Sequencing And GE Gene Deletion Strains Construction And Identification Of IBRV

Infectious bovine rhinotracheitis virus (IBRV), also known as bovine herpes virus type I and necrotizing rhinitis virus, can invade a variety of tissues and organs of the cattle. This virus has a wide range of tropism and thus can cause a variety of clinical diseases such as infectious bovine rhinotracheitis, encephalitis, enteritis, vaginitis, and secondary infection of the lungs which can cause death. When infections happen on adult cattle, it would result in a decrease of milk production, abortion and slow growth in beef bovine; Even for the cattle that recovers after infection, it still have the potential of detoxification and could be a threat to susceptible cattle. In recent years, this disease has gradually become epidemics in China, and thus caused great losses to the cattle industry. Therefore, study of IBR is extremely important.Infectious bovine rhinotracheitis virus belongs to linear double-stranded DNA virus. The total length of the DNA is about130Kb, in which a unique area, a short unique region and two inverted repeats exist in order. There are two isomers due to heterogeneous flip of the short unique region. In addition, genetic variation in the inverted repeat sequence appears frequently. The infectious bovine rhinotracheitis virus (IBRV), which isolated and reserved by our laboratory, was named GD0109. This virus is amplified by infected into MDBK cell line. Using the phenol-chloroform extraction method, virus genome was obtained.20meaningful Contigs were found by high-throughput sequencing of the virus genome. Comparing and splicing these with the reference strains Bovine herpesvirus type1.1complete genome (GenBank:AJ004801.1), there were still some gaps existed. According to the Contigs’sequence, we continue to design primers to amplify the gaps afterwards to gain most of the genome sequence. Comparative analysis found that virus isolates are not identical to the published sequence, indicating that it is a new strain of infectious bovine rhinotracheitis virus. This found would lay a foundation for the study of the variability of the virus and the development of new vaccines against different strains basis. The analysis of this study showed that the homology of isolated virus and reference sequence is more than90%, but there are still obvious mutation between them such as inverted repeats, which means that this region prone to mutate. After all it is necessary to do some further research for the isolated infectious bovine rhinotracheitis virus is a new strain.There is still no effective approach for the prevention and control of infectious bovine rhinotracheitis. The current prevention method mainly depends on vaccines, novel vaccines were developed with an alarming rate. The first DNA vaccine in the history is aiming against this disease; and immediately, gene-deleted vaccine which produced by deletion of the IBRV virμLence gene was proposed. In addition, virus as a carrier was combined with the recombinant live vector was made for the recombinant live vector vaccine. In this work, based on the sequence of infectious bovine rhinotracheitis virus (GD0109), we designed specific primers to amplify two of the gE gene homologous arms, and then design primers according to pIRES-EGFP plasmid to obtain CMV-EGFP selectable marker gene by fusion PCR method. After that, these fragments were ligated into pUC19vector respectively to produce a transfer plasmid pUC-gEE. The plasmid was then transfected into the MDBK cells which was infected with isolated virus. Recombinant virus was produced and purified through Homologous Recombination and named rIBRV-A7. In this experiment, the titer reduced to105.6after recombination while the parent strains’titer is106.7;and the measurement of plaque forming units(PFU) size of recombinant virus was compared with parent strain showed that the virμLence of recombinant virus is much lower; according to the growth curve of recombinant viral and genetic stability test confirmed that the recombinant virus less prone to mutation; the recombinant virus and the parent strains were used to cross neutralizing test with the antiserum obtained by mice which immunized with parent strains and recombinant strains respectively, the titer of neutralization test reach27showed that the obtained recombinant viruses have immunogenicity. Taking all these resμLts into consideration, the recombinant virus has genetic stability and immunogenicity, and virμLence decline, which pointed out that the recombinant virus have potential to be developed into a secure virulence gene deleted vaccine. we present comparations of the recombinant and wild viruses in immunogenicity and other characteristics and prove its possibility to be developed into a gene-deleted vaccine.