| At present,the control of Bovine respiratory disease com-plex is mainly based on the prevention of bovine herpes virus type I(BoHV-1)and bovine viral diarrhea virus(BVDV)infections.Currently,vaccination is the main effective measure to prevent BoHV-1 and BVDV.BoHV-1 live attenuated vaccine,BoHV-1/BVDV bivalent live attenuated vaccine and BoHV-1,BVDV-1 and BVDV-2 trivalent live attenuated vaccine are the mainstream vaccines today.This study aimed to use BoHV-1 as a vector to develop an effective BoHV-1/BVDV-1 bivalent genetically engineered live vector vaccine as a control method for BoHV-1 and BVDV-1,and provide theoretical basis for the prevention and control of BRDC.The main research contents and results are divided into the following five parts:(1)Rapid construction of BoHV-1 gE/EGFP~+,BoHV-1(?)gE recombinant virus using CRISPR/Cas9 gene editing systemUsing CRISPR/Cas9 and homologous recombination technology,3 targeted BoHV-1gE donor plasmids and CRISPR/Cas9 system were co-transfected into Vero E6 cells for targeted gene editing,and the construction of green fluorescent protein(EGFP)-labeled Recombinant BoHV-1,which named BoHV-1 gE/EGFP~+.The sequencing results showed that insertion,deletion and inversion of the 5’and 3’ends of the sg RNA cleavage site occurred at the sg RNA nick position in terms of gene structure.When using CRISPR/Cas9and homologous recombination at the same time to delete the gE,the non-coding region between the gE and the US9 and part of the US9,the US4,US6 and part of US7 sequences will be inserted into the EGFP sg RNA nick position,resulting the loss of EGFP 3’end sequence.However,this did not affect the morphological structure of the virus,and these mutant viruses induced neutralizing antibody similar to the parental virus in mice.In terms of mediated recombination efficiency,the double sg RNA px459-gE-sg RNA1 and px459-gE-sg RNA2 was 0.5%,and the double sg RNA px459-EGFP-sg RNA1 and px459-EGFP-sg RNA2 was 3%.In terms of shearing efficiency,the shearing efficiency of double px459-EGFP-sg RNA1 and px459-EGFP-sg RNA2 is as high as 9%,and the shearing efficiency of single px459-EGFP-sg RNA1 is as high as 5.5%.(2)Construction and identification of BoHV-1 gE/E2-Linker-E2~+recombinant virusThe donor plasmid containing the signal peptide sequence of the BoHV-1 gD gene and the codon-optimized BVDV E2-linker-E2 and the shearing vector were co-transfected into Vero E6 cells and then infected with BoHV-1 gE/EGFP~+.The recombinant virus BoHV-1 gE/E2-linker-E2~+was obtained by CRISPR/Cas9 and homologous recombination technology.PCR and sequencing results showed that the E2-linker-E2 gene was successfully inserted into the recombinant virus gE gene.Using indirect immunofluorescence assay(IFA)and Western-blot to verify the expression of the E2 gene in inoculated cells.The expression of E2-linker-E2 could be detected vaguely at 24 h after inoculation,and the expression of E2 protein increased correspondingly with time,reaching a peak at 60 h,and the protein expression level of E2-linker-E2 was consistent with the m RNA level.The result showed that BVDV E2 protein was successfully expressed.The results of biological characterization showed that the growth curve of the recombinant virus was similar to that of the parental virus.(3)Establishment of BVDV-1 and BoHV-1 guinea pig challenge modelsUsing guinea pigs to conduct BoHV-1 and BVDV-1 challenge experiments to establish BVDV-1 and BoHV-1 guinea pig challenge models.The results showed that after intranasal inoculation of BoHV-1 virulent,the body temperature of the guinea pigs in the challenge group did not change significantly,and they were all within the normal range.One guinea pig in the BoHV-1 challenge group developed white secretions in the nasal cavity for about 3 days.Three guinea pigs had dark yellow secretions in the nasal cavity and eyes,which lasted for about 6 days.The nasal swabs of the guinea pigs in the immunization group were taken,and the antigen was detected by fluorescence quantitative PCR.BoHV-1 could not be detected in the challenge group in day 7 after challenge.The lungs of the challenged guinea pigs were collected,and the pathological section results showed that,compared with the control group,severe lesions were seen in the lungs of the guinea pigs after challenge,and typical symptoms of interstitial pneumonia appeared.After intranasal inoculation of BVDV-1 virulent,the body temperature of guinea pigs in the experimental challenge group did not change significantly,and they were all within the normal range.In the BVDV-1 challenge group,3 guinea pigs had white secretions in the nasal cavity,which lasted for about 8 days.Two guinea pigs had dark yellow secretions in the nasal cavity and eyes,which lasted for about 6 days.The anal swabs of the guinea pigs in the immunization group were taken,and the antigens were detected by fluorescence quantitative PCR.It was found that all guinea pigs in the challenge group were positive in days 1～14.The white blood cell count results showed that guinea pigs in the BVDV-1challenge group developed mild and transient leukopenia,with a maximum percentage decrease of 15.67%～38.36%,and an average percentage decrease of 17.94%in the group.(4)Immunogenicity study and serological detection of recombinant viruses BoHV-1(?)gE and BoHV-1 gE/E2-Linker-E2~+in guinea pigsUsing the BoHV-1(?)gE and BoHV-1 gE/E2-Linker-E2~+recombinant viruses constructed in this study,guinea pigs were used to conduct safety and immunogenicity research experiments.The results showed that the BoHV-1 antigen could not be detected in guinea pigs immunized with BoHV-1 inactivated antigen in day 6 after challenge.In guinea pigs immunized with BoHV-1 gE/E2-Linker-E2~+,BoHV-1 and BoHV-1(?)gE inactivated antigens,the viral shedding time and the level of viral shedding after challenge were smaller than those of the challenge control group.The final serum neutralization titer in the BoHV-1 gE/E2-Linker-E2~+immunized group was slightly higher than that in the BoHV-1 and BoHV-1(?)gE immunized groups.The pathological changes in the lungs of all immunized groups were milder than those of the challenge control group.BVDV-1inactivated antigen immunization group could not detect BVDV-1 antigen in day 8 after challenge.BoHV-1 gE/E2-Linker-E2~+immunized group could not detect BVDV-1 in day13 after challenge.The E2-Linker-E2 protein expressed by BoHV-1 gE/E2-Linker-E2~+can make guinea pigs produce serum neutralizing antibody against BVDV-1 infection.In the group inoculated with BoHV-1 gE/E2-Linker-E2~+inactivated recombinant virus,one guinea pig developed symptoms of leukopenia after challenge.(5)Establishment of BVDV-1 and BoHV-1 calf challenge modelsBoHV-1 and BVDV-1 challenge experiments were carried out with calves,and BVDV-1 and BoHV-1 calves challenge models were established.The results showed that there were 3 calves in the BoHV-1 challenge group,of which two(#106,#104)had elevated body temperature in day 2,the other one(#105)had an elevated body temperature in day 4,and the day 7 increased to 40.7°C,and obvious clinical symptoms such as dyspnea and purulent nasal fluid appeared in days 6 and 7,respectively.Serous nasal fluid appeared in the nasal cavity in day 4 and lasted for 10 days.Antigen test was performed on nasal swabs of calves,and it was found that the nasal swabs of the challenge group were positive by PCR in day 2,and BoHV-1 could not be detected until the day 12.In the BVDV-1 challenge group,1 calf(#124)had an increase in body temperature in day 1,and the other two calves had an increase in body temperature in day 4,and watery diarrhea in day 5,which lasted for about 5 days.Symptoms were relieved in day 10.Using 5’UTR F/R primers to detect antigens on the anal swabs of the calves in the immunized group,it was found that one of the calves in the challenge group had a longest viral shedding time of 12days.By 3 days before challenge(-2,-1,0 dpc)and within 14 days after challenge,we measured and recorded total leukocyte counts.The results showed that all calves in the challenge control group had leukopenia.Two of the calves had decreased leukocyte counts in days 4,5,6,9,and 10,and the other calves had decreased leukocyte counts on days5,6,7,9,11,and 12.It is worth noting that the maximum percentage of white blood cell decline in the control calves ranged from 28.62%～42.61%,and the average percentage decline in the group was 34.26%.(6)Immunogenicity study and serological detection of recombinant virus BoHV-1(?)gE and BoHV-1 gE/E2-Linker-E2~+on calvesThe BoHV-1(?)gE and BoHV-1 gE/E2-Linker-E2~+recombinant viruses constructed in this study were used to conduct safety and immunogenicity research experiments on calves.The results showed that after BVDV-1 and BoHV-1 challenge,the shedding time and shedding amount of the vaccinated calves were both smaller than those of the challenge control group.After 42 days of antigen inoculation,the average titer of neutralizing antibody in BoHV-1 serum in BoHV-1 gE/E2-Linker-E2~+group was slightly higher than that in BoHV-1(?)gE and BoHV-1 groups.And the BoHV-1 gE/E2-Linker-E2~+group could produce serum neutralizing antibody specific for BVDV-1.Leukopenia was seen in calves in the BVDV-1 challenge group,but not in the immunized group.In addition,the pathological tissue section results showed that compared with the challenge control group,the calf lung and small intestine lesions were reduced,and the inactivated antigen inoculation group provided a good protection effect of BoHV-1 and BVDV-1. |
PDF Full Text Request