Attenuation And Molecular Mechanism Of Infectious Bursal Disease Virus Field Strains

Infectious bursal disease (IBD) is an acute and highly contagious chicken and turkey infectious disease caused by infectious bursal disease virus (IBDV). The disease mainly damages young chickens in 3-6 weeks. In addition to causing chickens'death and production decrease, the disease could lead to serious immunosuppression. IBDV belongs to the genus Avibirnavirus in the family Birnaviridae. It is a small, non-enveloped virus, double-stranded RNA (dsRNA) virus. The virion has a single capsid shell of icosahedral symmetry. The genome of IBDV consists of two segments of dsRNA, designated A and B. The larger A segment contains two open reading frames (ORFs) which encode VP2, VP3, VP4 and VP5. The smaller genome B segment encodes VP1. VP2 and VP3 are major structural proteins of the viral capsid, forming the protective antigen of the virus. VP2 carries highly conformational epitopes responsible for the induction of neutralizing antibodies that confer protective immunity. Because the virus'character is highly related to the variation of amino acid sequences of VP2, the hypervariable region become the major object to study the antigenic and pathotypic variation. Since the late 1980s, variant IBDV and very virulent infectious bursal disease (vvIBDV) have emerged. Classical IBDV, variant IBDV and vvIBDV coexisted in China, which makes it very difficult to vaccination. Only if the pathogenicity and the molecular biological character are decided, can the vaccination be successful. The interference of maternally derived antibody (MDA) is another crucial problem in the establishment of the vaccination schedule. Serological monitoring is usually necessary to determine the optimal timing for vaccination. VvIBDV has broken out in many regions of China. Whether vvIBDV can be attenuated to make a vaccine which is apathogenic and non-immunosuppressive and can induce effective immune response is a huge challenge to all researchers on avian disease. The study on the molecular mechanism of attenuation of vvIBDV, especially the study of variety of amino acid sequence of VP2 hypervariable region, will promote study on viral morphogenesis, virulence markers, effective identification of vaccine strains and field strains and accelerating the attenuation of field strains. Based on the prevalence condition of IBDV in Shandong province, the following studies have been carried out: 1. A systemic analysis of the epidemiology of IBDV in Shandong province. Some field strains of IBDV were isolated from many regions in Shandong province where IBDV broke out within two years. According to the epidemiology and clinic symptom of infected chickens, virus isolation, results of tissue pathology and electron microscope analysis, AGID and IBDV detection with differential antibody, some chickens had been identified to be infected by IBDV. The test of pathogenicity on 3-6-week-old SPF chickens indicated that all the four isolated field strains had high pathogenicity and they could be called vvIBDV. The sequences analysis of VP2 hypervariable region showed that all the field strains had the known molecule character of vvIBDV, which caused the serious epidemic situation in recent years in Shandong province. 2. MTT colorimetry was used for detecting IBDV propagated in cell culture. It's the first time that MTT colorimetric method was applied to IBDV isolating. It showed that the method was practicable for detecting IBDV propagated in CEF. Two different fragments were added into materials to compare the effects on IBDV propagation. The results indicated that OD value between bursa materials and bursa lixiviums was significantly different (P<0.01). The mean OD value of bursa lixiviums was higher than that of bursa materials, which showed that the number of live cells decreased and IBDV propagated in CEF. When the OD value is much higher than control, it indicates that IBDV is propagating. The results showed that the method was simple and rapid, practicable for detecting IBDV passaged in CEF. It could also avoid unnecessary blind passages in cell culture. 3. The isolated vvIBDV was attenuated successfully. Through several passages in SPF chickens embryo and CEF, vvIBDV becomes apathogenic and non-immunosuppressive and can induce effectual immune response without virulence reversion. Sequence analysis showed that VP2 hypervariable region changed regularly in the course of adaptation to tissue culture. The amino acid residue 284 mutated from A to T when IBDV began to cause CPE. When the amino acid residue 253 mutated from Q to H, IBDV could adapt CEF better. Both residues may have close connection with the adaptation to tissue culture, the virulence and the pathotype of IBDV. 4. The immune procedure to IBDV was studied. Commercial young chickens were divided into several immune groups according to different immune doses and times. The chickens were finally challenged by vvIBDV GX 8/99 strain. The results showed that both the 21st and 25th cellular viruses offered good protection to chickens challenged with vvIBDV when the inoculation was carried with fit doses in 14-day-old chickens and didn't cause tissue damage to major immune organ and immunosuppression to immune response of NDV vaccine. When the chickens were inoculated within 7 days with the same doses, thecellular viruses caused tissue damage to major immune organs. Therefore, inoculation with chickens younger than 7days is not advocated.