The use of cells from ANPEP knockout pigs to evaluate the role of aminopeptidase N (APN) as a receptor for porcine deltacoronavirus (PDCoV)

Abstract

A B S T R A C TThe coronaviruses, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) represent important sources of neonatal diarrhea on pig farms. The requirement for aminopeptidase N (APN) as a receptor for TGEV, but not for PEDV, is well established. In this study, the biological relevance of APN as a receptor for PDCoV was tested by using CRISPR/Cas9 to knockout the APN gene, ANPEP, in pigs. Porcine alveolar macrophages (PAMs) from ANPEP knockout (KO) pigs showed resistance to PDCoV infection. However, lung fibroblast-like cells, derived from the ANPEP KO PAM cultures, supported PDCoV infection to high levels. The results suggest that APN is a receptor for PDCoV in PAMs but is not necessary for infection of lung-derived fibroblast cells. The infection of the ANPEP KO pigs with PDCoV further confirmed that APN is dispensable as a receptor for PDCoV.

CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance

“…Our results on the delayed PDCoV-specific neutralizing antibodies production, and a reduced extent of gross and histopathological lesions on small intestine in DKO pigs compared to WT pigs are consistent with this previous suggestion that pAPN may play a role but is not the only path for PDCoV cell entry. Interestingly, a recent study showed that PAMs, but not lung fibroblast-like cells, from pAPN knockout pigs showed resistance to PDCoV infection ( Stoian et al, 2020 ), a finding consistent with our in vitro experiments showing that DKO PAMs exhibit decreased susceptibility to PDCoV infection. In addition, pAPN knockout pigs are susceptible to PDCoV when virus levels were detected using qRT-PCR, and virus neutralization activity was measured, although the extent of tissue lesions between the KO and WT groups was not compared ( Stoian et al, 2020 ).…”

“…Interestingly, a recent study showed that PAMs, but not lung fibroblast-like cells, from pAPN knockout pigs showed resistance to PDCoV infection ( Stoian et al, 2020 ), a finding consistent with our in vitro experiments showing that DKO PAMs exhibit decreased susceptibility to PDCoV infection. In addition, pAPN knockout pigs are susceptible to PDCoV when virus levels were detected using qRT-PCR, and virus neutralization activity was measured, although the extent of tissue lesions between the KO and WT groups was not compared ( Stoian et al, 2020 ). Our findings are in line with this study reporting that pAPN knockout pigs are still susceptible to PDCoV.…”

“…PDCoV is a highly pathogenic virus that has recently been shown to cause diarrhea in newborn piglets, although the functional receptors for PDCoV have not yet been confirmed ( Li et al, 2018 ; Stoian et al, 2020 ; Zhu et al, 2018 ). Whether pAPN functions as a receptor or co-receptor in PDCoV infection of pigs remains controversial.…”

“… Li et al, 2018 suggested that PDCoV infection may require a co-receptor, in addition to pAPN ( Li et al, 2018 ). Using cells isolated from pAPN knockout pigs, however, Stoian et al, 2020 showed that these pig cells were still susceptible to PDCoV infection in vitro. It was suggested that pAPN may be one of the receptors for PDCoV, and an unknown receptor or factor may compensate for pAPN function in the absence of pAPN ( Stoian et al, 2020 ).…”

“…Using cells isolated from pAPN knockout pigs, however, Stoian et al, 2020 showed that these pig cells were still susceptible to PDCoV infection in vitro. It was suggested that pAPN may be one of the receptors for PDCoV, and an unknown receptor or factor may compensate for pAPN function in the absence of pAPN ( Stoian et al, 2020 ). However, whether pAPN knockout pigs may be resistant to PDCoV infection in vivo remains unknown.…”