Date Published: May 29, 2019
Publisher: Public Library of Science
Author(s): Ching-Fu Tu, Chin-kai Chuang, Kai-Hsuan Hsiao, Chien-Hong Chen, Chi-Min Chen, Su-Hei Peng, Yu-Hsiu Su, Ming-Tang Chiou, Chon-Ho Yen, Shao-Wen Hung, Tien-Shuh Yang, Chuan-Mu Chen, Xiuchun Tian.
The porcine epidemic diarrhoea virus (PEDV) devastates the health of piglets but may not infect piglets whose CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene is mutated (knockouts, KO) by using CRISPR/Cas9 gene editing techniques. This hypothesis was tested by using KO piglets that were challenged with PEDV. Two single-guide RNAs targeting the CMAH gene and Cas9 mRNA were microinjected into the cytoplasm of newly fertilized eggs. Four 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, n = 6, and III, n = 15) and 3-day-old (in exp. II, n = 9) KO and wild-type (WT, same ages in respective exps.) piglets were inoculated with TCID50 1×103 PEDV and then fed 20 mL of infant formula (in exps. I and II) or sow’s 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 either deceased or euthanized were necropsied and 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.
Porcine epidemic diarrhoea (PED) was first recognized as an enteric disease in 1971 by the British veterinarian Oldham ; subsequently, the PED virus (PEDV) was isolated by Pensaert and de Bouck  at Ghent University in Belgium. Since then, PEDV-associated diarrhoea has been widely detected in Europe. In Asia, it was reported in 1982 , and it has subsequently greatly impacted the Asian pork industry. In China during 2010 and 2011, over one million nursing piglets were lost due to PEDV-associated diarrhoea ; in 2013, PEDV emerged in Korea and the USA [5–7] as well as in Taiwan , causing great economic losses and continuing to spread as an epidemic.
Currently, gene editing is widely used in both basic and applied studies, e.g., in studies of the disease resistance of farm animals . One convincing report showed that CD163 gene-edited pigs generated by CRISPR/Cas9 exhibited physiological normality and showed little vulnerability to porcine reproductive and respiratory syndrome virus (PRRSV) infection either in vitro  or in vivo [35–37]. However, other attempts, including putative receptors of CD169 KO and CD163 KO, failed to produce evident resistance to PRRSV  or African swine fever , respectively. These failures may have occurred because the mechanism of viral infection involves other receptors or because it does not involve receptors .