Arch Virol (2001) 146: 791799 Feline and canine coronaviruses are released from the basolateral side of polarized epithelial LLC-PK1 cells expressing the recombinant feline aminopeptidase-N cDNA Brief Report J. W. A. Rossen1,2, J. Kouame1, A. J. W. Goedheer1, H. Vennema1, and P. J. M. Rottier1 1Institute of Virology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands 2Pediatric Gastroenterology and Nutrition, Erasmus University Medical Center, Rotterdam, The Netherlands Accepted November 24, 2000 Summary.Inthisstudyfeline(FECVandFIPV)andcanine(CCoV)coronavirus entry into and release from polarized porcine epithelial LLC-PK1 cells, stably expressing the recombinant feline aminopeptidase-N cDNA, were investigated. Virusentryappearedtooccurpreferentiallythroughtheapicalmembrane,similar to the entry of the related porcine coronavirus transmissible gastroenteritis virus (TGEV) into these cells. However, whereas TGEV is released apically, feline and canine coronaviruses were found to be released from the basolateral side of the epithelial cells. These observations indicate that local infections as caused by TGEV,FECVandCCoVdonotstrictlycorrelatewithapicalrelease,assuggested by earlier work. * Coronaviruses have been shown to infect humans and animals. The viruses have a narrow host range and the consequences of infection range from subclinical to lethal, symptoms including respiratory and enteric disease (most commonly) as wellashepatitis,peritonitisandencephalomyelitis5,6.Theprimarytargetcells of coronaviruses are the epithelial cells of the respiratory and/or gastrointestinal tract. Epithelial cells are organized in a layer and have a polarized organization, i.e.,theyhaveanapicalsidefacingtheexternalenvironmentandabasolateralside facing the inner environment 17, 18. Previously, we have shown that corona- viruses, after being assembled intracellularly, are released from a specifi c side of thesecellsdependingonthevirusandthecells.Thus,transmissiblegastroenteritis virus(TGEV)wasreleasedfromtheapicalsideofporcinekidneyLLC-PK1cells 792J. W. A. Rossen et al. 8,whereasmousehepatitisvirus(MHV)wasreleasedbasolaterallyfrommurine kidneyepithelialcells(mTAL;9)aswellasfromLLC-PK110andfromhuman colon carcinoma cells (Caco-2; 11) both stably expressing the recombinant MHV receptor cDNA. We suggested that the apical release of TGEV and the basolateral release of MHV might be factors contributing to the difference in pathogenesisfoundbetweenTGEVandMHVintheirrespectivenaturalhosts,the former causing mainly a localized enteric infection, the latter spreading through the body to other organs. However, MHV appeared to be released from the apical membrane when tested in canine kidney cells (MDCK) that stably expressed the recombinant MHV receptor cDNA 11, an observation incompatible with our hypothesis. To fi nd out whether this was just an exception, we extended our studies to includefelinecoronaviruses(FCoVs)andcaninecoronavirus(CCoV)that,unlike MHV,belongtothesamecoronavirusgroupasTGEV4.Oftheseviruses,CCoV and feline enteric coronavirus (FECV) cause local, enteric infections whereas feline infectious peritonitis virus (FIPV) affects many organs usually resulting in fatal peritonitis. Because no well-characterized epithelial cell line was available to us that can be infected by these viruses, porcine kidney cells (LLC-PK1) cells were made susceptible by introducing the virus receptor. To this end, LLC-PK1 cells were transfected with pCR3-fAPN (a kind gift of Dr. Kay Holmes, Department of Mi- crobiology,UniversityofColoradoHealthSciencesCenter,Colorado,USA)con- taining the neomycin resistance gene and the recombinant feline aminopeptidase N (fAPN) cDNA. The latter encodes the protein that has been shown to function as a receptor for feline, canine, porcine and human coronaviruses. G418-resistant colonies were propagated and functionally tested for fAPN expression by assess- ing their susceptibility to FIPV (results not shown). One of the positive cell lines was selected for further experimentation and was named PKFA-2. This cell line remainedsusceptibletoFIPVinfectionforatleast20passages.PKFA-2cellswere grownonglasscoverslipsandinfectedwithFIPVstrain1146,FECVstrain1683, CCoV strain I-71 or with the Purdue strain of TGEV. At 6h p.i. cells were fi xed withice-coldmethanolandpreparedforindirectimmunofl uorescenceanalysisus- ingforTGEVaporcineanti-TGEVserum(akindgiftofDr.LuisEnjuanes,Centro NacionaldeBiotecnologia,CSIC,UniversidadAutnoma,CantoBlanco,Madrid, Spain) and for FCoVs and CCoV a combination (1:1) of antiserum G73 15 and ascitic fl uid A40, both obtained from experimentally FIPV-infected cats. The c Fig. 1. Susceptibility of PKFA-2 cells to CCoV, FECV, FIPV and TGEV infection. To prepare a cell line stably expressing the FIPV receptor glycoprotein, LLC-PK1 cells were transfectedwithpCR3-fAPN,aplasmidcontainingthefelineAPNreceptorgene14.G418- resistan