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.

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.

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.

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.

Circulation of Immunosuppressive Viruses and Avian Encephalomyelitis Virus in Backyard Chicken Flocks

The objective of this study was to evaluate the circulation of Chicken Anemia Virus (CAV), Infectious Bursal Disease Virus (IBDV), Avian Reovirus (ARV) and Avian Encephalomyelitis virus (AEV) in properties of backyard chickens and carry out an epidemiological analysis. We evaluated 200 samples of chickens from 19 backyard chicken property. Only one property (P10) did not present serological titers for the diseases evaluated. This property is close to industrial farms as well as the other properties, however, P10 remained a few years without the breeding of chicks and these were the first poultry to be housed on site. This reinforces the importance of the fallow period for poultry production. The prevalence of virus-seroreactive birds was 78% (156/200), 64.5% (129/200), 78% (156/200), 78% (156/200) for CAV, IBDV, ARV and, EA, respectively. All the free-range farms studied are within a radius of 500 meters to 6 Km away from some establishments of industrial poultry. There was a correlation between serological titers for CAV and the frequency of disease in poultry (r = 0.6178). In places where birds are frequently sick, 30.76% reported that the disease occurs in chicks, 30.76% in broilers, 23.07% in broiler chickens and 7.69% in birds of all ages. Birds get sick more often in the summer period. The owners reported that the most common signs of disease were respiratory signs (snoring and nasal discharge) (46.15%), diarrhea (30.76%), and paralysis of wings and/or paws (38.46%). There was a correlation between the presence of untreated water in the property and serological titers for ARV (r = 0.5576). This report draws attention not only to high serological prevalence for the viruses studied but also important epidemiological aspects of backyard chicken diseases that may indirectly influence the industrial production.

Effects of Qishen Ultrafine Powder on Immune Efficacy in Chickens

[Objective] This paper aimed to observe the effects of Qishen ultrafine powder on immune efficacy of infectious bursal disease vaccine in chickens. [ Method] 360 1-day-old chickens were randomly divided into six groups equally, and group I -V were vaccinated with IBD live virus vaccine on the 21 th and 35th day, respectively. From 14th to 21th day, group I - Ⅲ were added with 1%, 0.5% of Qishen ultrafine powder and 2% of Qishan powder in basel diet, respectively, group IV was given Yupingfeng oral liquid and group VI was as a control group without vaccination. Be- fore the expedment （14-day-old chickens as DO ） and 7 （ D7 ）, 14 （ D14 ）, 21 （ D21 ）, 28 （ D26 ）, 35 （ D36 ）, 42 （ D42 ） days after the experiment, the dynamic change of serum IBD specific antibody was determined, respectively. On D7, D21 and D36, the dynamic change of peripheral T lymphocyte proliferation and IL-2 in serum were determined, respectively. The weight change was also observed. [Results] Compared with the immune control group, the immune effects of IBD vaccine of each administration group were significantly improved, and there was no significant difference between each other. [ Condusion] Qishen ultrafine powder could increase immune function of chickens at lower dosage.