Production Of APN Gene-edited Pigs And Their Resistance To TGEV And PEDV

Swine enteric diseases have caused significant economic loss and have been considered as the major threat to the global swine industry.Several coronaviruses,including transmissible gastroenteritis virus(TGEV)and porcine epidemic diarrhea virus(PDEV),have been identified as the causative agents of these diseases.Effective measures to control these diseases are lacking.The major host cells of TGEV and PDEV have thought to be epithelial cells on small intestine villi.Aminopeptidase-N has been described as the putative receptor for entry of TGEV and PDEV into cells in vitro.Currently,studies on the interaction between TGEV/PEDV and Aminopeptidase-N(APN)mainly focus on model cells,and whether Aminopeptidase-N is the cell receptor of PEDV remains controversial.Therefore,this study focused on the ability of Aminopeptidase-N-null piglets to resist TGEV and PEDV infection,so as to determine the role of Aminopeptidase-N in antiviral infection and shed insight into disease-resistant breeding.The CRISPR/Cas9n gene editing system was employed in this study with screening of transfection positive cells by flow cytometry,and monoclonal cell lines obtained by cell culture.Nineteen monoclonal cells were randomly selected,sixteen of which were modified at APN for both alleles,with an editing efficiency of 84.3%.Gene editing forms include insertion,deletion and mismatch.Then,APN gene editing cells were used as donor cells for somatic cell nuclear transfer.Reconstituted embryos can develop normally,indicating APN knockout does not cause embryonic lethality.Embryo transfer analysis showed the pregnancy rate of the recipient sows is comparable to the reported rate of somatic cell nuclear transfer pregnancy.Upon full-term pregnancy,twenty-one APN gene-edited piglets were born,eight of which died within 2 days due to low birth weight,and these APN gene-edited piglets had no obvious physiological defects,and survived with great vitality.Sanger sequencing and Western blotting analysis showed that all the 13 piglets were biallelic edited at APN,and no Aminopeptidase-N protein was detected.In the present study,thirteen live piglets were divided into two groups for TGEV and PEDV infection experiments.After TGEV challenge,APN-null piglets were resistant to highly virulent TGEV,Histopathological analysis indicated APN-null pigs exhibited normal small intestine villi while wildtype pigs showed typical lesions in small intestines.Immunochemistry analysis confirmed that no TGEV antigen was detected in target tissues in APN-null piglets.However,upon PDEV challenge,APN-null piglets are still susceptible with 100%mortality.Histopathological and immunohistochemical analyses showed no significant difference between APN-null piglets infected with PEDV and wild-type piglets.In conclusion,our study demonstrated that APN deletion has no negative effect on embryonic and fetal development in pig.APN-null newborn piglets can resist TGEV infection but not PEDV infection.The results of this study,using animal model,demonstrate that APN is essential for TGEV infection,but PEDV may have other cell surface receptors.In addition,our study not only enriches the means of antiviral research,but also providing a viable tool for disease resistance breeding in pigs.