Abstract
The alphacoronaviruses, transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV) are sources of high morbidity and mortality in neonatal pigs, a consequence of dehydration caused by the infection and necrosis of enterocytes. The biological relevance of amino peptidase N (ANPEP) as a putative receptor for TGEV and PEDV in pigs was evaluated by using CRISPR/Cas9 to edit exon 2 of ANPEP resulting in a premature stop codon. Knockout pigs possessing the null ANPEP phenotype and age matched wild type pigs were challenged with either PEDV or TGEV. Fecal swabs were collected daily from each animal beginning 1 day prior to challenge with PEDV until the termination of the study. The presence of virus nucleic acid was determined by PCR. ANPEP null pigs did not support infection with TGEV, but retained susceptibility to infection with PEDV. Immunohistochemistry confirmed the presence of PEDV reactivity and absence of TGEV reactivity in the enterocytes lining the ileum in ANPEP null pigs. The different receptor requirements for TGEV and PEDV have important implications in the development of new genetic tools for the control of enteric disease in pigs.
Highlights
Respiratory and enteric infections caused by coronaviruses have important impacts on both human and animal health
No amino peptidase N (ANPEP)-edits were observed in fibroblasts transfected with the remaining guide RNAs (gRNAs) plasmids
Mice made transgenic for human ANPEP, a receptor for human coronavirus-229E [HCoV-229E (Yeager et al 1992)], possess similar levels of expression in the same tissues as mouse ANPEP, including high levels of hANPEP expression in epithelial cells of the intestines (Wentworth et al 2005). hANPEP-transgenic mice are resistant to infection by intragastric inoculation with HCoV229E, but cell lines derived from embryos and bone marrow of hANPEP mice support HCoV-229E replication in vitro
Summary
Respiratory and enteric infections caused by coronaviruses have important impacts on both human and animal health. TGEV typically causes less destruction in swine herds due to a deletion mutant of TGEV, porcine respiratory corona virus (PRCV) that replicates in the respiratory tract (Kim et al 2000). Pigs typically recover from PRCV exposure and produce neutralizing antibodies that neutralize TGEV resulting in a less severe infection in TGEV exposed piglets. (2009, 2012 and 2016) three distinct chimeric viruses containing the S gene and 3a sequences of PEDV on a TGEV backbone have been described in Europe. These viruses, named swine enteric coronaviruses, cause clinical signs similar to PEDV but their impact is unknown since standard diagnostic techniques would not distinguish them from their parental viruses (Belsham et al 2016)
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