Molecular Characteristics And DNA Vaccine Research Of Avian Infectious Bronchitis Virus Isolated In China

Infectious bronchitis virus(IBV) is a world-wide viral disease of poultry, which poses a major economic threat to the poultry industry. Because of relatively high variation of IBV, new variant strains continue to emerge. The cross-protection between different strain is weak. In spite of routine vaccination, the outbreaks of IB have been reported with high frequency. Based on the initial work, studys were carried out for the molecular characterization and DNA vaccine of avian infectious bronchitis virus isolated, which is important to study the epidemiology of IBV at the molecular level and to develop a DNA vaccine for control the IB.1.19 pair primers were designed according to the complete genome sequences of IBV strains published on GenBank. The genome of SAIB_K strain was amplified by RT-PCR and all the RT-PCR products were cloned into the pMD 18-T vector and sequenced. The SAIB_K strain is embryo dwarfing, and its virus has the typical features of coronavirus when purified by difference speed centrifugation and discontinuous centrifugation on sucrose gradients and observed in the electron microscope. The genome sequence of SAIB_k strain was 27520 bp in length and the ratio of A+T in the genome was 61.74%. The genome of SAIB_k strain has the typical genome structure of coronavirus. The gene of RNA-dependent RNA polymerase which is the region from 528 to 20410 bp encode two polyproteins, namely, ORF1a and ORF1b.The gene which is the region from 20361 to 27155 bp encode main structure proteins of IBV including the spike glycoprotein(S protein), the membrane glycoprotein(M protein) and the nucleocapsid protein(N protein). The homology was between 85.6% and 87.6% compared with the other five published genome sequences of IBV strains in GenBank. There was a notable difference among these IBV strains, which indicated that there was not relationship at the origin between the SAIB_k strain and the other IBV strains and the SAIB_k strain was relatively independent IBV strain. The 3'UTR and 5'UTR were the most conservative region in the genome, the similarity was between 90%-98%, and S1 gene has the most variation, the similarity was between 74.8%-83.2%.2. The 3'UTR adjacent to the nucleocapsid(N) gene of eleven infectious bronchitis virus (IBV) strains, which isolated from natural outbreaks of IB in different types of commercial chicken flocks, were sequenced and analyzed. Comparison of these sequences with other fifteen sequences deposited in Genbank revealed deletions or insertions in the 3'UTR. The size of the PCR products of eleven isolated viruses are different from each other, ranging from 328bp to 512bp. According to the length of sequences, 26 strains were clearly divided into four groups. Phylogenetic analysis based on nucleotide sequences of the 3'UTR showed that the majority field strains isolated from China were clustered into each other. It has indicated these isolated strains have close genetic relationship due to the geographical relationship.3. To study the S1, M and N gene expressions and distributions, we construct these fusion expressing plasmids including pS1-EGFP, pM-EGFP, pN-EGFP, pS1-DsRed, pM-DsRed and pN-DsRed. These recombinant fusion protein expression vectors were verified by restriction enzyme digestion and DNA sequencing. Restriction enzyme digest and DNA sequencing showed that the EGFP fusion expression plasmids (pS1-EGFP, pM-EGFP, pN-EGFP) and DsRed fusion expression plasmids (pS1-DsRed, pM-DsRed, pN-DsRed) were constructed successfully. The sequences of S1, M and N structure protein were analyzed by Prosite and PredictNLS server to identify nuclear localization signals (NLS). The analysis results showed that the S1, M and N structural protein genes have not the nuclear localization signal region. Then these fusion expressiong plasmids were transfected to the Vero cells by using the liposome transfection method, respectively. Vero cells were examined by fluorescence microscope after being transfected with EGFP fusion expression plasmids (pS1-EGFP, pM-EGFP, pN-EGFP) and DsRed fusion expression plasmids (pS1-DsRed, pM-DsRed, pN-DsRed). 48h later, red and green fluorescent were observed by fluorescent microscope in transfected cells. S1 fluorescent fusion protein was distributed in both cytoplasm and nucleus and the fluorescence of nucleus was more intensive than that of cytoplasm. M fluorescent fusion protein was distributed in the cytoplasm. But the distribution of fluorescence was not uniform, which indicated that the M fluorescent fusion protein accumulated. It is associated with the M protein retained in the Golgi complex. N fluorescent fusion protein also accumulated in the cytoplasm or cell nucleus, which leads to the ununiform fluorescent in the cells.4. The S1, M and N gene were amplified from the nephropathogenic IBV strain SAIB_k by PCR and then inserted into the expression plasmid pcDNA3.1(+) to yield the plasmids pIBVS1, pIBVM and pIBVN. These eukaryotic expression recombinant plasmids were verified by restriction enzyme digestion and DNA sequencing. The eukaryotic expression recombinant plasmids including pIBVS1, pIBVM, pIBVN were then transfected to Vero cells by using the liposome transfection method, respectively, and the expressed products were examined by RT-PCR and indirect immunofluorescent assay. Restriction enzyme digest and DNA sequencing showed that the eukaryotic expression plasmids (pIBVS 1, pIBVM, pIBVN) were constructed successfully. The genes encoding S1, M and N structural protein were transcrited and expressed correctly as shown by RT-PCR and indirect immunofluorescent assay. These results might provide basis for the further development of DNA vaccine against IBV.5. Chickens were immunized by individual recombinant plasmid pIBVS1, pIBVM, pIBVN, or their admixture. Specific antibody were induced in all test groups that inoculated with DNA vaccines, the results of challenging chicken with nephropathogenic IBV strain SAIB_k showed that the chickens could obtain the ability against IBV infection. The protection rate of pIBVS1, pIBVM and pIBVN is 50%, 37.5% and 50% respectively, and the protection rate and antibody titer induced by three genes mixture DNA vaccine group showed higher than individual recombinant plasmid. DNA vaccine mixtures expressing three antigens combining with liposome as an adjuvant was injected into health chickens, there was no significant difference of specific antibody in test groups with or without liposome, but the titers of specific antibody in liposome/DNA vaccine group were higher than DNA vaccine group; the number of CD4+ and CD8+ T lymphocyte of liposome/DNA vaccine induced was significant (P＜0.05 or P＜0.01) higher than DNA vaccine group. The protection rate of liposome/DNA vaccine group was 72.7%, higher than DNA vaccine group which was 54.5%.