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Contents lists available at ScienceDirect Virology journal homepage The use of cells from ANPEP knockout pigs to evaluate the role of aminopeptidase N APN as a receptor for porcine deltacoronavirus PDCoV Ana Stoian b Raymond R R Rowland b Vlad Petrovan b Maureen Sheahan b Melissa S Samuel a Kristin M Whitworth a Kevin D Wells a Jianqiang Zhang d Benjamin Beaton c Mark Cigan c Randall S Prather a a Division of Animal Science University of Missouri Columbia MO 65211 USA b Department of Diagnostic Medicine and Pathobiology Kansas State University Manhattan KS USA c Genus plc DeForest WI USA d Veterinary Diagnostics and Production Animal Medicine Iowa State University Ames IA USA ARTICLE INFO Keywords Aminopeptidase N Porcine deltacoronavirus APN CRISPR ANPEP CD13 ABSTRACT The coronaviruses porcine epidemic diarrhea virus PEDV transmissible gastroenteritis virus TGEV and porcine deltacoronavirus PDCoV represent important sources of neonatal diarrhea on pig farms The re quirement 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 broblast 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 broblast cells The infection of the ANPEP KO pigs with PDCoV further con rmed that APN is dispensable as a receptor for PDCoV 1 Introduction Coronaviruses belong to the family Coronaviridae order Nidovirales and are important pathogens of humans and animals Coronaviruses are divided into four genera Alphacoronavirus Betacoronavirus Gammacoronavirus and Deltacoronavirus Infection of neonatal pigs with the alphacoronaviruses transmissible gastroenteritis virus TGEV or porcine epidemic diarrhea virus PEDV results in mal absorptive diarrhea which can lead to dehydration and death Madson et al 2016 Saif et al 2012 The rst outbreak of PEDV on U S farms in 2013 resulted in the death of nearly 7 million pigs or about 10 of U S pig production for that year Stevenson et al 2013 In 2014 a diar rhea causing porcine deltacoronavirus PDCoV was isolated from ve pig farms in Ohio Wang et al 2014 The M and N gene sequences were 99 identical to another PDCoV HKU15 from China Since 2014 PDCoV has rapidly spread throughout pig producing regions in the U S To date four host proteins aminopeptidase N APN angiotensin converting enzyme 2 ACE2 dipeptidyl peptidase 4 DPP4 and car cinoembryonic antigen related cell adhesion molecule 1 CEACAM1 have been described to function as coronavirus receptors Delmas et al 1992 Li et al 2003 Raj et al 2013 Dveksler et al 1991 Porcine APN pAPN a 963 amino acid type II membrane metallopeptidase participates in the removal of N terminal amino acids from protein substrates during digestion Delmas et al 1994 were the rst to characterize an APN peptide sequence located between amino acids 717 and 813 as required for infection of cells with TGEV The corresponding receptor region on TGEV is located on the S1 subunit C terminal domain S1 CTD of the TGEV spike S protein Godet et al 1994 Recent studies have focused on the potential role of APN as a re ceptor for PDCoV The data have yielded three di erent observations and conclusions In 2018 Wang et al showed that cell lines such as Vero and BHK 21 were resistant to infection with PDCoV and TGEV Permissiveness was acquired after transfection of both cell lines with a plasmid expressing a pAPN cDNA PDCoV and TGEV growth curves on the transfected cells showed a yield greater than 5 log 10 TCID 50 ml The authors concluded that pAPN is a functional receptor for PDCoV Also in 2018 Zhu et al reported similar results However HeLa cells which were pAPN negative and resistant to TGEV supported PDCoV infec tion Furthermore a knockout of pAPN expression in the porcine IPI https doi org 10 1016 j virol 2019 12 007 Received 10 October 2019 Received in revised form 16 December 2019 Accepted 17 December 2019 Corresponding author E mail address PratherR missouri edu R S Prather Virology 541 2020 136 140 Available online 24 December 2019 0042 6822 2019 The Authors Published by Elsevier Inc This is an open access article under the CC BY NC ND license http creativecommons org licenses by nc nd 4 0 T 21 cell line was prepared by using CRISPR to inactivate ANPEP the gene which codes for APN The ANPEP KO IPI 21 cells were completely resistant to TGEV but retained permissiveness for PDCoV The authors concluded that pAPN is likely not a critical functional receptor for PDCoV although it is involved in PDCoV infection The third outcome related to pAPN and PDCoV is found in Li et al 2018 who evaluated the permissiveness of ANPEP knockout KO ST cells to infection with TGEV and PDCoV ST cells lacking pAPN were completely resistant to TGEV but retained a small capacity to support PDCoV infection The authors concluded that PDCoV utilizes pAPN as a primary receptor for virus attachment but the presence of a second co receptor contributes to the permissiveness of cells for infection Furthermore the PDCoV co receptor can retain function independent of pAPN In this study we investigated the role of pAPN as a receptor for PDCoV by evaluating the permissiveness of di erent cell populations derived from the lungs of ANPEP KO and wild type WT pigs Porcine alveolar macrophages from ANPEP KO pigs were resistant to PDCoV and TGEV However lung broblast like cells which appeared fol lowing the outgrowth of ANPEP KO PAM cultures were susceptible to PDCoV but remained resistance to TGEV Furthermore ANPEP KO pigs supported PDCoV infection The results support a role for pAPN as a receptor for PDCoV but the presence of a second unknown receptor or factor can substitute for pAPN function 2 Results 2 1 Permissiveness of cells from ANPEP KO and WT pigs for PDCoV infection Porcine alveolar macrophages PAMs from ANPEP KO and WT pigs were used to evaluate the permissiveness of cells for infection with PDCoV and TGEV As shown in gure panels 1A and 1B the ANPEP WT PAMs were permissive for infection with PDCoV and TGEV while no infected TGEV or PDCoV cells were detected in PAMs from the ANPEP KO pigs The results showed that PAMs from pigs lacking a functional ANPEP gene are resistant to TGEV and PDCoV infection The long term culture of PAM cultures typically results in the out growth of a minor population of lung mesenchymal stem cells MSCs which exhibit a broblast like morphology Khatri et al 2015 By two weeks the PAM cultures were completely overgrown with broblast like cells along with the disappearance of macrophages The broblast like cells from the WT and KO pigs were passaged at least two times and then infected with PDCoV or TGEV The broblast like cells derived from the ANPEP WT pigs were permissive for both TGEV and PDCoV Fig 1C and D However the ANPEP KO broblast like cell cultures showed no evidence of TGEV infection but showed several PDCoV infected cells all possessing a broblast like morphology These data con rmed the requirement of APN for the permissiveness of the bro blast cells to TGEV however the absence of APN had no e ect on in fection of broblast like cells with PDCoV The permissiveness of ANPEP WT and KO PAMs and broblast like cells for TGEV and PDCoV infection was also evaluated by determining percent virus antigen positive cells after infection with di erent MOIs of virus The results for WT PAMs are shown in Fig 2A An MOI 1 produced 20 and 80 antigen positive cells for PDCoV and TGEV respectively The corresponding virus dilution endpoints were 0 0001 and 0 1 MOI Increasing the MOI to 10 increased the percent PDCoV Fig 1 Coronavirus infection of porcine alveolar macrophages PAMs and lung derived bro blast like cells from ANPEP wild type WT and knockout KO pigs A C PAMs and lung bro blasts were infected with PDCoV at an MOI of 0 1 the infected cells were xed and stained with AlexaFluor488 labeled anti PDCoV antibody and nuclei were counterstained with propidium iodide PI at two days after infection B D PAMs and lung broblasts were infected with TGEV at an MOI of 1 the infected cells were xed and stained with AlexaFluor488 labeled anti TGEV antibody and nu clei were counterstained with PI at 48 h post infec tion Representative pictures are shown from ex periments performed on cells derived from three WT and three KO pigs Fig 2 PDCoV and TGEV infection of PAMs and lung derived broblast like cells A ANPEP WT and KO PAMs were infected with di erent MOIs of TGEV or PDCoV After 24 h from infection the cells were stained and xed with AlexaFluor488 labeled anti TGEV or anti PDCoV antibodies and the percent antigen positive cells recorded B ANPEP WT and KO lung derived broblast like cells were infected with TGEV or PDCoV at an MOI of 0 0001 10 After 24 h from infection the cells were stained and xed with AlexaFluor488 la beled anti TGEV or anti PDCoV antibodies and the percent antigen positive cells determined Results are shown as the mean and standard deviation of results from three ANPEP WT and three KO pigs A Stoian et al Virology 541 2020 136 140 137 antigen positive WT PAMs to 40 For the KO PAMs an MOI of 1 showed no TGEV or PDCoV antigen positive cells However increasing the MOI to 10 resulted in 2 PDCoV antigen positive PAMs Results for the infection of the WT broblast like cells presented in Fig 2B showed that an MOI 1 produced 80 TGEV and 40 PDCoV antigen positive cells The dilution endpoints were 0 0001 MOI for TGEV and 0 001 MOI for PDCoV For the KO broblast like cells TGEV infection was not detected whereas PDCoV infection resulted in 35 antigen positive cells at an MOI 1 with an endpoint of 0 01 The results for virus yields on ANPEP WT and KO broblast like cells are shown in Fig 3 At an MOI 1 PDCoV grew to near equal levels on broblast like cells derived from the ANPEP WT and KO pigs whereas TGEV could only be propagated on broblast like cells de rived from the ANPEP WT pigs 2 2 PDCoV infection of ANPEP WT and KO pigs Previous work from our lab showed that ANPEP KO pigs are com pletely resistant to infection with TGEV Whitworth et al 2018 We tested the ability of PDCoV to infect ANPEP KO pigs The results for virus infection are summarized in Table 1 All pigs were positive for PDCoV nucleic acid at one day after infection On the second day two ANPEP WT and three KO pigs remained RT PCR positive By three days after infection only one pig an ANPEP KO pig was positive for PDCoV nucleic acid By four days after infection all pigs were negative for virus nucleic acid in feces Serum samples obtained at three days after infection were negative for virus nucleic acid data not shown At four days after infection one WT and one KO pigs were removed from the study and necropsied RT PCR ampli cation of PDCoV nucleic acid in intestines and mesenteric lymph nodes showed that both pigs were negative for PDCoV The presence of PDCoV infection was also assessed by virus speci c neutralizing activity measured in serum at the termi nation of the study 14 days after infection The results summarized in Table 1 showed that two of the remaining six pigs seroconverted one WT and one KO pig Clinical signs such as diarrhea and fever were not detected in any of the infected WT or KO pigs The absence of clinical signs is likely a result of the older age pigs used in the infection study Together the RT PCR and serological results showed that ANPEP KO pigs are not resistant to infection with PDCoV 3 Discussion The results from this study showed no PDCoV infection of PAMs obtained from ANPEP KO pigs see Fig 1A which supports the ob servations of Wang et al 2018 who concluded that APN is a receptor for PDCoV In contrast the PDCoV positive infection results for bro blast like cells derived from the ANPEP KO cultures support the ob servations of Zhu et al 2018 who concluded that APN is not a functional receptor for PDCoV see Fig 1C When taken together our data support the observations of Li et al 2018 who concluded that there are APN dependent and APN independent receptors for PDCoV We hypothesize that PAMs possess only the APN receptor while the lung derived broblast like cells possess both receptors However since the APN independent receptors for PDCoV have not been identi ed we currently cannot easily prove this hypothesis and it remains to be elu cidated in future studies Ultimately the importance of pAPN as a receptor for PDCoV is determined by the permissiveness of ANPEP KO pigs for infection The results in Table 1 showing positive results for the presence of PDCoV nucleic acid in the feces of infected ANPEP KO pigs con rms the results obtained following the infection of KO broblast like cells These data support the notion that APN is not required for PDCoV infection of pigs However the conclusion that ANPEP KO pigs retain permissiveness for infection with PDCoV comes with a few caveats For example all fecal samples on day 1 after infection were positive for PDCoV nucleic acid and by 3 days the virus was only detected in one KO pig Intestinal tissues from the two pigs tested one WT and one KO were negative for PDCoV The positive results obtained for feces can be explained by the presence of environmental contamination caused by the residual in oculum used for infection However support for a productive infection of the KO pigs comes from the seroconversion of one KO pig at 14 days after infection see Table 1 A second caveat comes from the transient nature of PDCoV infection in the WT and KO pigs used in this study which made it di cult to determine if APN plays a role in the disease process Previous studies show that neonatal piglets are more suscep tible than weaned pigs to enteric virus infection Thomas et al 2015 Jung et al 2015 The relationship of pig age and PDCoV infection outcome has not been studied in detail however it is speculated that PDCoV infection may be age dependent similar to PEDV with more severe outcomes in younger piglets In this study 28 33 day old ANPEP KO and WT pigs were used for PDCoV inoculation In future studies a more sensitive neonatal pig infection model can be used which may yield a more accurate determination regarding the role of APN in PDCoV infection of its natural host 4 Materials and methods 4 1 ANPEP KO pigs viruses and cells The ANPEP edited pigs used in this study were derived by breeding founder animals created using direct zygote injection of CRISPR Cas9 along with two CRISPR guides directed at exon 2 of ANPEP Whitworth et al 2017 Along with sequencing of the ANPEP gene the APN phenotype for each knockout allele was con rmed by the absence of CD13 expression in intestines and by the resistance of ANPEP KO pigs to infection with TGEV Whitworth et al 2018 Fig 3 Yields of PDCoV and TGEV on broblast like cells Fibroblast like cells were infected with an MOI 1 Virus concentration was measured by titration of virus on ST cells at two days after infection Table 1 RT PCR for PDCoV nucleic acid in feces and virus neutralizing activity in serum a Pig ANPEP Genotype RT PCR Virus Neutralization Day after infection 1234 18 Wild Type 38 1 40 40 40 1 64 b 19 Wild Type 35 2 37 3 40 40 40 40 ND c 20 Knockout 37 1 36 0 40 40 1 64 21 Knockout 35 1 37 1 40 40 40 ND a Ct 40 negative for detectable quantities of PDCoV nucleic acid Virus neutralization assays were performed at 14 days after infection A tite 1 16 was considered negative for detectable neutralizing activity All pigs at day 0 were negative for neutralizing activity b Neutralizing activity was measured on day 10 after infection c ND Not Done A Stoian et al Virology 541 2020 136 140 138 The PDCoV isolate South Dakota was used for the infection of cells and pigs Vitosh Sillman et al 2016 PDCoV stocks were prepared on ST cells maintained in MEM supplemented with 7 FBS Pen Strep 80 Units ml and 80 g ml respectively 3 g ml Fungizone 25 mM HEPES MEM and 0 2 g ml L 1 Tosylamide 2 phenylethyl chlor omethyl ketone TPCK trypsin as described in Chen et al 2015 After 2 h the media was replaced with MEM FBS with antibiotics TGEV Purdue strain was obtained from Iowa State University TGEV was maintained in the same media without TPCK Cells were maintained at 37 C and 5 CO 2 After 48 h the virus was harvested and titrated on ST cells Serial 1 10 dilutions of virus in triplicate were performed on a 96 well plate containing ST cells Twenty four h later the cells were xed for 10 min in 80 acetone and air dried For detection of PDCoV cells were stained with anti N protein mAb SD110 121 kindly provided by Steve Lawson at South Dakota State University which was diluted 1 500 in PBS with 5 goat serum PBS GS Detection of TGEV was performed using anti FIPV3 70 mAb Custom Monoclonals Interna tional USA diluted 1 700 in PBS GS After 1 h incubation with anti body at 37 o C the cells were washed with PBS and bound antibody detected with AlexaFluor488 labeled goat anti mouse IgG Cat No A 11001 Invitrogen diluted 1 400 in PBS GS After 1 h incubation at 37 C the plates were washed with PBS nuclei counterstained with propidium iodide PI and cells viewed under a uorescence micro scope The 50 tissue culture infectious dose TCID 50 ml was calcu lated according to the method of Reed and Muench 1938 The recovery of porcine alveolar macrophages is described in Wells et al 2017 Lungs were removed from euthanized pigs and lavaged by pouring 100 ml of cold PBS into the trachea PAMs were sedimented by centrifugation at 1200 g for 10 min at 4 C and cells re suspended and washed once in cold sterile PBS The nal cell pellet was re suspended in freezing medium containing 45 RPMI 1640 with antibiotics 45 FBS 10 dimethylsulfoxide DMSO and stored in liquid nitrogen until use For the infection approximately 10 4 PAMs were added to each well of a 96 well plate and incubated overnight at 37 o Cin5 CO 2 The cells were gently washed to remove non adherent cells Serial 1 10 dilutions of virus in media were added to wells in triplicate After an overnight incubation the cells were washed with PBS and xed for 10 min in 80 acetone Presence of either PDCoV or TGEV in the infected PAMs was con rmed as described above For enrichment of lung mesenchymal stem cells PAMs were cul tured in RPMI 1640 supplemented with 10 FBS L glutamine and antibiotics The continuous maintenance of PAMs for two weeks re sulted in the overgrowth of cultures by broblast like cells which comprise only about 1 of the lung lavage material The con uent cells were removed by trypsinization and passaged at least 2 times The in fection of broblast like cells was carried out as described for the PAM cultures 4 2 Infection of pigs All experiments involving animals and viruses were performed in accordance with the Federation of Animal Science Societies Guide for the Care and Use of Agricultural Animals in Research and Teaching the USDA Animal Welfare Act and Animal Welfare Regulations and after approval by the Kansas State University and University of Missouri institutional animal care and institutional biosafety committees Three KO piglets were obtained from an ANPEP sow mated with an ANPEP boar The ANPEP KO genotype of each piglet was con rmed by PCR and DNA sequencing To avoid unnecessary stress the weaned piglets were transported from the University of Missouri rearing facility to Kansas State University at three weeks of age Three ANPEP WT piglets obtained from a separate mating were included as positive infection controls At the time of virus infection the KO and WT pigs were 28 and 33 days old respectively Pigs were infected with 6 2 log 10 TCID 50 of PDCoV administered as a single oral dose in 20 ml of culture medium A 5 cm tube was attached to the end of the inoculating syringe to allow the virus to ow down the back of