Evaluation Of Immune Efficiencies Of Recombinant Bovine Herpesvirus-1 Expressing E2 Glycoprotein Of Bovine Viral Diarrhea Virus

Bovine viral diarrhea (BVD) is an infectious disease of cattle and other related animals caused by the bovine viral diarrhea virus (BVDV) with viral diarrhea mucosal disease and reproductive disorders as the main clinical features. Infectious bovine rhinotracheitis (IBR) is an infectious disease of cattle caused by the bovine herpes virus type Ⅰ (BHV-1) with the main clinical features of respiratory disease and reproductive tract disease. Currently, the two infectious diseases are endemic all around the world, causing decreased milk yield, pregnant cow abortion, fetal immune tolerance and vaccine failure, persistent infection, and animal morbidity and mortality, which seriously impact both the global dairy industry and beef cattle industry with a conservative estimate loss of billions of dollars every year. Based on serological investigation of cattle in China, BVDV (including genotype 1 and genotype 2) and BHV-1 have been widely prevalent in China now, So far, a standardized vaccine has not been achieved yet, So the development of a corresponding vaccine is a matter of extreme urgency.Based on an attenuated BHV-1 commercial vaccine used for many years abroad, genes of major immunogen E2 of BVDV genotype 1 and 2 circulating in China were cloned and expected, sequentially recombined with efficient expressions of the target proteins simultaneously to construct a recombinant multivalent active BHV-I vaccine candidate. Upon constructs success, immune efficiencies of this vaccine were verified in vivo through cows clinical trial to pave the way for the later clinical promotion of the vaccine. There are two parts as following:Study I:Based on the successful construction of recombinant BHV-1 virus expressing E2 of BVDV-1(v31B) and recombinant BHV-1 virus expressing both E2 genes of BVDV-1 and BVDV-2(L1F), the two recombinant virus were proliferated in the MDBK cell line. The stable presence of BVDV E2 gene was verified in the genome of the recombinant virus by RT-PCR and the expression of E2 gene in the recombinant virus-infected cells was confirmed by Western blot and IFA.50% tissue culture infective doses (TCID50) were determined for v31B and L1F, which were 1066 TCID50/mLand 1080 TCID50/mL, respectively.Study II:Fourteen 6-8 months old cows negative for both BVDV and BHV-1 antigens and antibodies were screened and randomLy divided into five groups, of which two for the control group and three for each of the other 4 groups. Using neck muscle injection inoculation method, Groups 1 and 2 were inoculated with the recombinant virus v31B at doses of 2mL/cow and 4mL/cow, respectively. Groups 3 and 4 were inoculated with the recombinant virus L1F at doses of 2mL/cow and 4mL/cow, respectively. The control group was injected with 2mL/cow of DMEM. The immunized animals were boosted at the same doses on the fourth week after the first immunization and their clinical symptoms were monitored and rectal temperatures were measured for two consecutive weeks following each immunization. Their white blood cells and lymphocytes were counted at scheduled time points and antibodies against BVDV and BHV-1 were detected, respectively, on a weekly basis. On the 13rd week after the first immunization, two cows from the v31B immunization group were inoculated with virulent BVDV-1(NADL strain) and two cows from L1F immunization group were inoculated with virulent BVDV-1(NADL) and virulent BVDV-2 (XJ-04), respectively. The control group was treated the same as the L1F group. Clinical symptoms were monitored, rectal temperatures were measured, white blood cells were counted, and the existence of BVDV viremia was detected.Experimental results showed that a transient mild stress occurred in the cattle after the first immunization with no clinical symptoms observed, but such phenomenon didn’t appear in the booster immunization; while serum anti-BHV-1 Ab could be detected at the first week, the production of anti-BVDV Ab could be detected at the second week. The level of antibody increased steadily, up to the 5th week,the levels of them will be same to the level of positive contonl serum provided in the ELISA kit (IDEXX) and maintained high till the end of this study(the 12th week after first immunization);Vaccination challenge protection test results showed that the control group appeared clinical symptoms,had fever,reduced the count of WBC,BVDV viremia,the groups treated with vaccine candidates did not present these symptoms, recombinant virus immunization provided good immunological protection for the immunized cattle.