Molecular Epidemiology Of Infectious Bursal Disease Virus With Novel Vaccine Development

Infectious bursal disease?IBD?is an acute,highly contagious infectious disease caused by infectious bursal disease virus?IBDV?that harms young chickens.IBD can directly cause death of chickens,and more importantly,early infection of chicks will cause severe long-term immunosuppression,leading to secondary diseases and reduced vaccine response levels.Therefore,the disease has important economic significance for the poultry industry.Currently,vaccination is the main means of controlling the disease.IBDV is classified into attenuated,classical,variant and vv IBDV according to antigenicity and pathogenicity.Due to mutations in the viral genome and recombination between strains,the emergence of new epidemics of IBDV has brought new challenges to IBD control.Therefore,this study conducted a molecular epidemiological study on the IBDV isolates in Guangxi from 2012 to 2015.Then,IBDV strains isolated in China from 1990 to 2017 were selected to analysis the temporal and spatial evolution characteristics.Finally,novel vaccines were developed based on the dominant genotype of IBDV strains.This research mainly includes the following contents:1.Molecular Epidemiology of IBDV in Guangxi during 2012-2015In this study,29 IBDV isolates were isolated from IBD suspected chickens in Guangxi from September 2012 to December 2015.The v VP2 gene and VP1b gene of 29 IBDV isolates were subjected to gene amplification,sequence analysis.The sequences of v VP2 and VP1b genes of 29 isolates showed high similarity??89.6%?nt?,?92.4%?aa??.Among the 29 isolates,there are 27isolates have the higher similarity with the vv IBDV reference strains??94.9%?nt?,?97.5%?aa??comprared with the v VP2 gene sequences.While the 27isolates had lower similarity with non-vv IBDV reference strains?classical strain,attenuated strains,variant strains???94.1%?nt?,?96.8%?aa??comprared with the v VP2 gene sequences.Comparing the VP1b gene sequence,the 29 isolates had similarity with the vv IBDV reference strain(86.8%-98.2%?nt?,93.7%-99.6%?aa?.While the 29 isolates had similarity with non-vv IBDV reference strains?classical strain,attenuated strains,variant strains??89.4%-99.9?nt??95.3%-100%?aa??comprared with the VP1b gene sequences.Based on the v VP2 and VP1b gene sequences,phylogenetic trees were constructed.29isolates could be divided into 4 genotypes:vv-A/Att-B,vv-A/Uniq-B,Classical-A/Uniq-B and Att-A/Uniq-B,of which vv-A/Uniq-B were dominant?75.9%,22/29?.2.Temporal and spatial analysis of IBDV strains in ChinaIn this study,we searched the Gen Bank database,consulted the literature,and obtained the sequence of the v VP2 gene of 29 IBDV isolates in the first part of the study.A data set containing v VP2 genes of 255 IBDV strains from 1990to 2017 was constructed.The data set was subjected to evolutionary analysis using Bayesian method.The results showed that 255 strains of IBDV were divided into three branches based on the v VP2 gene.Cluster 1 contains 217strains,which are vv IBDV strains.Cluster 2 contains 15 strains,which are subdivided into 2 subcluster,one of which is variant strains and the other is classical strains.Cluster 3 contains 23 strains,which are attenuated strains.At the same time,the substitution rate of the IBDV v VP2 gene was analyzed to be7.9001×10^-4substitutions/site/year.Based on the Bayesian skyline plots constructed by the IBDV v VP2 gene sequence data set,the IBDV population experienced a stable?1950-1988?,rapid growth?1989-1992?,moderate growth?1993-2008?,and a rapid decline?2009-2012?,and then smooth?2013-2017?period.3.Ubiquitin mediated IBDV and IBDV-IBV DNA vaccineIn this study,ubiquitin?Ub?was fused with the VP2,VP1 gene of IBDV strain NN1172 and the N,S1 genes of IBV strain GX-YL5 to obtain four recombinantplasmidsp VAX1-VP2,p VAX1-Ub-linker-VP2,p VAX1-Ub-linker-VP2-VP1 and p VAX1-Ub-linker-N-S1-VP2.Recombinant plasmids were identified by PCR,double digestion and sequencing.Four recombinant plasmids were transfected with Vero cells in vitro to detect m RNA and protein expression.RT-PCR analysis showed that the transient transfection of vero cells with the four recombinant plasmids could correctly express the m RNA of the target gene.Indirect immunofluorescence assay?IFA?indicated that the four recombinant plasmids correctly expressed the target protein.Animal experiments showed that the percentage of CD4⁺,CD8⁺T lymphocytes,B lymphocytes,the level of anti-IBDV antibodies in the four recombinant plasmid immunized groups were higher than that in vector control group and the PBS control group.And the level of anti-IBV antibodies in the p VAX1-Ub-linker-N-S1-VP2 immunized group was higher than that in vector control group and the PBS control group.The results of the challenge test showed that the p VAX1-Ub-linker-VP2 immunization group can against the IBDV attack,and the p VAX1-Ub-linker-N-S1-VP2 immunization group can resist the IBDV and IBV attacks.4.Reverse genetic modification and rescue of IBDV B87 vaccine strainIn this study,the IBDV whole genome c DNA clone was obtained by segmental amplification of the genome of IBDV B87 vaccine strain.The eukaryotic expression plasmids of the A and B segments of the B87 vaccine strain were obtained by multi-fragment fusion.The hammerhead ribozyme structure?Ham Rz?and the hepatitis D virus ribozyme structure?Hdv Rz?were introduced at both ends of the genome segment.Subsequently,the molecular tags Eco R V and Pst I were introduced into the genome segment A and B of the B87 vaccine strain,respectively.Then obtained infectious clones p VAX1-m B87A and p VAX1-m B87B.The recombinant plasmids were co-transfected into CEF cells,and rescued virus r B87 was obtained.The r B87was passaged on SPF chicken embryos.The r B87 identified by RT-PCR,IFA and sequencing.The B87 genome segment B was modified by multi-fragment fusion method,and finally the five rescue viruses r B87A-NN1172B,r B87A-NN1172VP1,r B87A-NN1172VP1N,r B87A-NN1172VP1M and r B87A-NN1172VP1C were obtained.All rescue viruses can cause CPE on CEF,and make SPF chicken embryos dead.All rescue viruses were indentified by IFA on CEF.Green fluorescences were detected on all infected cells.The replication kinetics of the parental virus B87,r B87 and r B87A-NN1172VP1C on CEF were similar.The average titers of r B87A-NN1172VP1 on CEF was the highest,followed by r B87A-NN1172VP1M.r B87A-NN1172VP1C has the lowest average titers on CEF.Conclusion:The IBDV strains in Guangxi evolved mainly through genetic mutation and genome reassortment.The vv-A/Uniq-B genotype is the dominant genotype of Guangxi IBDV strains.IBDV population dynamics experienced a smooth-fast growth-smooth growth-rapid decline-flat process.The ubiquitin mediated IBDV DNA vaccine could be against IBDV challenge.The ubiquitin mediated IBDV-IBV DNA vaccine can resist the attack of IBDV and IBV.VP1gene in the B segment of vv IBDV genome can enhance the replication ability of the virus.