Prokaryotic Expression Of Porcine BVDV-2 Nonstructural Protein N^pro And Its Effect On PBMCs And Some Cytokine Productions In Pigs

Bovine Viral Diarrhea Virus (BVDV) is the pathogen of bovine virus diarrhea-mucosal disease (BVD-MD) which is a positive stranded RNA virus. The whole genome of the virus is about 12.3 kb, and it belongs to Pestivirus genus of Flavividae family. According to the genetic characteristics of BVDV genome, it can be divided into two genotypes:genotype 1 (BVDV-1) and genotype 2 (BVDV-2). Previous epidemiologcial surveys showed that BVDV-1 spreaded more widely. However, the incidence of BVDV-2 in China has been increasing in recent years. BVDV has a wide host range, including bovine, swine, goats, sheep, deer and some wild ruminants. It is worth noting that, more and more pigs get infected with BVDV. It is reported that the nonstructural protein Npro of BVDV plays an important role in anti-viral response which could assist the virus to escape host innate immune response. The preliminary study had explored the function of BVDV Npro protein at the cellular level, which lays a foundation for further studying its effect on immune regulation and pathogenicity. The purpose of the study is to explored the influence of porcine BVDV-2 nonstructural protein Npro on porcine immune response which will be helpful for understanding its role in BVDV infection and give warning and prevention of porcine BVDV.This study is mainly divided into the following three parts:Part one:Prokaryotic expression of the nonstructural protein Npro of porcine BVDV-2. To ensure that the following experiments can be carried out smoothly, the nonstructural protein Npro of porcine BVDV was expressed and then anti-Npro polyclonal antibody was prepared. Porcine BVDV-2 strain SH-28 was inoculated on MDBK cells and its total RNA was extracted. The Npro gene was amplified by RT-PCR and cloned into pMD19-T simple vector to obtaine the positive recombinant plasmid pMD19-T-Npro. After inserting the target fragment into pET-28a(+), a prokaryotic expression system with E. coli BL21 (DE3) as the host was constructed. SDS-PAGE confirmed that the recombinant portein Npro was expressed successfully, about 26 KDa. Concentration of the purified fusion protein was 1.23 mg/mL detected by BCA method. Then the polyantiserum of Npro was prepared by immuning mice three times. Indirect ELISA indicated that the titer of the antiserum was 1:12800. The anti-Npro polyclonal antibody could specifically react with the purified Npro protein in western blot assay. It is demonstrated that the anti-Npro polyclonal antibody was effective and could be used for the following experiment.Part two:The impact of porcine BVDV-2 nonstructural protein Npro on swine peripheral blood cells. To explore the influence of porcine BVDV-2 nonstructural protein Npro on porcine peripheral blood cells,30-day-old post-weaning piglets with no BVDV and CSFV pathogens infection were inoculated with 4 mL(107 TCID50/0.1 mL) SH-28 and deletion strain vASHANpro, respectively. The control group was given intramuscular injection with 4 mL DMEM. Blood of the infected pigs were collected to isolate the viruses, and this would confirm whether the pigs were infected BVDV successfully. Results showed that both SH-28/p and vASHΔNpro/p viruses isolated from the infected pigs could specifically reacted with Bz-53 MAb which is targeted to BVDV E2. However, Npro antiserum can only recognize SH-28/p rather than vASHANpro/p. This confirmed that the pigs were infected with BVDV successfully. Clinical scores and rectal temperature of three experimental groups were observed and recorded everyday, the results showed that clinical scores of the group infected with SH-28 gradually increased within 14 days and then began to decline while the pigs infected with deletion strain vASHΔNpro were asymptomatic within 10 days and reached the highest score on 16 dpi, then began to decrease; during the late phase of infection period, both of the two infection groups significantly increased than control group (P<0.01). Rectal temperature of isolate SH-28 infection group gradually increased during 3-10 dpi, reaching the peak on 10 dpi, about 41.5 ℃, then began to decline; and rectal temperature changes of the group infected with deletion strain vASHANpro were not obvious within 8 days, but reaching the peak on 12 dpi, about 40.5 ℃, and then began to decline; during the middle phase of infection period, both of the two infection groups significantly increased than control group (P<0.01). They were all recovered during the late phase of infection period.The level of red blood cells (RBC), white blood cells (WBC), platelets (PLT) in porcine peripheral blood of three experimental pigs after challenge was detected by blood routine examination. The results showed that the number of WBC in porcine peripheral blood of the three experimental groups began to fluctuate widely after 6 dpi, and compared to deletion strain vASHANpr0 infection group, the level of PLT in SH-28 infection group was significantly reduced (P<0.01) on 18 dpi and 20 dpi; the number of RBC of the three experimental groups had no significant difference during the whole infection period; during the early and middle phase of infection period, the level of PLT fluctuate widely, and compared to deletion strain vASHANpr0 infection group, the level of PLT in SH-28 infection group was significantly reduced (P<0.01) from 12 dpi to 20 dpi. Considering CD4T and CD8T cells were two of the important indexs in immune response, so flow cytometry were used to analyze the changes of the two lymphocyte subsets in peripheral blood mononuclear cells (PBMCs) of experimental pigs after challenge. The results showed that, compared to SH-28 infection group and control group, the proportion of CD4T cells in PBMCs of deletion strain vASHANpro infection group significantly increased (P<0.05) and the proportion of CD8T cells significantly increased (P<0.01) during the middle and late phase of infection period.In conclusion, this study illustrated that the porcine BVDV-2 infection could decrease the level of WBC and PLT in porcine peripheral blood, but lack of Npro protein could rebound them. This initially confirmed that deficiency of the Npro protein could attenuate the pathogenicity of porcine BVDV-2. In addition, it could increase the proportion of CD8T cells and CD4T cells which suggested that the porcine immune response could get enhanced. It provides important clues to explore the pathogenesis and immunologic mechanism of pig BVDV-2.Part three:Influence of porcine BVDV-2 nonstructural protein Npro on productions of some cytokines in porcine PBMCs. In order to explore the antiviral immunologic mechanism of pig BVDV-2 Npro mediated by cytokines, the transcriptional levels of the seven cytokines (IL-1β、IL-8、IL-10、IFN-α、OAS、Mx1、PKR) in pig PBMCs after infection of SH-28 and vASHANpr0 were detected by qRT-PCR. These data could be useful to illustrate the regulation mechanism of Npro to host anti-viral immunity and understand its role in BVDV infection of pigs. After challenge, the results showed that the transcriptional levels of IL-1β and IL-8 of the two infection groups both first increased and then decreased, and the level of the pigs infected with SH-28 had a rising trend and both reached the peak at 14 dpi which was significant higher than that of the other two groups during the whole infection period (P<0.01). Though IL-10 production in both of the two infection groups presented a declined trend, vASHANpro infection group was higher than SH-28 infection group and both of them were tower than the control group (P<0.01). Further detection of IFN and its downstream antiviral cytokines productions indicated that IFN-a, OAS and Mxl productions in both of the two infection groups decreased while they were still higher than the control group (P<0.05), especially for OAS. And that in vASHANpr0 infection group was higher than SH-28 infection group (P<0.01). Besides, during the early and middle phase of infection period, the expression of PKR in infection pigs was significant higher than the control group (P<0.01), but there was no significant difference between the two infection groups.In conclusion, this study demonstrated that the porcine BVDV-2 nonstructural protein Npro had certain influence on the expression of cytokines in pig PBMCs and confirmed its role in host anti-viral response.