Lessening of porcine epidemic diarrhoea virus susceptibility in piglets after editing of the CMP-N-glycolylneuraminic acid hydroxylase gene with CRISPR/Cas9 to nullify N-glycolylneuraminic acid expression

Abstract

Porcine epidemic diarrhoea virus (PEDV) devastates the health of piglets but may not infect piglets whose CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene mutated (knockouts, KO) by using CRISPR/Cas9 gene editing techniques. This hypothesis was tested by using KO piglets challenged with PEDV. Two single-guide RNAs targeting the CMAH gene and Cas9 mRNA were microinjected into the cytoplasm of newly fertilized eggs; 4 live founders generated and proven to be biallelic KO, lacking detectable N-glycolylneuraminic acid (NGNA). The founders were bred, and homozygous offspring were obtained. Two-day-old (in exps. I and III) and 3-day-old (in exp. II) KO and wild-type (WT) piglets were inoculated with TCID50 1x103 PEDV and then fed 20 mL of infant formula (in exps. I and II) or sows colostrum (in exp. III) every 4 hours. In exp. III, the colostrum was offered 6 times and was then replaced with Ringer/5% glucose solution. At 72 hours post-PEDV inoculation (hpi), the animals were euthanized for necropsy, and their intestines were sampled. In all 3 experiments, the piglets showed apparent outward clinical manifestations suggesting that infection occurred despite the CMAH KO. In exp. I, all 6 WT piglets and only 1 of 6 KO piglets died at 72 hpi. Histopathology and immunofluorescence staining showed that the villus epithelial cells of WT piglets were severely exfoliated, but only moderate exfoliation and enterocyte vacuolization was observed in KO piglets. In exp. II, delayed clinical symptoms appeared, yet the immunofluorescence staining/histopathologic inspection (I/H) scores of the two groups differed little. In exp. III, the animals exhibited clinical and pathological signs after inoculation similar to those in exp. II. These results suggest that porcine CMAH KO with nullified NGNA expression are not immune to PEDV but that this KO may lessen the severity of the infection and delay its occurrence.\n\nAuthor summaryThe infection of villus epithelial cells by PEDV has been suggested to occur via putative sialic acid and aminopeptidase N (APN) receptors. Thus, CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene-mutated pigs that lack N-glycolylneuraminic acid (NGNA) receptors should exhibit resistance to PEDV infection even when APN, which is also responsible for peptide digestion and amino acid absorption and should not be tackled and remains intact. This hypothesis was tested in the present study by generating animals of this type; however, after PEDV challenge, they still showed clinical manifestations of infection. Although the hypothesis could not be verified by the results of the study, some of the immunological and histopathological evidence obtained suggested that this genetic alteration may lessen the severity of infection and delay its occurrence. The results also suggested that binding to NGNA is not a sufficient and necessary condition for PEDV infection of enterocytes. The null expression of CMAH by gene editing induced insignificant resistance to PEDV infection in neonatal piglets.