| Porcine reproductive and respiratory syndrome（PRRS）is a highly contagious disease,which can cause severe reproductive failure in sows and respiratory failure in pigs of all ages.Outbreak of the disease has resulted in significant economic losses in the swine industry globally.The etiological agent PRRS virus（PRRSV）is a positive,single-stranded RNA virus,belonging to Arteriviridae family,Nidovirales order.When infected by PRRSV,the pig host can be immunologically inhibited and shows a much-delayed production of neutralizing antibodies against PRRSV.PRRSV is highly prone to mutation,which makes it one of the most difficult viruses to be controlled in the pig production using traditional approaches.Three cellular factors with a PRRSV entry mediator activity have been identified,and named heparan sulphate,CD163 and CD169.CD163 belongs to the scavenger receptor cysteine-rich（SRCR）family of membrane proteins,containing nine SRCR domains.SRCR domain deletion and substitution experiments revealed that the SRCR5 of porcine CD163（pCD163）is essential for PRRSV infection in vitro.Substitution of pCD163 SRCR5 with human CD163L1（hCD163L1）SRCR8 resulted in a loss of infectivity.In this thesis,we were trying to generate pigs resistant to PRRSV in which CD163 SRCR5 was precisely replaced through gene editing of CD163,and to investigate whether these pigs are resistant to PRRSV infection.To substitute the porcine endogenous CD163 SRCR5 with the corresponding domain of hCD163Ll,we designed and screened single-guide RNA（sgRNA）to target the exon 7 of pCD163.Meanwhile,a circular donor vector was used as a template to repair the DSB by homologous recombination.Modification of the target gene was assessed by PCR,Sanger sequencing and Southern blotting.We finally generated CD163 biallelic-modified pigs using the CRISPR/Cas9 system combined with the donor vector.Both Hpt in serum and the red blood cell in blood were similar in wild-type and gene-modificated pigs,indicating that the modification of CD163 gene did not alter its function.To show whether the gene-editing pigs are resistant to PRRSV at cell level,CD163MuI/Mut porcine alveolar macrophages（pAMs）and wild-type PAMs were both challenged with highly pathogenic PRRSV（HP-PRRSV）strains JXwn06 and WUH3.The results showed that the modification of CD163 could induce significant inhibition of PRRSV.At various MOIs（0.X05,0.025,0.1,0.25）,this antiviral effect was sustained.To further explore how the modification of CD163 gene influences PRRSV infection,we carried out immunofluorescence to observe the entry and replication of PRRSV virion.The result showed that the modification of CD163 gene inhibited PRRSV uncoating and releasing genome but exhibited no effect on PRRSV binding and internalization.Finally,we chose 4 cloned CD163Mut/Mut pigs from F0 generation and 6 wild-type pigs of the same age,for the in vivo challenging experiments.Pigs were infected with the HP-PRRSV strain JXAI at 106.5 TCID50 per headage and followed for 21 days after infection.PRRSV-induced clinical signs including respiratory distress,inappetence,lethargy and fever were recorded using a scoring system.The results demonstrated that CD163Mut/Mut pigs showed substantially relief from PRRSV-induced clinical signs compared to wild-type pigs.Although PRRSV viremia was apparent in all of the ten pigs,whereas it disappeared quickly from 7 dpi in the CD163Mut/Mut pigs.Moreover,the other data such as the viral RNA in lungs,lung histopathology and survival rate also demonstrated that CD163Mut/Mut pigs were resistant to PRRSV infection.In summary,we have successfully generated pigs in which the endogenous CD163 exon 7 were substituted with the corresponding exon of hCD163L1.Our data demonstrated that the modification of CD163 effectively inhibited PRRSV replication and protected pigs from PRRSV infection,thus laying a good foundation for breeding of PRRSV resistant pigs through gene editing technology. |
