Infectious Bursal Disease Virus VP2 Research Articles

Isolation and molecular characterization of very virulent infectious bursal disease virus from Egypt

In Egypt, the poultry industry is plagued by the significant economic losses caused by the infectious bursal disease virus (IBDV). In the present study, a total of 30 diseased commercial chicken flocks aged 23–41 days old in three Egyptian governorates (Fayoum, Beni-Suef, and Minya) having symptoms consistent with IBDV were examined. The collected bursal samples (n=30, ten pooled bursae for each flock) were screened by RT-PCR to amplify the IBDV VP2 gene. Results revealed that 20 out of 30 samples were positive, as indicated by the amplification of 620 bp of the VP2 gene. The twenty positive samples were inoculated into specific pathogen-free embryonated chicken eggs (SPF-ECEs) via the chorioallantoic-membrane (CAM) route to isolate the suspected virus. Eight samples were successfully isolated and were subjected to a partial sequence of VP2 (targeting the HVR). The nucleotide sequences and phylogenetic analysis revealed that the examined isolates exhibited amino acids A222, I256, I294, and S299, which are extremely conserved among very virulent (vv)IBDV. The amino acid sequence analysis of the eight isolates revealed a high degree of similarity (97-100%) in between and 90-100% similarity compared to other Egyptian vvIBDV strains. In contrast, lower similarity was observed with attenuated IBDV vaccine strains D78, Lukert, and Bursavac (91-93%, 91-93%, and 92-94%, respectively). In conclusion, this study underscores the importance of ongoing surveillance of the IBDV situation in the field, as well as the need for further research to investigate the efficacy of current vaccination strategies to curb IBDV infection.

Development of a Highly Efficient CRISPR/Cas9-Mediated Herpesvirus of Turkey-Based Vaccine against Novel Variant Infectious Bursal Disease Virus.

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is an extremely contagious immunosuppressive disease that causes major losses for the poultry industry worldwide. Recently, the novel variant IBDV (G2d) has been highly prevalent in Korea, but the current vaccines against this very virulent IBDV have limited efficacy against this novel variant. To develop a vaccine against this variant IBDV, a recombinant virus designated rHVT-VP2 was constructed by inserting the IBDV (G2d) VP2 gene into herpesvirus of turkeys (HVT) using CRISPR/Cas9 gene-editing technology. The PCR and sequencing results obtained showed that the recombinant virus rHVT-VP2 was successfully constructed. Vaccination with rHVT-VP2 generated IBDV-specific antibodies in specific pathogen-free chickens starting from 2 weeks post-immunization. Seven days after the challenge, the autopsy results showed that the bursa atrophy rates of the rHVT-VP2, HVT, vaccine A, and positive control groups were 0%, 100%, 60%, and 100%, respectively, and the BBIX values were 1.07 ± 0.22, 0.27 ± 0.05, 0.64 ± 0.33, and 0.32 ± 0.06, respectively. These results indicate that rHVT-VP2 can provide 100% protection against a challenge with the IBDV (G2d), whereas vaccine A only provides partial protection. In conclusion, vaccination with the recombinant virus rHVT-VP2 can provide chickens with effective protection against variant IBDV (G2d).

Isolation and genomic characteristics of the novel variant infectious bursal disease virus in China.

The infectious bursal disease virus (IBDV) is a member of the viruses that can induce immunosuppression in chickens. In recent years, more and more IBDV-infected cases by the novel variant IBDV were reported in China, and it has been demonstrated that currently used vaccines could not provide complete protection against these new IBDV variants. However, a lack of comprehensive analysis of the genomic characteristics of the novel variant strain IBDV has hampered its vaccine development. In this study, a strain of IBDV, designated HB202201, was phylogenetically analyzed, and it was found that the hypervariable region (HVR) of VP2 belonged to the novel variant strain. Furthermore, the 5'- and 3'-ends of segments A and B were analyzed using the rapid amplification of cDNA end (RACE) method. After the full-length of segment A and segment B were determined, the phylogenetic analysis of the segment A and segment B showed that the isolated HB202201 belonged to A2dB1 genotype, which demonstrated the HB202201 belonged to the novel variant strain. In addition, the specific mutations in VP1-VP5 amino acids were analyzed, which showed that there were multiple typical mutations in novel variant IBDV proteins, including VP1 (G24, I141, V163, and E240), VP2 (K221, and I252), VP3 (Q167 and L196), and VP5 (R7, P44, R92, G104, and E147), whereas there was no typical mutation in VP4. This study provides insights into the genomic and antigenic characteristics of the novel variant IBDV, which will promote the development of novel vaccine against the novel variant IBDV.

Protective effects of a novel chimeric virus–like particle vaccine against virulent NDV and IBDV challenge

Newcastle disease (ND) and infectious bursal disease (IBD) pose significant threats to the chicken industry, causing substantial economic losses. Currently, immunization through vaccination is the most effective strategy to prevent ND and IBD but currently used traditional vaccines, including inactivated or attenuated vaccines, face challenges in achieving a balance between immunogenicity and safety. To develop a green and efficient novel vaccine for ND and IBD, we developed a bivalent chimeric virus-like particle vaccine (ND-IBD cVLPs) displaying the ND virus (NDV) HN protein and the IBD virus (IBDV) VP2 protein based on the ND VLPs carrier platform and insect baculovirus expression system. This study aimed to evaluate the immunogenicity and protective efficacy of ND-IBD cVLPs in specific pathogen-free chickens. Chickens were immunized with 50 µg of purified ND-IBD cVLPs at 7 days old, boosted at 21 days old, and challenged at 42 days old. The results demonstrated that ND-IBD cVLPs stimulated highly effective hemagglutination inhibition antibody levels against NDV HN protein and enzyme-linked immunosorbent assay antibody levels against the IBDV VP2 protein. Furthermore, ND-IBD cVLPs provided complete protection against virulent NDV and IBDV challenges and mitigated pathological damage to the lung caused by NDV infection and the bursa of Fabricius caused by IBDV infection. These findings suggest that ND-IBD cVLPs hold promise as a safe and efficient novel vaccine candidate for the effective prevention of ND and IBD, extending the development of a foreign protein delivery platform of ND VLPs.

Virus-like Particle Vaccines of Infectious Bursal Disease Virus Expressed in Escherichia coli Are Highly Immunogenic and Protect against Virulent Strain.

Infectious bursal disease virus (IBDV) is a highly contagious, acutely infectious agent that causes immunosuppression in chickens. We expressed IBDV VP2 proteins in Escherichia coli (E. coli) to develop an effective virus-like-particles (VLPs) vaccine and evaluated its immunogenicity. The VLPs produced in E. coli were used as an immunogen mixed with a water-in-mineral-oil adjuvant (MontanideTM ISA 71 VG, ISA 71 RVG) or a white oil (7#) adjuvant. VLPs without an adjuvant, commercial subunit vaccine, inactivated vaccine, and attenuated vaccine were used as controls. These test vaccines were intramuscularly injected into 19-day-old SPF chickens, which were challenged with the IBDV virulent strain at 30 days after vaccination. The adjuvants boosted antibody production, and the adjuvant groups (except white oil) produced higher antibody levels than the non-adjuvanted controls and the commercial vaccine groups. In terms of cellular immunity, the VLPs plus adjuvant combinations produced higher levels of cytokines, IL-2, IL-4, and IFN-γ than the controls. IBDV VLPs plus the ISA 71 RVG adjuvant can be used as an optimal vaccine combination for improving the immune efficacy of IBD subunit vaccines, which can protect against the virulent strain.

Protein kinase Cdc7 supports viral replication by phosphorylating Avibirnavirus VP3 protein.

The Avibirnavirus infectious bursal disease virus is still an important agent which largely threatens global poultry farming industry economics. VP3 is a multifunctional scaffold structural protein that is involved in virus morphogenesis and the regulation of diverse cellular signaling pathways. However, little is known about the roles of VP3 phosphorylation during the IBDV life cycle. In this study, we determined that IBDV infection induced the upregulation of Cdc7 expression and phosphorylated the VP3 Ser13 site to promote viral replication. Moreover, we confirmed that the negative charge addition of phosphoserine on VP3 at the S13 site was essential for IBDV proliferation. This study provides novel insight into the molecular mechanisms of VP3 phosphorylation-mediated regulation of IBDV replication.

PLGA nanoparticle with Amomum longiligulare polysaccharide 1 increased the immunogenicity of infectious bursal disease virus VP2 protein

ABSTRACT 1. The purpose of this study was to create ALP1-VP2-PLGA nanoparticle (AVPN) and to study the immunogenicity of AVPN. AVPN was prepared and observed by scanning and transmission electron microscopies. 2. Chickens were divided into five groups and vaccinated with normal saline, VP2 protein, ALP1 and VP2 protein, AVPN or PLGA, respectively. After 28 days, the immune organ indexes were calculated; specific antibody levels in blood were detected by enzyme-linked immunosorbent assay (ELISA). Additionally, the spleen and bursa of Fabricius were determined by HE staining, immunological cytokine mRNA levels in bursa of Fabricius were detected by qPCR andchicken body weight was determined. 3. The results indicated that AVPN was a spherical nanoparticle with a diameter of about 85 nm. It increased bursal indexes and IBDV-specific antibody levels and promoted the expression of IL-2 mRNA in blood and TNF-α and IgG mRNA in bursa of Fabricius. This promoted growth. 4. This study suggested that AVPN can increase immunogenicity of VP2 protein, and it could possibly be used as an IBDV subunit vaccine.

Research Note: Application of reverse-transcription recombinase-aided amplification-lateral flow dipstick method in the detection of infectious bursal disease virus

Infectious bursal disease (IBD) is a highly contagious viral disease caused by infectious bursal disease virus (IBDV) in chickens. The consequent immunosuppression and secondary infection affect the healthy development of chicken industry. In this study, specific primers and probes were screened in the conserved region of IBDV VP2 gene sequence, and reverse transcription-recombinase-aided amplification (RT-RAA) was combined with lateral flow dipstick (LFD) for establishing RT-RAA-LFD method for detection of IBDV in chickens. The reaction conditions of RT-RAA-LFD assay were optimized, and the specificity, sensitivity, and repeatability were verified. The results showed that the RT-RAA-LFD method could amplify the IBDV target fragment at 37°C for 15 min, and the required primer and probe concentration was 1,250 nmol/L. The detection results were directly observed by the dipstick, the lowest detectable limit (LDL) for IBDV was 10 copies/μL, and there was no cross reaction with several common immunosuppressive pathogens in poultry. The total coincidence rate of sample test results between RT-RAA-LFD and reverse transcription-polymerase chain reaction (RT-PCR) was 95.83%. Due to advantages of high sensitivity, strong specificity, easy operation, fast detection, the established RT-RAA-LFD method can provide some technical support and new solutions for local laboratory to detect IBDV.

Comparative Safety, Immunogenicity, and Efficacy of CEF Cell-Based and DF-1 Cell Line Adapted Infectious Bursal Disease Vaccines in Specific-Pathogen-Free Chickens.

Infectious bursal disease (IBD) is an immunosuppressive and economically important disease of young chickens caused by infectious bursal disease virus (IBDV). The National Veterinary Institute (Bishoftu, Ethiopia) produces intermediate IBDV vaccine using primary chicken embryo fibroblast (CEF) cells, a method with technical and economical cumbersome. This study assessed the safety, immunogenicity, and efficacy of DF-1 cell line-adapted IBDV LC–75 vaccine strain in reference to the CEF-based vaccine. Confluent monolayer of DF-1 cells was infected with IBDV and cells with cytopathic effects were passaged until 3rd passage. Viral growth was confirmed using a one-step RT-PCR targeting IBDV VP2 gene. Viral titer increased from 1st passage through 3rd passage. Safety was assessed in 30 specific-pathogen-free chickens (15 chickens/group) injected with 10-fold field dose of each vaccine intraocularly and monitored for 21 days. For immunogenicity and efficacy, 60 specific-pathogen-free chickens were grouped into 3 (20 chickens/group). First and 2nd group received DF-1 cell and CEF-based IBDV vaccines, respectively. The 3rd group served as unvaccinated control. Antibody response was measured using iELISA. Chickens were challenged 4 weeks postvaccination with very virulent IBDV (vvIBDV) intraocularly and followed-up for 10 days. Vaccination did not cause any adverse reactions during the 21 days of follow-up. In addition, both vaccines induced higher antibody titer 14 and 24 days-post-vaccination as compared to unvaccinated controls (p < 0.05). Moreover, DF-1 and CEF-based IBDV LC–75 vaccines rendered a complete protection against vvIBDV. Contrarily, morbidity and mortality in unvaccinated chickens was 50% and 30%, respectively. The results indicated that DF-1 and CEF cell-based IBDV vaccines are comparably immunogenic and efficacious. Therefore, DF-1 cell-line can be considered an affordable and convenient alternative to the CEF-based approach. The suitability of DF-1 cells to grow other IBDV strains and safety of these vaccines on bursa of Fabricius should further be investigated.

Construction of a Novel Infectious Clone of Recombinant Herpesvirus of Turkey Fc-126 Expressing VP2 of IBDV.

The increased virulence of infectious bursal disease virus (IBDV) is a threat to the chicken industry. The construction of novel herpesvirus of turkey-vectored (HVT) vaccines expressing VP2 of virulent IBDV may be a promising vaccine candidate for controlling this serious disease in chickens. We generated a novel infectious clone of HVT Fc-126 by inserting mini-F sequences in lieu of the glycoprotein C (gC) gene. Based on this bacterial artificial chromosome (BAC), a VP2 expression cassette containing the pMCMV IE promoter and a VP2 sequence from the virulent IBDV NJ09 strain was inserted into the noncoding area between the UL55 and UL56 genes to generate the HVT vector VP2 recombinant, named HVT-VP2-09. The recovered vectored mutant HVT-VP2-09 exhibited higher titers (p = 0.0202 at 36 h) or similar growth kinetics to the parental virus HVT Fc-126 (p = 0.1181 at 48 h and p = 0.1296 at 64 h). The high reactivation ability and strong expression of VP2 by HVT-VP2-09 in chicken embryo fibroblasts (CEFs) were confirmed by indirect immunofluorescence (IFA) and Western blotting. The AGP antibodies against IBDV were detected beginning at 3 weeks post-inoculation (P.I.) of HVT-VP2-09 in 1-day-old SPF chickens. Seven of ten chickens immunized with HVT-VP2-09 were protected post-challenge (P.C.) with the virulent IBDV NJ09 strain. In contrast, all chickens in the challenge control group showed typical IBD lesions in bursals, and eight of ten died P.C. In this study, we demonstrated that (i) a novel HVT BAC with the whole genome of the Fc-126 strain was obtained with the insertion of mini-F sequences in lieu of the gC gene; (ii) HVT-VP2-09 harboring the VP2 expression cassette from virulent IBDV exhibited in vitro growth properties similar to those of the parental HVT virus in CEF cells; and (iii) HVT-VP2-09 can provide efficient protection against the IBDV NJ09 strain.

Tween 80 improves the infectivity of BCL1 cell-adapted infectious bursal disease virus

Non-ionic surfactants have the ability to alter the cell membrane's permeability for enhancing virus replication. The impact of non-ionic surfactant Tween 80 (TW80) on the infectivity of infectious bursal disease virus (IBDV) was studied in BCL1 cells. The toxicity of different concentrations of TW80 for BCL1 cells was determined for five-time passages. The confluent monolayer of BCL1 was infected by IBDV and subsequently passaged. The adaptation was confirmed by virus titration and RT-PCR assay. Replication kinetics of the cell-adapted IBDV was evaluated in pre-treatment and simultaneous treatment with TW80 at 0.01% concentration. The IBDV infectivity patterns were determined by virus titration, FRAP assay, and transmission electron microscopy. Sequence analysis, RNA secondary structure, and potential N-glycosylation site were conducted for IBDV VP2. Despite the similar cytopathic effects found in both TW80-treated cells and similar ROS levels, the IBDV titer was higher in TW80 pre-treated cells compared to the simultaneous treatment one. Such an increase in IBDV titer did not associate with changes in the VP2 sequence and RNA secondary structure. The possible antioxidant capacity of TW80 can attenuate the ROS damage and improve the cell viability, thereby improving IBDV infectivity.

A novel inactivated bivalent vaccine for chickens against emerging hepatitis-hydropericardium syndrome and infectious bursal disease

The emerging hepatitis-hydropericardium syndrome (HHS) caused by the novel genotype of fowl adenovirus 4 (FAdV-4) and the infectious bursal disease (IBD) caused by the infectious bursal disease virus (IBDV) are important avian diseases, both cause huge economic losses to the poultry industry. Therefore, it is necessary to develop an efficient and convenient FAdV-4/IBDV bivalent vaccine to prevent the spread of FAdV-4 and IBDV infections. Given that VP2 is the main structural protein and protective antigen of IBDV, we constructed a recombinant FAdV-4 expressing IBDV VP2 in our previous study. In the current study, an inactivated bivalent FAdV-4/IBDV vaccine was developed from the recombinant strain. The inactivated bivalent vaccine elicited effective and specific neutralizing antibodies against both FAdV-4 and IBDV in specific-pathogen-free chickens. Furthermore, the novel vaccine not only protected chickens from death caused by FAdV-4 and the very virulent IBDV (vvIBDV) infection, but also ameliorated target organ damage and reduced viral load. The FAdV-4-vectored vaccine developed in this study provides new options for the development of avian polyvalent inactivated vaccines and is a powerful tool for the prevention of both emerging HHS and IBD.

Sequence analysis of VP1, VP2 and VP3 genes of Infectious Bursal Disease Virus from a field outbreak in Kerala, India

Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is one among the top five infectious diseases of poultry that discernibly affects commercial poultry industry. The mutating viral genome of IBDV accounts for disease outbreaks in fields even after following stringent biosecurity measures and vaccination protocols. The present study is focussed on the characterisation of an IBDV field virus, IBD/CVAS/6, from a vaccinated flock in Kerala, based on the sequence analysis of VP1, VP2 and VP3 genes. Bursa of Fabricius samples collected from a 26 days-old chicken flock from a suspected outbreak in the Thiruvananthapuram district of Kerala formed the subject of the study. Reverse transcriptase-polymerase chain reaction (RT-PCR) targeting VP2 gene confirmed the presence of virus in the sample. The sequence analysis revealed that the deduced amino acid sequence of VP1 gene of IBD/CVAS/6 was 100 per cent homologous with an attenuated very virulent vaccine strain of Israel, mb and the VP2 gene was 100 per cent homologous with mb and Ventri IBDV plus vaccine strain of India. The analysis of VP3 gene also revealed the similarity with vaccine strains except for a single variation S745N in its deduced amino acid sequence. The phylogenetic analysis of IBD/CVAS/6 revealed it’s close relation with mb, Ventri IBDV plus and a very virulent strain of Israel, ks. The characteristic virulent marker amino acid motifs ‘SWSASGS’ and ‘TDN’ were present in the VP2 and VP1 genes, respectively. Hence, the study revealed that the obtained virus has emerged from an attenuated very virulent vaccine strain and hence the present study is a report of involvement of the intermediate plus vaccine strain in field outbreaks in Kerala. The role of S745N in virulence cannot be accounted from the present study, however the involvement of IBD/CVAS/6 in the outbreak might be related to S745N variation or variations in other genes of the virus or due to the inefficiency of the vaccine or vaccination protocol followed, which can be defined only after further studies. The present report is the first characterisation study in Kerala focussing on the analysis of VP1, VP2 and VP3 genes of IBDV.

Single-Dose Vaccination of Recombinant Chimeric Newcastle Disease Virus (NDV) LaSota Vaccine Strain Expressing Infectious Bursal Disease Virus (IBDV) VP2 Gene Provides Full Protection against Genotype VII NDV and IBDV Challenge.

Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) are the two most important and widespread viruses causing huge economic losses in the global poultry industry. Outbreaks of genotype VII NDV and IBDV variants in vaccinated poultry flocks call for genetically matched vaccines. In the present study, a genetic matched chimeric NDV LaSota vaccine strain expressing VP2 gene of IBDV variant, rLaS-VIIF/HN-VP2 was generated for the first time, in which both the F and HN genes of LaSota were replaced with those of the genotype VII NDV strain FJSW. The cleavage site of the FJSW strain F protein in the rLaS-VIIF/HN-VP2 genome was mutated to the avirulent motif found in LaSota. Expression of IBDV VP2 protein was confirmed by western blot. The rLaS-VIIF/HN-VP2 maintained the efficient replication ability in embryonated eggs, low pathogenicity and genetic stability comparable to that of parental LaSota virus. One dose oculonasal vaccination of one-week-old SPF chickens with rLaS-VIIF/HN-VP2 induced full protection against genotype VII NDV and IBDV lethal challenge. These results indicate that the rLaS-VIIF/HN-VP2 is a promising bivalent vaccine to prevent infections of IBDV and genotype VII NDV.

Development of a Novel Assay Based on Plant-Produced Infectious Bursal Disease Virus VP3 for the Differentiation of Infected From Vaccinated Animals.

Infectious bursal disease virus is the causative agent of Gumboro disease, a severe infection that affects young chickens and is associated with lymphoid depletion in the bursa of Fabricius. Traditional containment strategies are based either on inactivated or live-attenuated vaccines. These approaches have several limitations such as residual virulence or low efficacy in the presence of maternally derived antibodies (MDA) but, most importantly, the impossibility to detect the occurrence of natural infections in vaccinated flocks. Therefore, the development of novel vaccination strategies allowing the differentiation of infected from vaccinated animals (DIVA) is a priority. Recently, commercial vectored and experimental subunit vaccines based on VP2 have been proved effective in protecting from clinical disease and posed the basis for the development of novel DIVA strategies. In this study, an engineered version of the VP3 protein of IBDV (His-VP3) was produced in plants, successfully purified from Nicotiana benthamiana leaves, and used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-VP3 antibodies. The His-VP3 ELISA was validated with a panel of 180 reference sera and demonstrated to have 100% sensitivity (95% CI: 94.7–100.0) and 94.17% specificity (95% CI: 88.4–97.6). To evaluate the application of His-VP3 ELISA as a DIVA test, the novel assay was used to monitor, in combination with a commercial kit, detecting anti-VP2 antibodies, the immune response of chickens previously immunized with an inactivated IBDV vaccine, a recombinant Turkey herpes virus carrying the VP2 of IBDV (HVT-ND-IBD) or with plant-produced VP2 particles. The combined tests correctly identified the immune status of the vaccinated specific pathogen free white-leghorn chickens. Moreover, the His-VP3 ELISA correctly detected MDA against VP3 in commercial broiler chicks and showed that antibody titers fade with time, consistent with the natural decrease of maternally derived immunity. Finally, the novel assay, in combination with a VP2-specific ELISA, demonstrated its potential application as a DIVA test in chickens inoculated with VP2-based vaccines, being able to detect the seroconversion after challenge with a very virulent IBDV strain.

Six decades of infectious bursal disease in poultry: The journey so far and challenges ahead

&#x0D; &#x0D; &#x0D; Despite six decades of concerted efforts, Infectious bursal disease (IBD) still remains a major threat to the poultry industry worldwide. Most importantly, the emergence of variant and very virulent strains of infectious bursal disease virus (IBDV) has dramatically changed the epidemiology of the disease, thus resulting in the renewed efforts in the search for effective control measures. Currently, live attenuated, inactivated, and immune-complex vaccines are among the immune-therapeutic approaches employed for the control of IBD in the field alongside adequate biosecurity, albeit with various degrees of success and limitations. Progress in genetic engineering has allowed the generation of reverse genetic IBDV mutants, recombinant live viral vectors expressing the IBDV VP2 immunodominant protein, intra-serotypic recombinant IBDV viral-like particle co-expressing the outer capsid protein structures derived from 2 or more serotype 1 strains or the incorporation of either VP2 or VP2-4-3 polyprotein sequences alongside molecular adjuvants that can be used as IBD vaccine candidates to elicit an immune response. However, despite these advances, outbreaks are still reported even in flocks that have up to date vaccination records and somewhat excellent management practices. This paper reviews aspect of genetic characteristics of IBDV and reflects on the progress and future challenges in providing effective IBD vaccine to achieve effective control of both classical and very-virulent IBDV serotypes that constitute a major devastation to poultry production and health.&#x0D; &#x0D; &#x0D;

Characterizations of glucose-rich polysaccharides from Amomum longiligulare T.L. Wu fruits and their effects on immunogenicities of infectious bursal disease virus VP2 protein

The aim of this study is to explore the characterization of Amomum longiligulare T.L. Wu fruits polysaccharide (ALP) and their immune enhancement effects. Two homogeneous polysaccharides (ALP1 and ALP2) were isolated from the fruits. The structural characterization results showed that ALP1 (26.10 kDa) and ALP2 (64.10 kDa) were both mainly composed of glucose. Furthermore, ALP1 was consisted of (1,2)-α-D-Glcp, (1,2,3)-α-D-Glcp and T-α-D-Glcp, while ALP2 was consisted of T-α-D-Glcp, (1,3)-α-D-Glcp and (1,3,6)-α-D-Glcp. Afterwards, the immune enhancement effects of two polysaccharides were evaluated by determining their effects on immunogenicities of infectious bursal disease virus (IBDV) VP2 protein. Chickens were immunized with IBDV VP2 protein accompanied with ALP1/ALP2. And the results indicated both ALP1 and ALP2 promoted the weights and bursa of fabricius indexes of chickens. In addition, both two polysaccharides increased specific IBDV antibody levels, while ALP1 possessed higher immune enhancement ability and was expected to be an adjuvant for IBDV VP2 protein.

Development and evaluation of a recombinant VP2 neutralizing epitope antigen vaccine candidate for infectious bursal disease virus.

Infectious bursal disease (IBD) is one of the most economically important infectious diseases. Currently, vaccination is the most effective method to prevent IBD. Medium-virulence vaccines can damage the bursa of Fabricius and result in immunosuppression. Therefore, it is essential to develop a safe and effective vaccine against infectious bursal disease virus (IBDV). In this study, the five neutralizing epitopes of the IBDV VP2 protein were confirmed by neutralizing single chain variable fragment antibodies. Then, the neutralizing epitopes antigen (NEA) protein was constructed with five neutralizing epitopes and expressed by pET-27b. Furthermore, the immune effect and protective immunity of the NEA protein with the following adjuvants were evaluated in specific-pathogen-free chickens: oil emulsion adjuvant (OEA), double emulsion adjuvant (DEA), granulocyte-macrophage colony-stimulating factor (GM-CSF) adjuvant and complete Freund's adjuvant (CFA). The experimental results demonstrated that chickens immunized with NEA vaccines elicited stronger humoral and/or cellular immune responses and inflammatory responses than those in the NEA protein group. Chickens were protected in OEA, CFA and GM-CSF adjuvant groups, which were challenged with virulent IBDV BC6/85. Furthermore, IBDV RNA was not measured, and there appeared to be little apoptosis in the bursa of Fabricius based on TUNEL histology and the expression of Bax and Bcl-2 in the OEA, CFA and GM-CSF adjuvant groups. Based on the experimental results, the advantages and disadvantages of adjuvants and industrial production methods, GM-CSF was found to be the optimal adjuvant. Therefore, NEA with GM-CSF adjuvant is a promising vaccine candidate against IBDV, and it provides a framework for developing other vaccines against infectious viral diseases.

Recombinant Lactococcus Expressing a Novel Variant of Infectious Bursal Disease Virus VP2 Protein Can Induce Unique Specific Neutralizing Antibodies in Chickens and Provide Complete Protection.

Recent reports of infectious bursal disease virus (IBDV) infections in China, Japan, and North America have indicated the presence of variant, and the current conventional IBDV vaccine cannot completely protect against variant IBDV. In this study, we constructed recombinant Lactococcus lactis (r-L. lactis) expressing a novel variant of IBDV VP2 (avVP2) protein along with the Salmonella resistance to complement killing (RCK) protein, and Western blotting analysis confirmed that r-L. lactis successfully expressed avVP2-RCK fusion protein. We immunized chickens with this vaccine and subsequently challenged them with the very virulent IBDV (vvIBDV) and a novel variant wild IBDV (avIBDV) to evaluate the immune effect of the vaccine. The results show that the r-L. lactis-avVP2-RCK-immunized group exhibited a 100% protection rate when challenged with avIBDV and 100% survival rate to vvIBDV. Furthermore, this immunization resulted in the production of unique neutralizing antibodies that cannot be detected by conventional ELISA. These results indicate that r-L. lactis-avVP2-RCK is a promising candidate vaccine against IBDV infections, which can produce unique neutralizing antibodies that cannot be produced by other vaccines and protect against IBDV infection, especially against the variant strain.

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.