Characterisation and separation of infectious bursal disease virus-like particles using aqueous two-phase systems

Infectious bursal disease(IBD)causes considerable economic losses in the commercial poultry industry worldwide.The principal way to control IBD virus(IBDV),the causative agent of IBD,is still through vaccination programs.Virus-like particles(VLPs)are recognised as a safe and potent recombinant vaccine platform.This research work explores the characterisation and separation of infectious bursal disease virus-like particles(IBD-VLPs)from crude feedstock.Various characteristics were studied with highperformance size-exclusion chromatography(HP-SEC),sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS-PAGE)and transmission electron microscopy(TEM)analyses.Subsequently,the separation of IBD-VLPs using polyethylene glycol(PEG)/sodium citrate-based aqueous two-phase systems(ATPSs)was conducted and optimised.Moreover,a scale-up study of the best ATPS constituted of 15%PEG 6000,11%sodium citrate and 10%crude feedstock was performed to compare the separation performance of IBD-VLPs with and without centrifugation-assisted.The results indicated that the optimised ATPS with centrifugation-assisted for both 5 g and 50 g systems showed good recovery of IBDVLPs of>97%in the interphase between the PEG-rich top and salt-rich bottom phases.These optimised systems also showed high removal efficiencies of impurities of>95%.The results demonstrated that aqueous two-phase extraction could be a promising technology for efficient VLPs separation.

Genomic Sequencing and Molecular Characteristics of A Very Virulent Strain of Infectious Bursal Disease Virus Isolated in China

[Objective] The paper was to determine the genomic sequence of a very virulent strain of infectious bursal disease virus（IBDV）,and study its molecular characteristics.[Method] A very virulent strain（vvIBDV）（HLJ-0504） of infectious bursal disease virus（IBDV） with special characters was isolated in China and its genome was sequenced.[Result] Sequence analysis showed that segment A of HLJ-0504 was derived from vvIBDV,while segment B was from a distinct ancestor.The morbidity and mortality of HLJ-0504 was 100% and 86.7%to SPF chickens,respectively.[Conclusion] vvIBDV with distinct segment B were still circulating and the evolution of IBDV was diversified in China.Besides,it is hard to imagine that the virulence of IBDV is determined solely by segment A or B.

Genetic Analysis of the VP2 Hypervariable Region of Thirty-six Infectious Bursal Disease Virus Isolates in China during 2009-2012

[Objective] Infectious bursal disease （IBD） is a highly contagious immuno- suppressive disease caused by infectious bursal disease virus （IBDV）. IBDV is ge- netically prone to mutation, which results in challenges to the disease prevention and control. Thus, it is necessary to continuously monitor the prevalence of IBDV. [Method] 36 IBDVs were identified from ten provinces in China from 2009 to 2012. Partial fragments of VP2, including the hypervariable region （HVR）, from new iso- lates were sequenced and analyzed through comparisons with published sequences of IBDV, including 18 strains isolated previously by our lab and 24 reference strains from China and around the world. [Result] Phylogenetic analysis showed a co-exis- tence of IBDV strains belonging to classic, variant, attenuated, and very virulent IB- DV （wlBDV） in China. wlBDVs remain the predominant strains in China and the new subgroup was emerging. Alignment analysis revealed several distinct amino acid mutations that might be involved in virulence or antigenicity variation. [Conclu- sion] The results offered evolutionary clues showing the emerging trend of obvious variations and diversity of IBDV in major poultry-producing regions of China particu- larly in recent years. These findings will contribute to a better understanding of the genetic evolution of IBDV.

Comparative Studies on Detection of Antibodies against Infectious Bursal Disease Virus with Test Strips and Agar Gel Immunodiffusion Method

[Objective] This study aimed to compare the detection results of antibodies against infectious bursal disease virus with test strips and agar gel immunodiffusion method. [Method] Antibodies against infectious bursal disease virus in chicken serum were detected using test strips developed in our laboratory, and the results were comparad^with that using traditional agar diffusion method. [Result] The comparative study of the two methods showed that the sensitivity of test strips was eight times over agar gel immunodiffusion; test strips showed higher detection rate in the deter- mination test of 216 clinical samples, with high specificity, easy preservation, and simple and rapid operation, thereby being more suitable for the monitoring of clinical antibodies. [Conclusion] Test strips could replace the existing serological methods, having great promotion and application value in antibody monitoring.

Effect of Astragalus polysaccharides on Erythrocyte Immune Adherence of Chickens Inoculated with Infectious Bursal Disease Virus

Two hundred and forty specific pathogen free leghorn chickens were randomly divided into four groups and reared in isolated pens. The tested chickens were negative to infectious bursal disease virus （IBDV） at 25 d old. Group 1 was treated with saline, whereas Groups 2, 3, and 4 were inoculated with 0.3 mL IBDV suspension intranasally the next day. Groups 3 and 4 were also administered with Astragalus polysaccharides （APS） intramuscularly twice daily at 5 or 10 mg kg-1 BW, respectively, until 31 d old. The erythrocyte-C3b receptor rosette rate （E-C3bRR） and the erythrocyte-C3b immune complex rosette rate （E-ICRR） were measured at 25, 29, 32, 35, and 38 d old. The results showed that IBDV significantly reduced E-C3bRR and E-ICRR when compared with the control group （P 〈 0.05）, while simultaneous administration of APS with 1BDV maintained E-C3bRR at similar levels to the control group （P 〉 0.05） and increased E-ICRR when compared with the control group and the group non-treated with APS （P 〈 0.05）. APS treatment reduced the morbidity and mortality of chickens inoculated with IBDV （P 〈 0.05）. The results suggest that APS may enhance the immune adherence of chickens erythrocytes by affecting the activity and/or the number of complement receptors on the erythrocyte membrane. These findings can be beneficial in providing an understanding of the basic mechanisms required for the rational application of APS in modern medicine.

Pathogenic Antigenic and Molecular Characterization of the Very Virulent Strain(Gx) of Infectious Bursal Disease Virus Isolated in China

The very virulent infectious bursal disease virus (vvIBDV) strain Gx was isolated from a poutl-try farm in Guangxi Province, China, during 1996. The mortality in the infected flock was 80% and occurred 5 days after immunization with serotype I IBD vaccine. The results of antigen-capture ELISA (AC-ELISA), pathogenicity testing, cloning and sequence analysis of the VP2 gene showed that the deduced amino acid sequence of strain Gx VP2 was the same as vvIBDV UK661, which is considered as a reference strain for European vvIBDVs. The antigenicity of the Gx strain was the same as an European vvIBDV strain 849. The EID50 of Gx virus was 10-8.25/0. 2 ml, and the mortality was 64% when 4 week-old SPF chickens were challenged at dosage of 2×10~3EID50. We have demonstrated that the IBDV strain Gx isolated in China is vvIBDV according to European standards.

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 A1 B1,A2 B1,A3 B2,and A8 B1 genotypes of the proposed classification scheme.The novel variant IBDVs including the strains identified in this study were classified as belonging to genotype A2 d B1.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.

Immunogenicity of formaldehyde and binary ethylenimine inactivated infectious bursal disease virus in broiler chicks

Infectious bursal disease virus (IBDV) was inactivated by two different chemicals—formaldehyde and binary ethylenimine (BEI). Formaldehyde was used at 0.1% and 0.2%, while BEI was used at concentrations of 0.001 and 0.002 mol/L. These four vaccines were tested for their efficiency in generating humoral immune response in different groups of broiler chicks. Both BEI-inactivated vaccines gave relatively higher antibody titers and were almost twice as efficient as formaldehyde-inactivated ones.

Inactivation of infectious bursal disease virus by binary ethylenimine and formalin

In this experiment conducted to study the inactivation dynamics of infectious bursal disease virus （IBDV） by binary ethylenimine （BEI） in comparison with formalin, IBDV was isolated from the bursa of infected chickens and its confirmation was done by agar gel precipitation test. Viral suspensions were subjected to inactivation with BEI and formalin for pre-set time in- tervals. BEI was employed at concentrations of 0.001 and 0.002 mol/L while formalin was used at 0.1% and 0.2%. Sampling was done at 6, 12, 24, 36 and 48 h of incubation and samples were tested for their inactivation status in 9-day-old embryonated eggs and 3-week-old broiler chickens. IBDV was completely inactivated by 0.001 and 0.002 mol/L BEI after 36 h of incubation at 37℃, whereas formalin at 0. 1% and 0.2% concentrations inactivated IBDV in 24 h.

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 poulty industry,the nature of predominant strains of IBDV in Pakistan remained l-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 segmentA from vvIBDV and segment B from one unique ancestor,were identifed 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.

Analysis of the function of D279N mutation of VP2 of infectious bursal disease virus

Infectious bursal disease virus（IBDV）is responsible for the highly contagious infectious bursal disease of chickens.Further understanding the gene-function is necessary to design the tailored vaccine.The amino acid residue 279,located on strand P_F of VP2,is one of the three residues that have been reported to be involved in cell-tropism but with some inconsistency.In this study,to further clarify the amino acids involved in the cell tropism of IBDV,a series of mutations about residue 279were introduced into the VP2 of vv IBDV Gx strain.With the reverse genetic system,we found single mutation of D279N,double mutations of D279N/A284T or Q253H/D279N were not enough to adapt IBDV to chicken embryo fibroblast（CEF）cell.To evaluate whether residue 279 could influence the replication and virulence of IBDV,the virus r Gx HT-279 with three mutations（Q253H/D279N/A284T）was rescued and evaluated.Results showed that the mutation of residue 279 in VP2had no efficient effects on both the replication efficiency in vitro and the virulence to SPF chickens of IBDV.In summary,the results demonstrated that residue 279 of VP2 did not contribute efficiently to cell tropism,replication efficiency,and virulence of IBDV at least in some strains.These findings provided further information for understanding the gene function of IBDV.

Study on Propagation of Chicken Infectious Bursal Disease Virus on Vero Cells Using Microcarriers in Fermentor

It was in flask optimization tests proved that 2% serum, pH 7.0, 5:10 000 inoculation concentration of infectious bursal disease virus (IBDV) and 108 hours cultivation for IBDV harvest after its inoculation were the optimal conditions when IBDV was propagated on Vero cells. 250 ml self-made spinner bottle and 5 L stirring fermentor tests proved that IBDV could maintain higher liters for a long time and the highest liters of IBDV in a spinner bottle and a fermentor were 8.875 and 8.58 ( - lgTCID50/0.1 ml) respectively when IBDV was proliferated on Vero cells using 2 g/L microcarriers in a spinner bottle and a fermentor and was cultivated under the optimum conditions obtained from flask tests after Vero cells had developed a confluent monolayer on microcarriers, which were at least one titer higher than the highest titer in the traditional rolling bottle. All these results suggested that this technology could be applied to large scale production for IBDV.

Comparison on Infectious Bursal Disease Monoclonal Antibodies Prepared with Two Different Immunogens

[ Objective] To compare the characteristics of monoclonal antibodies against infectious bursal disease virus （IBDV） prepared with two different immunogens, VP2 protein expressed by prokaryotic system and purified IBDV. [Methed] IBDV VP2 gene was amplified by RT-PCR and expressed in a prokaryotic system. The recombinant protein was purified by affinity chromatography. IBDV was pudfied by ultracentrifugation. Balb/c mice were immunized with the purified recombinant protein and IBDV, respectively. The monoclonal antibodies were screened by ELISA. [ Result] Two cell lines secreting antibodies against IBDV VP2 protein were obtained, and their ELISA titers were 1：2 × 10^4. Four cell lines secreting antibodies against I BDV were produced, and their ELISA titers were 1：2× 10^6, 1：6 × 10^4, 1：1× 10^5 and 1：4 × 10^3, respectively. All monoclonal antibodies specifically bound to their own immunogen but did not react with other viruses or proteins. After 10 -20 passages, these cell lines still secreted antibodies stably. [Condusion] The monoclonal antibodies prepared with the recombinant IBDV VP2 protein or purified IBDV can induce immune resoonse in mice. and VP2 soecific monoclonal antibodies can be obtained with VP2 orotein expressed in the Drokarvotic system as immunoQen.

Titration Method Validation of Live Vaccines against Infectious Bursal Disease

Validating a method of analysis goes through different steps, which aims at testing the normality of measurements distribution, estimating the uncertainty of the components of a measurement （i.e., accuracy and correctness）, and finally, define the control tests of non degradation of the method performances. This paper outlines the steps for validating a biological method of analysis. It involves the construction of an experimental design, a statistical model, and the preparation of an interne laboratory reference material （pilot vaccine）. The latter is used to study the impact of deviation and variation factors, in order to, optimize the analytical method, to evaluate the bias （random error）, and to calculate the uncertainty of measurement, and make the control charts. This method is applied in the titration of live viral vaccines of Gumboro disease on chicken＇s embryos fibroblasts. The experimental results show that potential influence factors related to the titration method had no significant influence on the obtained results. Taking into account these results, an operating mode has been elaborated. The finalized method proved to be faithful to standard deviation of repeatability and reproducibility of 0.21 and 0.22, respectively, with a confidence level of 95%. The calculated uncertainty of measurement is equal to 0.2, which represents the average error level of a titer. A homogeneous stock of interne laboratory reference vaccine （MRIL）, with an average titer of 5.9 log DIT 50, was produced and the control chart set in away to provide the laboratory with an important tool of control and monitoring of the viral titers evolution in time, as well as, the mastery of the validated titration method performances.

Molecular characteristics and evolutionary analysis of a very virulent infectious bursal disease virus

Infectious bursal disease virus （IBDV） poses a significant threat to the poultry industry. Viral protein 2 （VP2）, the major struc- tural protein of IBDV, has been subjected to frequent mutations that have imparted tremendous genetic diversity to the virus. To determine how amino acid mutations may affect the virulence of IBDV, we built a structural model of VP2 of a very virulent strain of IBDV identified in China, vvIBDV Gx, and performed a molecular dynamics simulation of the interaction between virulence sites. The study showed that the amino acid substitutions that distinguish vvlBDV from attenuated IBDV （H253Q and T284A） favor a hydrophobic and flexible conformation of β-barrel loops in VP2, which could promote interac- tions between the virus and potential IBDV-specific receptors. Population sequence analysis revealed that the IBDV strains prevalent in East Asia show a significant signal of positive selection at virulence sites 253 and 284. In addition, a signal of co-evolution between sites 253 and 284 was identified. These results suggest that changes in the virulence of IBDV may result from both the interaction and the co-evolution of multiple amino acid substitutions at virulence sites.

A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus

To test whether amino acid mutations in the PBC and PHI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus(IBDV), a pair of viruses, namely the moderately virulent IBDV(rG x-F9VP2) and the attenuated strain(rGt), were used. Residue mutations A222P(P_(BC)) and S330R(PHI), selected by sequence comparison, were introduced individually into r Gx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222 A or R330 S was introduced into r Gt. The four modified viruses were then rescued and evaluated in vitro(CEF cells) and in vivo(SPF chickens). Results showed that A222 P elevated the replication efficiency of rG x-F9VP2 while P222 A reduced that of rG t in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in PBC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.

Cryo-EM structures of infectious bursal disease viruses with different virulences provide insights into their assembly and invasion

Infectious bursal disease virus(IBDV)causes a highly contagious immunosuppressive disease in chickens,resulting in significant economic losses.The very virulent IBDV strain(vvIBDV)causes high mortality and cannot adapt to cell culture.In contrast,attenuated strains of IBDV are nonpathogenic to chickens and can replicate in cell culture.Although the crystal structure of T=1 subviral particles(SVP)has been reported,the structures of intact IBDV virions with different virulences remain elusive.Here,we determined the cryo-electron microscopy(cryo-EM)structures of the vvIBDV Gx strain and its attenuated IBDV strain Gt at resolutions of 3.3 Å and 3.2 Å,respectively.Compared with the structure of T=1 SVP,IBDV contains several conserved structural elements unique to the T=13 virion.Notably,the Nterminus of VP2,which is disordered in the SVP,interacts with the S_(F) strand of VP2 from its neighboring trimer,completing theβ-sheet of the S domain.This interaction helps to form a contact network by tethering the adjacent VP2 trimers and contributes to the assembly and stability of the IBDV virion.Structural comparison of the Gx and Gt strains indicates that H253 and T284 in the VP2 P domain of Gt,in contrast to Gx,form a hydrogen bond with a positively charged surface.This suggests that the combined mutations Q253 H/A284 T and the associated structural electrostatic features of the attenuated Gt strain may contribute to adaptation to cell culture.Furthermore,a negatively charged groove in VP2,containing an integrin binding IDA motif that is critical for virus attachment,was speculated to play a functional role in the entry of IBDV.

Oral administration of Allium sativum extract protects against infectious bursal disease in chickens

Garlic(Allium sativum,Liliaceae)has been safely used for more than 5000 years,and research on garlic extract is rapidly increasing because of its multiple biological functions.The in vivo effects of oral administration of garlic mixture(GM,water-soluble extract)on infectious bursal disease virus(IBDV)-infected specific pathogen free male white leghorn chicken were examined through histopathological,immunohistochemical,and Western blot analyses,and enzyme-linked immunosorbent assay.The results confirmed the protective effects of oral administration of 5 mg·kg^(–1) BW GM(Group GM1)on bursal lesions after IBDV infection.In particular,protein expression of IBDV in the bursa decreased in Group GM1,indicating that GM administration decreased IBDV replication in the bursa.Furthermore,immunoglobulin M-and A-bearing B lymphocytes significantly increased 7 days post infection in bursae in Group GM1(P<0.01),suggesting that the oral administration of 5 mg·kg^(–1) GM offers moderate protection against B cell destruction after IBDV infection.During infection,the concentration of bursal interferon gamma(IFN-g)increased and peaked in Group GM1 earlier than in Group T(IBDV-exposed),demonstrating that GM administration prompted the production of IFN-g to protect against IBDV infection.

Effect of Chicken Akirin2 Gene on Immune Response Induced by VP2 DNA Vaccine of Infectious Bursal Dis-ease Virus

[ Abstracts ] In order to investigate the effect of chicken Akirin2 gene on the immune response induced by VP2 DNA vaccine of infectious bursal disease virus （IBDV）. [ Methods] The 14-day-old SPF chickens were immunized with recombinant plasmids expressing VP2 protein and Akirin2 protein, and strength- ened immunization was conducted at the 14＇~ day after the first immunization. Finally, test chickens were challenged with IBDVBC6-85 virulent strain. [ Resultss ] Test results showed that Akirin2 gene could enhance the specific immune response induced by VP2 DNA vaccine, improve the proliferation of peripheral blood lym- phocytes and ＇affect the expressing of cytokines TNF-a, IFN-Y, IL-1β, IL-2, IL-4, IL 6, IL-9, IL-10, IL-17 and IL-18. Effects of recombinant plasmids co-ex- pressing Akirin2 protein and VP2 protein on cytokine expression showed some differences with the recombinant plasmids expressing Akirir/2 protein or VP2 protein along. [ Conclusions] Chicken Akirin2 gene could significantly enhance the humoral immune response and cellular immune response induced by VP2 DNA vaccine of IBDV.

Immune Effect of Combination of Universal Molecular Adjuvant and IBDV Subunit

In order to evaluate the immune effect of the protein expressed by the universal vector pET-mLTA-CTLA-4 plus IBDV subunit, the fusion protein mLTA-CTLA-4 was expressed and purified. Protein toxicity tests were carried out on rabbits.The VP2 gene of infectious bursal virus was amplified by RT-PCR, and lately used for pET-VP2 construction. Ten-day-old free healthy chickens were chosen for a grouped test, including the mLTA-CTLA-4（at different doses） plus VP2 groups, IBDV living vaccine group and control group. Serum and mucosal samples were collected regularly and the neutralization titers of IgG and IgA were assayed, while an animal protection test was conducted to determine the protection rate. The results showed that the protein m LTA-CTLA-4 was non-toxic and its protection rate was100%. IgG or IgA levels in the IBDV vaccine group were slightly higher than those in recombinant protein groups. These results indicated that the recombinant protein mLTA-CTLA-4 could be applied with IBDV subunit vaccine to protect chickens from infection.