Effects Of Classical Swine Fever Virus Infection On Expression Of Immune Response Genes In Pk-15Cells

Classical swine fever virus (CSFV), which is an enveloped, single-and positive-stranded RNA virus, with bovine viral diarrhea virus and border disease virus, belongs to the members of the Pestivirus genus within the family of Flaviviridae. The genome of CSFV is approximately12.3kb and contains a single large open reading frame encoding a polyprotein of3898amino acid, which undergoes co-and posttranslational processing and maturation into four structural and eight nonstructural proteins by viral proteases and host proteases. Domestic pigs and wild boars are the unique host of CSFV. CSFV causes a highly contagious infectious disease with high morbidity and mortality in pigs, resulting in substantial economic losses to the pig industry worldwide. Classical swine fever (CSF) is therefore classified as a List A disease by the Office International des Epizooties (OIE). In China, CSF is also listed as one of the severe animal epidemic disease and the State practices a system of immunization planning for execution of compulsory immunization against animal epidemics since2008.After implementation of strict immunization measures, ecological environment of CSFV were changed. CSF obviously presented changing in epidemiological forms and clinical signs. The large-scale CSF has rarely outbroken but sporadic epizootics still frequently occurred every year, and the clinical signs are dominated by typical to atypical forms of the disease, such as reproductive failures in pregnant sows and congenital infections in newborn piglets were observed. Virus is detected in semen, gonads, tonsil and lymph node even in certain vaccinated pigs, which confirmed CSFV have been established as persistent infection. Virus has to invade host defense mechanism and immune response and then establishes life-long, persistent infections. Therefore, the study on the interaction between host and CSFV is critical for understanding viral pathogenesis and immunity. For realizing the status of the cellular genes relative to immune response in vitro, PK-15cells were offered as a feasible model to investigate transcriptional changes during CSFV infection. The study may provide some useful data of insight into the interaction between CSFV and swine. In this study, real-time RT-PCR and Western blot approaches was applied to monitor the expression of immune response genes of host cells following CSFV infection. The results are mainly showed as follows. Experiment1. Development of the method for determination of several immune response genes mRNA levels in PK-15cells by real-time RT-PCR.According to the GenBank data, mRNA sequences of SLA-2, TAP1, SLA-DR, Ii, CD40, CD80, CD86, IFN-a, IFN-p, GAPDH and5’-UTR of CSFV genes were downloaded, eleven pairs of specific primers were designed by biological Oligo6.0/Primer5.0software and synthesized. Oligo(dT)18were used as primer and first strand cDNA was synthesized from total RNA, which was isolated from swine lymphocytes. Using the synthesized cDNAs as template, eleven target fragments were amplified by polymerase chain reaction (PCR), respectively. The PCR products were electrophoresed on2%agarose gels and extracted. The extracted PCR products were cloned into pMD18-T Vector and sequenced. The extracted plasmids were diluted with series10-folds and were used as standard templates, and real-time PCRs were performed. In addition, the primer concentration and annealing temperature for each pair of specific primers was optimized, respectively. The results showed that each gene was amplified using specific primer pair. The optimal primer concentration was202.5nmol/μl and annealing temperatures for eleven primer pairs was60℃. The melting curve showed the unique peak for each PCR product, indicating that the method for determination SLA-2, TAP1, SLA-DR, Ii, CD40, CD80, CD86, IFN-a, IFN-p and5’-UTR of CSFV mRNA levels in PK-15cells by real-time RT-PCR is successfully established.Experiment2. Transcriptional abundance of several immune response genes of PK-15cells following CSFV infection in vitro using real-time RT-PCR.CSFV evade the immune response and establish persistent infection under natural and experimental conditions. As we know, some genes relative to MHC and cytokine regulation are involved in persistent infection, but the extent of each gene responds to CSFV infection is unclear. In our study, nine immune response genes including SLA-2、TAP1、SLA-DR、Ii、CD40、CD80、CD86、 IFN-a and IFN-β were detected using real-time RT-PCR after the PK-15cells were infected with0.1Moi or1.0Moi Shimen or GXXJ01strain of CSFV, respectively. The results showed that these immune response genes basically presented down-regulation during CSFV infection, except IFN-a and IFN-β transcriptional levels of mRNA in CSFV-infected cells did not change significantly. The SLA-2, SLA-DR, and Ii genes were notably decreased. Our experiments concluded that CSFV maybe involved in the inhibition of immune response genes.Experiment3. Effects of classical swine fever virus infection on expression of SLA-DR gene in PK-15cells.Following Shimen strain of CSFV infection, the expression of SLA-DR presented down-regulation. SLA-DR is inhibited at24and36hpi and its expression restained in48hpi. In order to further exploration of the regulation between CSFV and SLA-DR, eukaryotic recombination expression plasmids P-C, P-Npro and P-E2were generated. The C, Npro and E2genes from Shimen strain of CSFV were cloned, then inserted into the expression vector pcDNA3.0. The eukaryotic recombination expression plasmids P-C, P-Npro and P-E2were constructed and transfected into PK-15cells with liposome infection protocol. The SLA-DR expression was detected by real-time RT-PCR and western blot. The results showed that SLA-DR mRNA expression showed a significantly up-regulation following P-C or P-E2transfection. SLA-DR presented up-regulation after transfection3to24hours, but in24hour SLA-DR become down-regulated, the SLA-DR mRNA expression was similar to controls in36and48hours. Western blot showed that SLA-DR expression presented an obviously up-regulation following P-C or P-E2transfection or P-C/P-E2co-transfection into PK-15cells. The co-transfection of three recombination vectors P-C/P-Npro/P-E2were carried out, SLA-DR expression presented slight up-regulated. However, SLA-DR expression showed an obviously down-regulation during P-Npro or P-C/P-Npro or P-Npro/P-E2co-transfection into PK-15cells. The results suggested that C or E2alone or synergistic did not showed significantly effect on the down-regulation of SLA-DR, Npro alone or synergistic showed slight effect on the down-regulation of SLA-DR. Current studies suggest that the incidence and development of virus disease is a multi-gene, multi-stage process participation, in which the relationship between CSFV and SLA-DR need to be further studied.