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.

Application of monoclonal antibodies against chicken infectious bursal disease virus(IBDV)

Preliminary study has been made to test wether three strains McAb（1B1,5D6, 6D8）are against the same antigen determinant.Througn ELISA additive and competition expeiments,itproved that these three strains are against different antigen determinant.The result of positiveserum antigen component analysis with 2 strains IBDV McAb showed that sample IBD positiveserum had obvious inhibition against combination of 5D6 McAb with corresponding antigen.Theresults of substitution of corresponding component in ELISA inhibition experiment and compari-son of non-IBD serum （SPF chicken serum,ND,MD,IA positive serum）proved that IBD anti-serum was the only one showing inhibition against 5D6 McAb.Comparison with AGP and electro-microscope observation showed that ELISA inhibition experiment was characterised by high-specificity,rapidity and sensitiveness.799 serum samples were tested with ELISA inhibition anddouble immunodiffusion（AGP） experiments.ELISA had gotten 486 positive,with positive rate of60.83%;and AGP 334。

Quantification of Protective Antibody Vaccine-Elicited in Chickens against Infectious Bursal Disease Virus

A study on infectious bursal disease virus(IBDV)on chickens of Cobb-500 strain broiler breed at Thakurgaon district of Bangladesh was performed.The protective antibody was measured on one day old chicks(DOC)and post-vaccinated(PV)flocks up to 75 weeks by indirect enzyme linked-immunosorbent assay(I-ELISA).The assays have included five flocks with vaccination historic against IBDV contained 45,000 birds with age ranging from DOC to 75 weeks,just before culling.Maternally derived antibody(MDA)mean titer(MT)ranged from 3,395 to 5,184.The antibody from serum samples(N=92 per flock)were titer tested by I-ELISA at age 14 d,5,8,23,50 and 75 weeks of each vaccinated flock.Antibody titer level gradually decreased before vaccination.Vaccination done by intermediate plus vaccine resulting titers level was increased and stayed at the same level.The antibody MT at the 14th day was 500,which supported Deventer method.The protective antibody MT was declined at growing,laying,mid laying and last stage of laying groups.So MDA titer was enough in offspring that could protect birds easily.

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.

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.

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.

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.

Develope Monoclonal Antibody against Foot-and-mouth Disease Virus A Type

In order to develop an anti-FMDV A Type monoclonal antibody （mAb）, BABL/c mice were immunized with FMDV A type. Monoclonal antibodies （mAbs） 7B 11 and 8H4 against Foot-and-mouth disease virus （FMDV） serotype A were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with A/AV88. The microneutralization titer of the mAbs 7Bll and 8H4 were 1024 and 512, respectively. Both mAbs contain kappa light chainS, the mAbs were IgG1. In order to define the mAbs binding epitopes, the reactivity of these mAbs against A Type FMDV, were examined using indirect ELISA, the result showed that both mAbs reacted with A Type FMDV. These mAbs may be used for further vaccine studies, diagnostic methods, prophylaxis, etiological and immunological research on FMDV. Characterization of these ncindicated that prepared anti-FMDV A mAbs had no cross-reactivity with Swine Vesicular Disease （SVD） or FMDV O, Asial and C Type antigens. Their titers in abdomen liquor were 1：5×10^6 and 1：2×10^6, respectively. 7B 11 was found to be of subtype IgGb 8H4 was classified as IgG2b subtype. The mAbs prepared in this study, are specific for detection of FMDV serotype A, and is potentially useful for pen-side diagnosis.

Molecular Differentiation of Different Pathogenic Phenotypes of Infectious Bursal Disease Viruses by RT-PCR Combined with Restriction Fragment Length Polymorphism(RFLP) Assay

Accurate differentiation of the pathogenic phenotypes of infectious bursal disease viruses(IBDVs) will instruct effective vaccination programs and improve the study of the molecular epidemiology of IBDVs. In this study, an 833 bp hypervariable nucleotide region was identified in VP2 genes of known IBDVs with different virulences through multiple sequence alignment.Moreover, using NEBcutter software analysis, two restriction enzyme sites, SpeⅠ(generating 531 and 302 bp fragments) and StuⅠ(generating 242 and 591 bp fragments) were found presented in very virulent but not attenuated IBDVs. Moreover, the restriction enzyme site SacⅠ(generating 218 and 615 bp fragments) presented in attenuated IBDVs but not very virulent IBDVs. Therefore,a reverse-transcription(RT)-PCR combined with a restriction fragment length polymorphism(RFLP) assay was developed to differentiate attenuated and very virulent IBDVs. The RT-PCR assay was used to confirm 282 IBDV positive samples from 310 suspicious dead chicken samples. The 60 IBDV positive samples were used to evaluate the assay, followed by confirmation via gene sequencing and histopathological examinations of the bursas of Fabricius from chickens infected by these IBDVs. The results showed that 24 viral strains with SpeⅠand StuⅠsites were very virulent, causing severe pathological damage in the bursas of Fabricius, while36 viral strains with the SacⅠsite were attenuated IBDVs, exhibiting only slight pathological damage. The combined RT-PCR and RFLP assay provided a useful approach for differentiating the pathogenic phenotypes of IBDVs.

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.

MDV-1 VP22 conjugated VP2 enhancing immune response against infectious bursal disease virus by DNA vaccination in mice

VP22 of Marek’s disease virus serotype 1 (MDV-1) could function in protein transduction. In this study, an infectious bursal disease virus VP2 gene was fused to the carboxyl termini of VP22. It showed that the fusion protein did not spread into the bystander cells from the cells transfected with pVP22-VP2, as the VP22 alone could. The VP22 proteins were found to be translocated into all the nuclei in the neighboring COS-1 cells, as analyzed by a fluorescence assay. Although mice were immunized with the recombinant DNAs mixed with polyethylenimine (PEI) at a dose of 1:2, it failed to enhance the antibody response against IBDV VP2, as measured by the indirect ELISA assay, yet the cell mediated immune response was significantly increased. The ratio of CD8+/CD4+ T cells was significantly increased in the immunized group with the fusion genes, compared with the group immunized with VP2 (P<0.05). Our results demonstrated that VP22 indeed enhances the cell-mediated response in the fused VP2 in a mice model system, possibly due to the fact that the IBDV VP2 could be carried into the surrounding cells at a limited level under pressure from MDV VP22.

Mortality rates from a Nigerian isolate of the <i>Infectious Bursa Disease Virus</i>and passive haemagglutination antibody titer that protects chicks against challenge with the virus isolate

To determine passive haemagglutination (PHA) antibody titer that would protect chicks against Nigerian isolates of the Infectious Bursa Disease Virus (IBDV), five groups of chicks aged 30 days which had different antibody titers were challenged with a Nigerian isolate of virulent IBDV. Mortality rates of the different groups were plotted against their respective mean PHA antibody titers. A group with zero antibody titer had a mortality rate of 75% while those with PHA antibody titers of 185.6, 243.2, 256 and 307.2 had mortality rates of 40%, zero, zero and zero respectively. Linear equation generated for a line of best fit of the graph of mortality rates of the chicks on their IBD antibody titers gave antibody titer (X) at which mortality (Y) would be zero as 300. A mortality of 75% and the high antibody level needed to protect chicks suggest that the isolate may be a hypervirulent strain.

Pathogenic Characterization and Full Length Genome Sequence of a Reassortant Infectious Bursal Disease Virus Newly Isolated in Pakistan

Dear Editor,Infectious bursal disease (IBD) is one of the most important diseases of the poultry. The IBD virus (IBDV), a nonenveloped virus belonging to the Birnaviridae family with a genome consisting of two segments of double-stranded RNA (segments A and B), targets B lymphocytes of bursa of Fabricious leading to immunosuppression. In Pakistan,poultry farming is the second biggest industry and IBD is the second biggest disease threating the poultry sector.However.