biological system revealed that (i) a higher viral diversity was generated in segment A than in segment B, regardless 7DMA treatment and viral strain, (ii) diversity in segment B was increased by 7DMA treatment in both viruses, (iii) passaging of IBDV in primary chicken B cells, regardless of 7DMA treatment, did not select cell-culture adapted variants of rvv, preserving its capsid protein (VP2) properties, (iv) mutations in coding and non-coding regions of rCu-1 segment A could potentially associate to higher viral fitness, and (v) a specific selection, upon 7DMA addition, of a Thr329Ala substitution occurred in the viral polymerase VP1. The latter change, together with Ala270Thr change in VP2, proved to be associated with viral attenuation in vivo. These results identify genome sequences that are important for IBDV evolution in response to selection pressures. Such information will help tailor better strategies for controlling IBDV infection in chickens.

A field study on the evaluation of day-of-hatch and in grow-out application of live infectious bursal disease virus vaccine in broiler chickens

Infectious bursal disease (IBD) is an acute, highly contagious, economically important disease of young chickens caused by Avibirnavirus, the infectious bursal disease virus (IBDV). The causative virus is highly resilient in poultry environments and vaccination is the most effective measure for IBDV control. However, the susceptibility of highly attenuated IBDV vaccine strains to neutralization by maternally derived antibodies (MDA) and overwhelming virulence of partly attenuated strains have limited the application of conventional live IBDV vaccines in pre- and posthatch chicks. Nevertheless, preliminary data have raised questions about the validity of this prevailing dogma. India is an IBD endemic country and the disease causes sizeable economic losses in the sector. To evaluate the feasibility of application of live IBDV vaccine strain, the IBDV MB-1, to the maternally immunized day-of-hatch chicks in Indian production environment, 4 large-scale field trials have been conducted. The 4 trials have measured the relative safety, IBDV immunization parameters, and production performances of MB-1 vs. the established live and immune complex IBDV vaccines in a variety of commercial broiler systems. The overall health and production performances in all 4 trials have been better in the MB-1 groups. The results challenge the prevailing notion that live IBDV strains may be neutralized or break through maternal immunity and induce permanent damage to the young broiler chick's immune response. A delayed replication phenomenon following parenteral administration of the live IBDV vaccine strain has been observed, while the delayed replication mechanism remains to be elucidated. This study warrants further research on the molecular mechanism of live IBDV vaccine strain, MB-1, and its interaction with the chicken immune system.

Experimental co-infection of variant infectious bursal disease virus and fowl adenovirus serotype 4 increases mortality and reduces immune response in chickens

Infectious bursal disease virus (IBDV) and fowl adenovirus serotype 4 (FAdV-4) cause infectious bursal disease (IBD) and hydropericardium-hepatitis syndrome, respectively. Recently, studies have reported co-infections of poultry with IBDV and FAdV-4, which is an important problem in the poultry industry. Here, the variant IBDV strain ZD-2018-1 and FAdV-4 isolate HB1501 were used to assess the pathogenicity of co-infection in 1-day-old specific pathogen-free (SPF) chickens. Compared with chickens infected with only FAdV-4, those coinfected with IBDV and FAdV-4 showed enhanced clinical symptoms, higher mortality, more severe tissue lesions, and higher biochemical index levels. Furthermore, the expression of interleukin (IL)-6, IL-1β, and interferon-γ mRNAs in the IBDV-FAdV-4 coinfected chickens was delayed, and the antibody response levels were significantly lower in those birds compared with the FAdV-4-infected chickens. These results indicate that co-infection with variant IBDV ZD-2018-1 and FAdV-4 HB1501 could significantly promote the pathogenicity of FAdV-4 and reduce the immune response in chickens. This study provides the foundation for further investigation of the interaction mechanism in IBDV and FAdV-4 co-infection.

Genetic Diversity of Recent Infectious Bursal Disease Viruses Isolated From Vaccinated Poultry Flocks in Malaysia.

Vaccination is an essential component in controlling infectious bursal disease (IBD), however, there is a lack of information on the genetic characteristics of a recent infectious bursal disease virus (IBDV) that was isolated from IBD vaccinated commercial flocks in Malaysia. The present study investigated 11 IBDV isolates that were isolated from commercial poultry farms. The isolates were detected using reverse transcription-polymerase chain reaction (RT-PCR) targeting the hypervariable region (HVR) of VP2. Based on the HVR sequences, five isolates (IBS536/2017, IBS624/2017, UPM766/2018, UPM1056/2018, and UPM1432/2019) were selected for whole-genome sequencing using the MiSeq platform. The nucleotide and amino acid (aa) sequences were compared with the previously characterized IBDV strains. Deduced aa sequences of VP2HVR revealed seven isolates with 94–99% aa identity to very virulent strains (genogroup 3), two isolates with 97–100% aa identity to variant strains (genogroup 2), and two strains with 100% identity to the vaccine strain (genogroup 1) of IBDV. The phylogenetic analysis also showed that the isolates formed clusters with the respective genogroups. The characteristic motifs 222T, 249K, 286I, and 318D are typical of the variant strain and were observed for UPM1219/2019 and UPM1432/2019. In comparison, very virulent residues such as 222A, 249Q, 286T, and 318G were found for the vvIBDV, except for the UPM1056/2018 strain with a A222T substitution. In addition, the isolate has aa substitutions such as D213N, G254D, S315T, S317R, and A321E that are not commonly found in previously reported vvIBDV strains. Unlike the other vvIBDVs characterized in this study, UPM766/2018 lacks the MLSL aa residues in VP5. The aa tripeptides 145/146/147 (TDN) of VP1 were conserved for the vvIBDV, while a different motif, NED, was observed for the Malaysian variant strain. The phylogenetic tree showed that the IBDV variant clustered with the American and Chinese variant viruses and are highly comparable to the novel Chinese variants, with 99.9% identity. Based on the sequences and phylogenetic analyses, this is the first identification of an IBDV variant being reported in Malaysia. Further research is required to determine the pathogenicity of the IBDV variant and the protective efficacy of the current IBD vaccines being used against the virus.

Infectious bursal disease in Nigeria: continuous circulation of reassortant viruses.

Outbreaks of infectious bursal disease (IBD), a highly contagious immunosuppressive disease of young chickens, are still reported globally despite vaccination efforts. This study investigated the genetic characteristics of infectious bursal disease virus (IBDV) from 26 reported outbreaks in 2019 in Nigeria. Nucleotide sequences of VP2 hypervariable (hvVP2) region (n=26) and VP1 (n=23) of Nigerian IBDVs were determined. Our results revealed the detection of reassortant strains with segment A related to very virulent IBDV (vvIBDV) having virulence marker (222A, 242I, 256I, 294I and 299S), whereas their segment B were closely related to previously detected IBDV strains having QEG substitution at positions 145-147. Phylogenetic analysis of the hvVP2 region revealed that all the Nigerian IBDV clustered with vvIBDV (genogroup 3) and were independent of the Asian/European lineage. Interestingly, in the hvVP2, all the viruses had a G-S substitution at residue 254. Additionally, one isolate had an A321T substitution at the PHI loop, which has been suggested to play a key role in antigenicity. Four of the viruses (Bauchi=3 and Plateau=1) had a unique A-T substitution at residue 144 on the VP1 region. We also observed a T174S substitution in nine of the Nigerian viruses from Bauchi and Plateau state that were not found in any outbreak viruses from Oyo and Akwa Ibom. This report demonstrates the circulation of reassortant strains in commercial and backyard poultry farms in Nigeria despite sustained vaccination efforts. Our data suggest that the Nigerian outbreak viruses have mutations that may affect antigenicity and contribute to antigenic drift.

An improved scheme for infectious bursal disease virus genotype classification based on both genome-segments A and B

Infectious bursal disease (IBD) is caused by infectious bursal disease virus (IBDV), which has a genome consisting of two segments of double-stranded linear RNA. IBDVs have been traditionally divided into four phenotypes based on their pathogenicity and antigenicity, including classic, variant, very virulent, and attenuated IBDV. With the emergences of divergent molecular characteristics of novel strains produced by continuous mutations and recombination, it is increasingly difficult to define new IBDV strains using the traditional descriptive classification method. The most common classification scheme for IBDV with segmented genome is based solely on segment A, while the significance of segment B has been largely neglected. In this study, an improved scheme for IBDV genotype classification based on the molecular characteristics of both VP2 (a viral capsid protein encoded by segment A) and VP1 (an RNA-dependent RNA polymerase protein encoded by segment B) was proposed for the first time. In this scheme, IBDV was classified into nine genogroups of A and five genogroups of B, respectively; the genogroup A2 was further divided into four lineages. The commonly used phenotypic classifications of classic, variant, very virulent, and attenuated IBDVs correspond to the A1B1, A2B1, A3B2, and A8B1 genotypes of the proposed classification scheme. The novel variant IBDVs including the strains identified in this study were classified as belonging to genotype A2dB1. The flexibility and versatility of this improved classification scheme will allow the unambiguous identification of existing and emerging IBDV strains, which will greatly facilitate molecular epidemiology studies of IBDV.

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.

The expression in plants of an engineered VP2 protein of Infectious Bursal Disease Virus induces formation of structurally heterogeneous particles that protect from a very virulent viral strain.

Infectious Bursal Disease Virus (IBDV), the etiological agent of Gumboro disease, causes mortality and immunosuppression in chickens and major losses to poultry industry worldwide. The IBDV major capsid protein VP2 is considered the best candidate for the production of novel subunit vaccines. This structural protein contains the major conformational epitopes responsible for the induction of IBDV neutralizing antibodies in chickens and has been demonstrated able to form supramolecular structures in yeast and insect cells. The aim of this study was to express an engineered version of the VP2 protein (His-pVP2) to verify its ability to self-assemble into virus-like particles in plants. The recombinant VP2 was transiently expressed by agroinfiltration in Nicotiana benthamiana and transmission electron microscopy of sucrose density gradient fractions revealed the presence of a mixed population of differently shaped particles ranging from spherical capsids, with a diameter between ~25 and ~70 nm, to tubular structures, with variable length (from 100 to 400 nm). The recombinant VP2-based particles when used for the intramuscular immunization of specific-pathogen-free chicks resulted able to induce the production of anti-IBDV specific antibodies at titers comparable to those induced by a commercial vaccine. Moreover, all the immunized birds survived to the challenge with a Moroccan very virulent IBDV strain with no major histomorphological alterations of the Bursa of Fabricius, similarly to what obtained with the commercial inactivated vaccine.

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

ABSTRACT 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.

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

ABSTRACT 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.

DEVELOPMENT AND PRODUCTION OF INFECTIOUS BURSAL DISEASE (GUMBORO) VACCINE IN NIGERIA

An infectious bursal disease virus strain obtained from an pathogenic strain that was attenuated in em-bryonated eggs, is produced in a primary culture of chicken embryo fibroblast, (CEF). This virus has been passaged 10 times further in CEF, and is now intended for use in the active immunisation of chickens against IBD. The vaccinę virus replicates well in CEF giving a titer of up to107.5 TCID50 per ml. In spite of its pathogenicity for CEF, the vaccine has no pathogenicity for chickens as shown by the absence of gross and histopathological lesions in the bursa of Fabricius (BF) of birds infected with it. Immunogenicity is retained and infact compares favourably with those of other IBD virus strains. The vaccine virus does not revert back to its original pathogenicity but can be adversely affected by storage at room temperature and at 37°C. It could however be stored at +2°C to +8°C or lower for up to six months without loss in potency, The vaccine can be administered by mouth or intramuscularly and as low as 50 TCID50 per bird guarantees full protection. However, as much as 125,000 times the guaranteed dose per bird has been administered without any observable changes in the BF of the affected birds. The field dosage is calculated so that at least one guaranteed dose (i.e. 50 TCID50 ) is still available to each bird even after incubation at 37°C for 7 days. The vaccine did not depress the immune response of chickens to ND vaccine intraocular when administered concurrently with it. The vaccine was tested for safety and immunogenicity in a population of two isolated flocks totalling 8504 birds. The immune status of a flock tested was significantly enhanced as a result of the vaccination (Table 6). More than 10.8 million doses have been issued to the field from 1979 to 1982 and the demand is increasing. Every batch of vaccine produced is tested for viability in CEF, sterility in bacterial culture media and for safety and potency in chickens.

Efficacy of a novel in ovo-attenuated live vaccine and recombinant vaccine against a very virulent infectious bursal disease virus in chickens.

Infectious bursal disease (IBD) causes severe economic damage to the poultry industry worldwide. To prevent IBD virus (IBDV) infection, live virus vaccines have been widely used in chickens having wide-ranging levels of maternally derived antibodies. But, the risks of infection with other pathogens because of lesions related to atrophy of the bursa of Fabricius in vaccinated chickens are a concern. To resolve the problems, a recombinant turkey herpesvirus (HVT) vaccine expressing IBDV-VP2 protein (rHVT-IBD) has been developed. However, the induction of neutralizing antibodies by rHVT-IBD against a virulent IBDV might be delayed compared with that by the live IBD vaccine, leading to the high risks of IBDV infection for young chickens. To find the best selection of IBDV vaccine for the onset of immunity, we examine the protective efficacy of a novel in ovo-attenuated live IBDV (IBD-CA) vaccine and the rHVT-IBD vaccine in young chickens challenged with a very virulent IBDV (vvIBDV) strain. We show that the protective efficacy of IBD-CA vaccine was higher than that of the rHVT-IBD vaccine in 14-day-old chickens challenged with the vvIBDV strain, leading to the risk of IBDV infection for young chickens when vaccinated with rHVT-IBD. Our results suggest that farmers should select the best vaccines to maximize vaccine efficacy in consideration of the vaccine characteristics, prevalence levels of IBDV in the areas, and initial MDA levels of the chickens since the attenuated live and recombinant vaccines play a role in the different vaccine efficacies.

Comparison of two attenuated infectious bursal disease vaccine strains focused on safety and antibody response in commercial broilers.

Background and Aim:Infectious bursal disease (IBD) or Gumboro disease is one of the most detrimental diseases in the poultry industry worldwide. Previous scientific studies have shown that live IBD vaccination might induce transient immunosuppression, leading to suboptimal vaccine responses, and therefore lack of protection against other infectious diseases; therefore, selecting an IBD vaccine in commercial farms is a concern. This study aims to compare two commercially attenuated IBD vaccines (intermediate and intermediate-plus strains) in terms of safety and antibody response to IBD and Newcastle disease viruses (NDV) in commercial broilers.Materials and Methods:Overall, 216 Cobb broiler chickens were divided into three groups based on the IBD vaccine strain administered: V217 strain (Group 1), M.B. strain (Group 2), and an unvaccinated group (Group 3). Groups 1 and 2 were orally vaccinated with Hitchner B1 NDV vaccine strain 7 days after IBD vaccination. Blood samples were collected at IBD vaccination day (15 days of age) and at 7, 14, 21, and 28 days post-IBD vaccination. The immunosuppressive effects of the IBD vaccination were determined by NDV antibody response, the bursa:body weight (B:BW) ratio, and the histopathological lesion scores of the bursa of Fabricius. Phylogenetic analysis was also performed.Results:Phylogenetic analysis revealed that the M.B. strain belonged to a very virulent IBD strain, whereas the V217 strain belonged to a classical IBD virus strain. NDV antibody titers of the two vaccinated groups increased after ND vaccination, reaching their maximum at 14 days post-ND vaccination and decreasing thereafter. The V217 group presented the highest NDV humoral response from 7 days post-vaccination (dpv) to the end of the study. The mean NDV antibody titer of the V217 group was significantly (p<0.05) higher than that of the M.B. group at 14 dpv. In addition, the V217 strain-induced lower bursal lesions post-IBD vaccination and a higher B: BW ratio at 7 and 21 dpv compared to the M.B. group. The higher B: BW ratio, lower bursal lesions, and higher ND antibody response present in the V217 group indicate that the V217 strain induces lower immunosuppressive effects compared to the M.B. strain.Conclusion:The results of this study indicate that IBD vaccine selection merits consideration, as avoiding the immunosuppressive effects induced by live IBD vaccination and the consequent impact on response to other vaccines is important.

Full-length genome sequencing of a very virulent infectious bursal disease virus isolated in Tunisia

Infectious bursal disease (IBD), an acute, highly contagious, and immunosuppressive avian disease, is caused by infectious bursal disease virus (IBDV) and constitutes one of the main threats to the poultry industry, worldwide. This study was performed to isolate and characterize IBDV isolates circulating in Tunisia. Eleven collected bird samples were identified using an SYBR Green–based one-step real-time reverse transcriptase polymerase chain reaction. The full-length genome sequencing of 7 of the 11 IBDV isolates has been realized. VP2 gene data showed limited sequence variations for all the 7 tested samples. The few nucleotide changes were silent and the deduced amino acid sequences were identical with the exception of a unique and characteristic nonsilent mutation (C1203) detected for the TN37/19 isolate, with a change of amino acid (L) to (F) at position 401. In addition, the serine-rich heptapeptide SWSASGS, characteristic of virulent IBDV, as well the amino acid residues, conserved in most very virulent IBDV (vvIBDV) strains, were detected in all the Tunisian tested isolates. Nucleotide sequences of VP5 gene revealed the presence of 5 substitutions leading to changes in the amino acid sequences of the virus. Two of these mutations were unique and characteristic of the Tunisian isolates. Besides, the alternative AUG start codon, characteristic of vvIBDV, was observed in all obtained VP5 gene sequences. The Tunisian protein sequences of VP1 showed E242 and the TDN triplet at positions 145, 146, and 147, a motif specific of vvIBDV. Phylogenetic analyses of the 5 genes confirmed the sequence alignment results and showed that the Tunisian strains are closely related to the very virulent Algerian IBDV strains.

Efficacy of a turkey herpesvirus double construct vaccine (HVT-ND-IBD) against challenge with different strains of Newcastle disease, infectious bursal disease and Marek’s disease viruses

ABSTRACT A double construct vaccine of turkey herpesvirus (HVT) was prepared that contains the fusion (F) gene from Newcastle disease virus (NDV) and the viral protein 2 (VP2) gene from infectious bursal disease virus (IBDV). Safety of the vaccine (HVT-ND-IBD) was confirmed and efficacy was evaluated after subcutaneous (SC) vaccination at 1 day of age or the in ovo route of vaccination. Challenges were performed with velogenic NDV strains (Texas GB and Herts Weybridge 33/56), with different strains of IBDV (classical strain STC; very virulent strain CS89 and variant E strain) and with Marek’s disease virus (MDV) strain RB1B. Vaccination with HVT-ND-IBD induced a high level of protection against these challenges. Vaccination with HVT is often combined with Rispens CVI988 vaccine and live ND vaccines for higher and earlier, MD and ND protection, respectively. HVT-ND-IBD vaccination in combination with these vaccines showed MD protection as early as 4 days post vaccination and ND protection as early as 2 weeks post vaccination. The long protection as seen with HVT vaccination was confirmed by demonstrating protection against NDV up to 60 weeks. Finally, to evaluate the performance of the vaccine in commercial birds with maternally-derived antibodies, two field trials were performed, using in ovo vaccination in broilers and SC vaccination in combination with Rispens CVI988 vaccine in layer-type birds. The efficacy was confirmed for all components by challenges. These results demonstrate that HVT-ND-IBD is a safe and highly efficacious vaccine for simultaneous control of ND, IBD and MD. RESEARCH HIGHLIGHTS A double construct HVT vaccine with the NDV F and the IBDV VP2 genes was prepared. The vaccine protects against three important diseases: MDV, NDV and IBDV. In ovo and sub-cutaneous vaccination was evaluated in the field in commercial chickens.

Transcriptome analysis of chicken intraepithelial lymphocyte natural killer cells infected with very virulent infectious bursal disease virus

The infectious bursal disease (IBD) is an acute immunosuppressive viral disease that significantly affects the economics of the poultry industry. The IBD virus (IBDV) was known to infect B lymphocytes and activate macrophage and T lymphocytes, but there are limited studies on the impact of IBDV infection on chicken intraepithelial lymphocyte natural killer (IEL-NK) cells. This study employed an mRNA sequencing approach to investigate the early regulation of gene expression patterns in chicken IEL-NK cells after infection with very virulent IBDV strain UPM0081. A total of 12,141 genes were expressed in uninfected chicken IEL-NK cells, and most of the genes with high expression were involved in the metabolic pathway, whereas most of the low expressed genes were involved in the cytokine-cytokine receptor pathway. A total of 1,266 genes were differentially expressed (DE) at 3 day-post-infection (dpi), and these DE genes were involved in inflammation, antiviral response and interferon stimulation. The innate immune response was activated as several genes involved in inflammation, antiviral response and recruitment of NK cells to the infected area were up-regulated. This is the first study to examine the whole transcriptome profile of chicken NK cells towards IBDV infection and provides better insight into the early immune response of chicken NK cells.

Very virulent infectious bursal disease virus-induced immune injury is involved in inflammation, apoptosis, and inflammatory cytokines imbalance in the bursa of fabricius

Infectious bursal disease virus (IBDV) can cause a highly contagious disease in young chickens, resulting in bursal necrosis that causes severe damage to the immune system. The effects of various IBDV strains on the bursa of Fabricius (BF) have been extensively studied; however, few studies have investigated the effects of IBDV strain LJ-5, a newly discovered very virulent IBDV (vvIBDV), infection on young chicken BF. In this study, three-week-old specific pathogen-free (SPF) chickens were infected with vvIBDV for one to five days. LJ-5 decreased the bursa index, B lymphocyte viability and immunoglobulin (Ig) levels, including IgM and IgA in the bursa and IgY in the sera. Histopathological analysis revealed necrosis and depletion of the lymphoid cells and complete loss of bursal architecture in the BF, and transmission electron microscopy revealed mitochondrial vacuoles, cristae breaks, and nuclear damage in vvIBDV-infected bursa tissue. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei significantly increased following IBDV infection. Cytokine levels increased in the bursa after IBDV infection, promoting inflammation and causing an inflammatory imbalance. Apoptotic gene expression confirmed that vvIBDV infection promotes the apoptosis of bursal cells. These results suggest that vvIBDV infection attenuate immune responses by reducing B lymphocyte activity of secretion Ig in the bursa or sera and triggers inflammation, apoptosis, and an imbalance of inflammatory cytokines in the BF, resulting in immune injury in SPF chickens, which offered basic data for further study of vvIBDV pathogenesis.

Ex vivo rescue of recombinant very virulent IBDV using a RNA polymerase II driven system and primary chicken bursal cells

Infectious Bursal Disease Virus (IBDV), a member of the Birnaviridae family, causes an immunosuppressive disease in young chickens. Although several reverse genetics systems are available for IBDV, the isolation of most field-derived strains, such as very virulent IBDV (vvIBDV) and their subsequent rescue, has remained challenging due to the lack of replication of those viruses in vitro. Such rescue required either the inoculation of animals, embryonated eggs, or the introduction of mutations in the capsid protein (VP2) hypervariable region (HVR) to adapt the virus to cell culture, the latter option concomitantly altering its virulence in vivo. We describe an improved ex vivo IBDV rescue system based on the transfection of an avian cell line with RNA polymerase II-based expression vectors, combined with replication on primary chicken bursal cells, the main cell type targeted in vivo of IBDV. We validated this system by rescuing to high titers two recombinant IBDV strains: a cell-culture adapted attenuated strain and a vvIBDV. Sequencing of VP2 HVR confirmed the absence of unwanted mutations that may alter the biological properties of the recombinant viruses. Therefore, this approach is efficient, economical, time-saving, reduces animal suffering and can be used to rescue other non-cell culture adapted IBDV strains.

The circulation of unique reassortment strains of infectious bursal disease virus in Pakistan

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is one of the most devastating and immunosuppressive diseases of the poultry and has been a constraint on the sustainable poultry production around the globe including Pakistan. While the disease is threatening the poultry industry, the nature of predominant strains of IBDV in Pakistan remained ill-defined. In this study, an epidemiology survey was conducted in the main chicken-farming regions of Pakistan. The batch of Pakistan IBDVs genes simultaneously covering both VP1 and VP2 were amplified, sequenced, and analyzed. The unique segment-reassortant IBDVs (vv-A/Uniq-B), carrying segment A from vvIBDV and segment B from one unique ancestor, were identified as one important type of circulating strains in Pakistan. The data also discovered the characteristic molecular features of Pakistan IBDVs, which will contribute to scientific vaccine selection and effective prevention of the disease.