The Development Of A Novel Vaccine Strain Of Infectious Bursal Disease Virus Using Reverse Genetics

Infectious bursal disease (IBD),mediatied by Infectious bursal disease virus (IBDV), is the causative agent of an acute and highly contagious disease of 3-8 week-old chickens. Since the disease was reported, it had been an important threat for the poultry industry. Before the emergence of variant and very virulent IBDV, IBD had been under well control with the vaccine for a long time. Vaccination is the primary means to prevent vvIBDV, but vvIBDV is pathogenic and not suitable for cell culture. To obtain the traditional live vaccine, vvIBDV were attenuated by passaging blindly in CEF cells or chick embryo, which waste a lot of time and effort. It is high time for us to develop a novel vaccine against vvIBDV and variant strains, especially sudden epidemic infectious bursal disease.In this study, the parental strain HLJ0504 of high mortality and antigenic drift was obtained from strains isolated in the epidemiological survey. The full length genomes of HLJ0504 were amplified and sequenced. The length of segment A and B were 3260bp and 2827bp, respectively. The number of GenBank accession were GQ451330 and GQ451331. With the biological tools, antigenic drift of VP2 of HLJ0504 was detected. Phylogenetic analysis showed that the segment A of HLJ0504 was located on the branch of vvIBDV, while the segment B formed an independent branch between attenuated and vvIBDV strains, indicating that natural reassortment might have occurred between different IBDV stains.To obtain the optimal novel vaccine strain of IBDV, many construction models were tried. First of all, two amino acid sites Q253H and A284T were introduced in the major protective antigen VP2 of HLJ0504 and the recombined virus rHLJ0504HT was rescued, which was adapted to cells, but of strong toxicity. To further reduce its pathogenicity, the recombined rHLJ0504HTâ–³VP5 was rescued with VP5 protein knocked out basing on rHLJ0504HT. The virulence of rHLJ0504HTâ–³VP5 to the SPF chickens had been significantly reduced, but the titer of replication on CEF cells was low.To obtain a novel vaccine strain of IBDV with high replication titer, low pathogenicity, good genetic stability and immunogenicity, the VP2 of Gt strain was exchanged with the corresponding gene of HLJ0504 with two nucleotide sites (Q253H and A284T) introduced in the VP2 gene and the corresponding gene-mosaic viruse rGtHLJVP2 were rescued with IBDV reverse genetics system. The recombinant virus rGtHLJVP2 had the following biological characteristics: 1) good replication characteristics: it had the similar replication kinetic in CEF cells as Gt, and the titer could reach 107.3 TCID50/ml. Besides, rGtHLJVP2 and Gt also had considerable viral loads in the bursa of SPF chickens. 2) non-pathogenic to SPF chickens: the inoculation SPF chickens had good spirit without any symptoms, and bursals without any obvious pathological demages within 14 days after vaccination. 3) good genetic stability: the virus replication titer remained stable, and there was no reversion in the genome and virulence after blindly passage for 20 and 5 generations on CEF cells and SPF chickens, respectively. 4) good immunogenicity: 80% serum were positive at 7d.i., 100% at 10d.i., and had 100% protection against vvIBDV. Currently, there are two types of the live IBDV vaccine on the market, the attenuated IBDV vaccine and the medium or plus medium vaccine. The immune effects were developed compared with Gt and B87 as representatives of these two types of vaccine. The results showed that: 1) the immune effects of rGtHLJVP2 was better than Gt (attenuated live IBDV vaccine) and comparable with B87 (medium virulence IBDV vaccine), and there are no differences between rGtHLJVP2 and B87 in antibody titres and the protective effect, which were better than Gt. 2) It was much more safety of rGtHLJVP2 compared with B87. As Gt group, rGtHLJVP2 caused no pathological damage to SPF chickens. However, B87 vaccine caused irreparable pathological damage to the bursas.The representative vvIBDV HLJ0504 strain with high mortality and antigenic drift was obtained in this study. The VP2 of Gt strain was exchanged with the corresponding gene of HLJ0504 with two nucleotide sites (Q253H and A284T) introduced in the VP2 gene and the corresponding gene-mosaic viruse rGtHLJVP2 were rescued with IBDV reverse genetic system, which has high replication titer, non-pathogenicity to SPF chickens, genetic stability, good immunogenicity and protection against vvIBDV. Moreover, the immune effect of rGtHLJVP2 was better than attenuated live IBDV vaccine on the market and the security was better than medium virulence IBDV vaccine. rGtHLJVP2 was a good candidate vaccine strain, which was efficient, safe, cheap and so on. This study laid a solid foundation for the further development of IBDV novel vaccine and had great significance for the control of sudden epidemic infectious bursal disease.