| Infectious bovine rhinotracheitis (IBR), caused by bovine herpesvirus type 1 (BHV-1), is known as an acute, pyrexic and contagious disease. It's a major pathogen of cattle causing respiratory and genital tract infections such as bovine rhinotracheitis, conjunctivitis, fever, infectious pustular vulvovaginitis, and abortion. Since BHV-1 was discovered, IBR has occurred all over the world, and causes great economic loss to the cattle and milk industry. IBR is ranked in list B diseases by OIE, and the main measure for eradication is to slaughter the infected cattle. In addition to this, Immunization is an effective way to control it. Many live BHV-1 vaccines has been developed such as BHV-1 TK null, BHV-1 gE null and so on.The genome of BHV-1 is a double-strand DNA of about 138 kb, with the G+C content of 72%. It contains two unique areas called the long unique (UL) and the short unique(US), and two reverse repeat units, e.t. IR and TR, flanking the US area. BHV-1 encodes 10 glycoproteins, including glycoprotein E (gE). gE is a non-essential gene for virus growth, but it influences cell-to-cell transmission in MDBK cell monolayer. On the other hand, thymidine kinase (TK) gene, another main virulence determing gene, is also non-essential for virus proliferation. It plays a very important role in the latency infection in the nervous system.The objective of this study is to construct a bovine herpersvirus type 1 live modified vaccine strain with TK gene and gE gene deletion, providing a platform for a live viral vector expressing exogenous genes. Safety and efficacy of the both TK gene and gE gene deleted vaccine strain was carried out. The research results are summarized as follows:1. Construction of BHV-1 TK-/gE-/EGFP+ double gene-deleted mutants1). Construction of BHV-1 TK-/EGFP+ gene-deleted mutantTwo pairs of primers of TK gene was synthesized based on the genomic DNA sequence of BHV-1 (GenBank Accession No. AJ004801). The upstream (1.0kb) and downstream (1.1kb) of TK gene homologous arms were amplified by PCR, sequenced, and were directly cloned into pBluescriptⅡSK(+) vector one by one by restriction enzymes. Then EGFP expression cassette was inserted in between the two homologous arms. The recombinant plasmid was linearized and co-transfect into MDBK cells together with pBICPO and BHV-1 wild-type genomic DNA. By green fluorescence screening and plaque purification, the recombinant BHV-1 TK-/EGFP+ was generated.2). Construction of BHV-1 TK- gene-deleted mutantThe pZF08-21 was linearized and co-transfected into MDBK cells together with pBICPO and BHV-1 TK-/EGFP+ genomic DNA. Through five rounds of flurescence selection and plaque purification, EGFP expression cassette was successfully removed, and BHV-1 TK- gene-deleted virus was generated.3). Construction of BHV-1 TK-/gE-/EGFP+ double gene-deleted mutantTwo pairs of primers of gE gene was synthesized based on the genomic DNA sequence of BHV-1 (GenBank accession No. AJ004801). The upstream (1.1kb) and downstream (1.0kb) of gE gene homologous arms were amplified by PCR, sequenced, and were directly cloned into pcDNA3.1(+) myc-His B vector one by one by restriction enzymes. The EGFP expression cassette was inserted in between the two homologous arms. The transfer plasmid was linearized and co-transfected into MDBK cells together with pBICPO and BHV-1 TK- genomic DNA. Through nine rounds of flurescence selection and plaque purification, BHV-1 TK-/gE-/EGFP+ double gene-deleted mutant was generated.2. The safety and efficacy of BHV-1 TK-/gE-/EGFP+ double gene-deleted vaccine strain in cattle1)The safety of BHV-1 TK-/gE-/EGFP+ double gene-deleted mutantThe safety experiment was divided into three parts.They were inoculation experiment, cohabitation experiment and reactivation experiment. Three different immune dose of vaccine strain (4×105PFU,4×106PFU,4×107PFU) were infected intranasally in 10 cattle. In addition, unvaccinated cattle was designated as cohabitation test in the inoculation group. Body temperature was mesured daily, serum samples and swabs were collected. Then, dexamethasone were injected for three days to reactivate the latent virus.The safe experiment shows that the TK-/gE-/EGFP+ gene-deleted mutant is quite safe for cattle.2)The efficacy of BHV-1 TK-/gE-/EGFP+ double gene-deleted mutantVaccination group and negative control group were classified in this experiment. Three different immune dose of vaccine strain (4×105PFU,4×106PFU,4×107PFU) were used to infect 9 cattle of two month old in the vaccination group, while DMEM were used in the negative control group (6 cattle). Four weeks post inoculation, both vaccination group and the control group were challenged with 4×107PFU BHV-1 wild-type virus intranasally. Body temperature was mesured daily, serum samples and swabs were collected on 1d,2d, 3d,5d,7d,14d,21d and 28d post challenging. The efficacy experiment shows that the BHV-1 TK-/gE- double gene-deleted virus could provide full immunoprotectivity to cattle.
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