Abstract
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 (PEDV), have been identified as the causative agents of these diseases. Effective measures to control these diseases are lacking. The major host cells of transmissible gastroenteritis virus and porcine epidemic diarrhea virus have thought to be epithelial cells on small intestine villi. Aminopeptidase-N (APN) has been described as the putative receptor for entry of transmissible gastroenteritis virus and porcine epidemic diarrhea virus into cells in vitro. Recently, Whitworth et al. have reported that APN knockout pigs are resistant to TGEV but not PEDV after weaning. However, it remains unclear if APN-null neonatal pigs are protected from TGEV. Here we report the generation of APN-null pigs by using CRISPR/Cas9 technology followed by somatic cell nuclear transfer. APN-null pigs are produced with normal pregnancy rate and viability, indicating lack of APN is not embryonic lethal. After viral challenge, APN-null neonatal piglets are resistant to highly virulent transmissible gastroenteritis virus. Histopathological analyses indicate APN-null pigs exhibit normal small intestine villi, while wildtype pigs show typical lesions in small intestines. Immunochemistry analyses confirm that no transmissible gastroenteritis virus antigen is detected in target tissues in APN-null piglets. However, upon porcine epidemic diarrhea virus challenge, APN-null pigs are still susceptible with 100% mortality. Collectively, this report provides a viable tool for producing animals with enhanced resistance to TGEV and clarifies that APN is dispensable for the PEDV infection in pigs.
Differential Detection of Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus by Duplex RT-PCR
