Screening Of Resistant Indicator Traits And Candidate Genes Based On Etong Two-End-Black Artificially Infected With PRRSV

Porcine Reproduction and Respiratory Syndrome?PRRS?was caused by Porcine Reproductive and Respiratory Syndrome Virus?PRRSV?with a respiratory disorder in pigs of all age,reproductive failure in sows and high mortality rate in piglets.At present,the PRRS prevention and control of China mainly depend on biosafety and vaccines.However,the current commercial inactivated PRRSV vaccines are mostly based on a single PRRSV strain,which is not good protective effect on PRRSV infection with high mutation.Therefore,by enhancing the resistance of the host to PRRSV from the genetic level,breeding of PRRSV resistant varieties,it provides a research approach for the prevention and control of PRRSV's large-scale epidemic,and is also the focus of pig genetic and breeding improvement.The laboratory found that the TC pigs showed strong resistance to disease compared with the Large White?LW?pigs.Through artificial infection of PRRSV,it was found that there were significant differences between the two breeds in clinical symptoms,cytokine level,blood routine indicators,lung pathological damage degree,viremia in serum et al.The laboratory constructed a athwart crossed population of high generation,which is formed by the cross of LW pigs and TC pigs.This population is an ideal resource for gene mapping and gene identification of disease resistance.Therefore,in this study,the self constructed athwart crossed population of high generation was used as the research object,and the viremia,blood routine indicators,body temperature,body weight,viability?survival or not and survival time?reflecting the resistance to PRRSV of host were measured after PRRSV infection,and the analysis was carried out for the measured characters.Resistance candidate genes and markers were screened by transcriptome sequencing in peripheral blood of resistant and susceptible individuals,and signal pathway analysis was carried out.The main results are as follows:1. PRRSV artificial infection was carried out by using the high generation athwart crossed population of LW pigs and TC pigs,at different time pionts the viremia,blood routine indicators,body weight were measured and the changes were analyzed.72 piglets in the high generation athwart crossed population constructed by resistant Tongcheng pig and susceptible big white pig were carried on PRRSV artificial infection.Their clinical symptoms were observed,body temperature and clinical symptoms were collected for 0-35 days after infection,blood routine indicators,viremia and weight.The results showed that all kinds of clinical symptoms appeared on 3dpi after PRRSV infection,and the body temperature continued to rise to 6dpi after PRRSV infection,and the maximum body temperature reached 41.2±1.1?.Death occurred 7dpi,and the dead individuals were mainly concentrated on 7 to 14 dpi.The death peak appeared on the 10dpi,and the number of dead individual was basically stable19dpi.Viremia increased from 0 to 4dpi,peaked on 4dpi and lasted until 7dpi,viremia decreased to 6.6.log₁₀copies/ml from 7dpi to 35dpi.The growth rate of body weight was slow from 0 to 4dpi,the body weight increased negatively from 4 to 21dpi,the loss of weight reached the maximum from 7 to 11dpi,the body weight increased positively and the growth increased gradually 21 to 35dpi.when compared to 0dpi,the WBC count,the lymphocyte percentage and the monocyte count were significantly reduced?P<0.01?,and the granulocyte percentage was significantly increased?P<0.01?at both 4dpi and 7dpi.According to the survival or not death on 14 dpi,the pigs were divided into two groups:resistance and susceptibility,resistance has 36 pigs and susceptibility also has 36 pigs.The difference of the viremia,blood routine indicators,body weight were analysed.It was detected that the viremia of resistance was significantly lower than susceptibility4,7,11dpi?P<0.05?,the weight gain of resistance was significantly higher than that of susceptibility at 4-7dpi,7-11dpi,11-14dpi?P<0.05?,the lymphocyte and the granulocyte percentage were significantly different at 7dpi between groups.The lymphocyte percentage was significantly higher than susceptible group?P<0.05?,and the granulocyte percentage was significantly lower?P<0.05?.The correlation among these traits was analyzed to screen the indicator traits of resistance to PRRS.2. Transcriptome analysis of resistance and susceptibility peripheral blood leukocytes after PRRSV infectionIn order to find the difference ofresistance and susceptibility in the transcriptome level,peripheral blood leukocytes?peripheral blood?at 0dpi before PRRSV infection and 4,7,11dpi after PRRSV infection were carried out transcriptome sequencing.First the time coruse was analysed for peripheral blood at different time points,among which 2422 DEGs were differentially expressed on the 4dpi compared with that 0dpi before PRRSV infection,1717 DEGs were differentially expressed on the 7dpi compared with that 0dpi before PRRSV infection,and 3372 DEGs were differentially expressed on the 11dpi ycompared with that 0dpi before PRRSV infection.By KEGG and GO functional enrichment,On the 4dpi,the specific DEGs were enriched in cell division and differentiation,which may have an effect on the growth and development of pigs,on the 7dpi,the specific DEGs were enriched in Wnt pathway and signal transduction,which may play a role in immune response,on the 11dpi,the specific DEGs were mainly enriched in post transcriptional processing and post-translational modification.At different stages of HP-PRRSV infection,the host plays different immune response pathways.Next Weighted Gene Co-Expression Network Analysis?WGCNA?was used to analysed peripheral blood RNA Resistance and Susceptibility at different time points.The transcripts in turquoise and blue modules have a strong correlation with viral load and weight growth.In these two modules,the number of specific DEGs in the resistant group is less,the common DEGs in the resistant group and the susceptible group are mainly enriched in the immune pathway,and the specific DEGs in the susceptible group are mainly enriched in the immune pathway and signal transduction pathway,in the susceptible group the specific RASGRP1,CD247,MALT1,NFATC14 DEGs are enriched in T cell receptor signaling pathway.