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Whittaker Ithaca Available online 18 May 2012 Keywords Canine coronavirus CCoV of CCoV A76 type II strain CCoV 1 71 that more efficiently replicates in feline cells CCoV A76 can use canine aminopeptidase N cAPN receptor for infection of cells but was unable to use feline APN fAPN In contrast CCoV 1 71 can utilize both Genomic analysis shows that CCoV A76 possesses a distinct spike RNA e mammals al 20 2003 due when coinfections occur with canine parvovirus or other pathogens II also exist as two serotypes type I FCoV and type II FCoV based Contents lists available at SciVerse ScienceDirect Virolo Virology 430 2012 90 99 genetic source Lorusso et al 2008 Also closely related to the 1 These authors contributed equally to this work on distinct spike proteins Haijema et al 2007 It has been proposed that type I CCoVs and FCoVs evolved from a common ancestral virus and that the canine and feline type II lineages arose from multiple recombination events with an unidentified 0042 6822 see front matter Decaro et al 2009 Erles and Brownlie 2009 Ntafis et al 2011 The emergence of such highly virulent isolates of CCoV has significantly renewed interest in this group of viruses Receptor binding domain Recombination Introduction Coronaviruses are single stranded humans and a variety of non primat Lai and Holmes 2001 Perlman et viruses has greatly increased since N and C terminal domains NTD and C domain of the S1 subunit These data suggest that CCoV A76 represents a recombinant coronavirus form with distinct host cell tropism Decaro et al 2007 Escutenaire et al 2007 Evermann et al 2005 Naylor et al 2002 Sanchez Morgado et al 2004 Zappulli et al 2008 In some of these cases these new pathogenic viruses are able Spike protein which is the result of a recombination between type I and type II CCoV that occurred between the Characterization of a recombinant canine receptor binding S1 domain Andrew D Regan a 1 Jean K Millet a 1 Long Ping V Tse Edward J Dubovi b Christopher D Town c Gary R a Department of Microbiology and Immunology Veterinary Medical Center Cornell University b Animal Health Diagnostic Center College of Veterinary Medicine Cornell University c J Craig Venter Sequencing Center Rockville MD United States article info Article history Received 29 February 2012 Returned to author for revisions 30 March 2012 Accepted 17 April 2012 abstract Canine alphacoronaviruses gastroenteritis Here we character 1976 Serological studies show 1 71 Efficient replication journal homepage www elsev coronavirus with a distinct a Zach Chillag a Vera D Rinaldi a Beth N Licitra a a n Ithaca NY 14853 United States NY 14853 United States exist in two serotypes type I and II both of which can cause severe ize a canine alphacoronavirus designated CCoV A76 first isolated in that CCoV A76 is distinct from other CCoVs such as the prototype CCoV is restricted to canine cell lines in contrast to the prototypical gy CCoV FCoV type II lineage is TGEV whose origin is unclear but which may have arisen from zoonotic transfer of a type II CCoV to pigs Lorusso et al 2008 The coronavirus spike protein is a major antigenic determinant and is also responsible for host cell receptor binding and viral entry Gallagher and Buchmeier 2001 Aminopeptidase N APN has been shown to act as a cellular receptor for FCoV CCoV and TGEV Wentworth and Holmes 2001 Although each virus would be assumed to utilize a species specific homolog in its respective host during in vivo infection the feline homolog fAPN can act in vitro as a common receptor for type II FCoV type II CCoV and TGEV Tresnan et al 1996 This situation is unlike the one found for most other coronaviruses which have highly species specific receptors This broad receptor binding ability likely plays a role in the zoonotic transfer and genetic recombination events that have defined the evolution of animal alphacoronaviruses In the case of the serotype I FCoV CCoV group the receptor determinants are much less certain While there is some evidence for fAPN as an further study Here we report data on in vitro growth antigenic and genomic analysis of CCoV A76 and show that it represents a recombinant CCoV Results In vitro cultivation of CCoV A76 A previous study indicated CCoV A76 to be antigenically distinct from other CCoV isolates Corapi et al 1992 however further characterization was not reported To perform more extensive analysis we obtained samples of CCoV A76 from the Animal Health Diagnostic Center Cornell University Ithaca NY and inoculated a canine cell line A 72 Supernatant and cells were tested for viral titer by plaque assay on A 72 cells demon strating that CCoV A76 infection produces large quantities of TGEV Purdue were all antigenically analyzed for comparison The A 72 A D Regan et al Virology 430 2012 90 99 91 FCoV type I receptor Tresnan et al 1996 other studies have concluded that there is a distinct receptor for FCoV type I Dye et al 2007 Hohdatsu et al 1998 Receptor determinants for CCoV type I remain essentially unknown In addition to a specific proteinaceous receptor there are indications that lectin based interactions via sugar moieties on either the virus or the host may play a role in the receptor binding complex for FCoV types I and II and TGEV Krempl et al 1997 Regan et al 2010 Regan and Whittaker 2008 Schultze et al 1996 Van Hamme et al 2011 Most CCoVs isolated to date are type II viruses These are easily cultivated in vitro Pollock and Carmichael 1983 with canine A 72 cells typically used for virus isolation In contrast type I CCoVs have yet to be easily cultivated in cell culture and were only recently discovered by reverse transcription polymerase chain reaction from canine fecal RNA Pratelli et al 2003 CCoV A76 was isolated from a closed breeding colony of Beagles at the James A Baker Institute for Animal Health Cornell University Ithaca NY in 1976 The dogs presented primarily with enteritis but the virus also appeared to cause additional clinical signs with significant morbidity in litters of newborn pups exposed to the virus and with abortions in some pregnant bitches Carmichael 1978 The virus was readily isolated and was later noted to possess distinct antigenic characteristics as compared to other type II CCoV isolates Corapi et al 1992 CCoV A76 was archived at the Animal Health Diagnostic Center College of Veterinary Medicine Cornell University Ithaca NY and further characterization was not performed In light of recent interest in CCoV we obtained this specimen from the archive for Fig 1 In vitro growth and antigenic identification of CCoV A76 A 72 cells were inoculated quantified 48 h p i for CCoV A76 and other closely related alphacoronaviruses A coronavirus N monoclonal antibody mAb FIPV3 70 B with CCoV A76 and extracellular virus EV or cell associated virus CAV was cells were infected with CCoV A76 and stained 12 hours p i with the anti distantly related betacoronavirus MHV A59 was also tested as an outlier control The polyclonal anti CCoV sera and all three anti nucleocapsid mAbs reacted strongly with all alphacoronavirus isolates tested including CCoV A76 but did not react with MHV A59 Fig 2B All six anti spike mAbs reacted with FCoV 1146 FCoV 1683 FCoV DF2 Fig 2B Four of the six anti spike mAbs 18A7 4 19G11 10 22G6 4 23A1 8 reacted with the CCoV type II isolates CCoV 1 71 CCoV S378 CCoV K378 and TGEV Purdue extracellular particles Fig 1A This is similar to results observed with other viruses such as type II CCoV 1 71 type II FCoV 1146 and TGEV Purdue but different than virus isolates such as type I FCoV TN406 FIPV Black which exclusively produce cell asso ciated virions Fig 1A The identification of A76 as a coronavirus was confirmed by immunofluorescence microscopy using the antibody FIPV3 70 a generally cross reactive CoV nucleocapsid specific monoclonal antibody Fig 1B Antigenic analysis of CCoV A76 The antigenic characteristics of CCoV A76 along with other alphacoronaviruses were further analyzed by immunofluorescence microscopy using polyclonal anti CCoV sera with mAb FIPV3 70 and with a panel of monoclonal antibodies previously tested against CCoV A76 anti nucleocapsid N 16C11 13 17B7 1 anti spike S 18A7 4 18H9 1 19G11 10 21D10 2 22G6 4 23A1 8 Corapi et al 1992 Fig 2A and B The CCoV type II isolates CCoV 1 71 CCoV S378 CCoV K378 the FCoV type II isolates FCoV 1146 FCoV 1683 FCoV DF2 the FCoV type I isolate FCoV TN406 FIPV Black and A D Regan et al Virology 430 2012 90 9992 Fig 2B None of the six anti spike mAbs cross reacted with CCoV A76 Fig 2B Cell tropism of CCoV A76 To further characterize the in vitro growth characteristics of CCoV A76 multiple cell lines including canine A 72 MDCK Cf2Th CDKE 2 feline CRFK AK D Fc2Lu FMEC porcine LLC PK1 and murine NIH 3T3 were inoculated with CCoV A76 at an MOI of 1 Fig 3A and B All cell lines were also inoculated with closely related CCoV type II isolates CCoV 1 71 CCoV S378 CCoV K378 FCoV type II isolates FCoV 1146 FCoV 1683 FCoV DF2 FCoV type I isolate FCoV TN406 and TGEV Purdue The CCoV type II isolates CCoV 1 71 CCoV S378 and CCoV K378 infected all feline cell lines tested but did not infect any canine or porcine cell lines except A 72 cells at a low efficiency 30 27 and 22 respectively Fig 3B The FCoV type II isolates FCoV 1146 FCoV 1683 and FCoV DF2 also efficiently infected all feline cell lines and A 72 cells but not other canine or porcine cell lines tested Fig 3B The FCoV type I isolate FIPV TN406 infected the feline cell line AK D only Fig 3B TGEV Purdue efficiently infected the porcine LLC PK1 cells and also infected all feline cell lines and A 72 cells at overall reduced efficiency CCoV A76 efficiently infected all canine cell lines tested but was unable toinfectany feline cell lines CCoV A76 Fig 2 Antigenic analysis of CCoV A76 CCoV A76 and CCoV 1 71 infected cells were 16C11 13 and the anti FCoV spike mAb 18A7 4 A Cells were infected with an assortment antisera the anti FCoV nucleocapsid mAbs FIPV3 70 16C11 13 and 17B7 1 or the anti FCoV represents a positive reaction C0 represents a negative reaction infected LLC PK1 cells at a reduced efficiency 24 None of the viruses tested infected the murine NIH 3T3 cell line Fig 3B Use of canine and feline APN as a CCoV A76 receptor Because of the distinct tropism for CCoV A76 for canine vs feline cells we examined the utilization of canine vs feline APN as a CCoV A76 receptor Fig 4 Non permissive BHK cells were transfected to express either fAPN or cAPN and infected with CCoV A76 along with CCoV 1 71 and FCoV 1683 as controls CCoV A76 was able to infect BHK cells expressing cAPN but was unable to infect cells expressing fAPN Fig 4A In contrast both CCoV 1 71 and FCoV 1683 were able to infect cells expressing either cAPN or fAPN Fig 4B and C Sequencing and phylogenetic analysis of CCoV A76 genome Viral RNA was purified from CCoV A76 particles and subjected to genome sequencing C Town unpublished results Most viral ORFs were successfully sequenced and annotated with the exception of ORF1ab where only the 3 0 end of ORF1ab sequence has been obtained In general the sequences for all non structural and structural proteins except for the spike were more related to type II CCoV than to type I Supplementary Fig 1 suggesting that stained by immunofluorescent microscopy with the anti FCoV nucleocapsidmAb of closely related alphacoronaviruses or MHV and stained with either anti CCoV spike mAb 18A7 4 18H9 1 19G11 10 21D10 2 22G6 4 or 23A1 8 B A D Regan et al Virology 430 2012 90 99 93 themostprobableancestorofCCoV A76wasatypeIIvirus Intriguingly this analysis has shown that the S protein has a sequence that would correspond to an intermediate between pro totypical type I and type II CCoVs CCoV Elmo 02 and CCoV 1 71 respectively Supplementary Fig 1 Phlyogenetic analysis of CCoV A76 spike Because the CCoV A76 spike sequence did not cluster well with either type I or II CCoVs and since the spike protein is a determinant of cell tropism and viral pathogenesis we focused on the spike protein sequence to perform a more extensive phylo genetic analysis Fig 5 Overall the complete spike protein was distinct from other alphacoronaviruses and did not cluster with either type I or type II CCoVs Fig 5A Analysis of the S2 fusion domain showed that it clustered closely with CCoV type II FCoV type II and TGEV Fig 5B In contrast the S1 receptor binding domain clustered with type I CCoV Elmo 02 and type I FCoV RM Fig 5C Notably the CCoV A76 spike sequence lacked a putative furin cleavage site RRARR previously shown to be present at the S1 S2 junction of the CCoV type I virus Elmo 02 de Haan et al 2008 Pratelli et al 2003 indicating it was more similar to CCoV type II viruses that do not contain consensus furin cleavage sites Supplementary Fig 2 Coronavirus S1 domains contain two independent functional sub domains the N terminal domain NTD and the C terminal domain C domain Peng et al 2011 To better characterize CCoV A76 S1 the NTD and C domain were independently analyzed by phylogenetic analysis The CCoV A76 NTD clustered closely with Elmo 02 and did not Fig 3 Cell tropism of CCoV A76 Canine MDCK cells were infected with either CCoV A76 coronavirus N mAb FIPV3 70 A A variety of cells lines were tested for their susceptibility are expressed as percent infected cells as determined by immunofluorescence assay The Felis catus cat and Mus musculus mouse are specified on top of cell line names 4500 represent standard deviation of the means cluster with 1 71 Fig 5D In contrast the CCoV A76 C domain was divergent and did not clearly cluster with any characterized alphacoronavirus Fig 5E Overall these data indicate that the CCoV A76 spike gene is probably a recombinant of a type I CCoV with a type II CCoV spike with a recombination site located between the NTD and C domain of S1 To determine possible recombination sites within S1 we used the BOOTSCAN RESCAN method to identify recombination events and breakpoints in the CCoV A76 S nucleotide sequence This analysis confirmed the presence of recombination between the NTD and C domain with a breakpoint at nucleotide 890 Fig 6 Discussion In a survey of canine coronaviruses stored at Cornell Univer sity we carried out a retrospective analysis of a CCoV first isolated in 1976 CCoV A76 which has previously been shown to be antigenically distinct from typical type II CCoVs Corapi et al 1992 Immunofluorescence experiments have shown that a panel of antibodies specific for the coronavirus N protein cross reacts with CCoV A76 but a panel of antibodies specific for the type II FCoV S protein fails to cross react with the virus Importantly while both CCoV A76 and CCoV 1 71 like viruses can infect canine A 72 cells CCoV A76 shows distinct and pronounced tropism for a variety of additional dog cell lines e g MDCK as well as pig cell lines e g LLC PK1 that the prototypical 1 71 like CCoVs fail to infect Additionally CCoV A76 fails to infect cells of cat origin e g CRFK AK D which are very efficiently infected by or CCoV 1 71 and stained for immunofluorescence microscopy with the anti to CCoV A76 and a number of closely related coronavirus isolates B Results mammalian species of each cell line used Canis familiaris dog Sus scrofa pig cells were quantified from each of three independent experiments Error bars A D Regan et al Virology 430 2012 90 9994 1 71 like CCoVs These serological data suggested that CCoV A76 possesses distinct characteristics setting it apart from other known CCoVs Phylogenetic analysis of the CCoV A76 genome shows that it is mostly related to type II CCoV however analysis of the spike sequence at the amino acid level places CCoV A76 as an outlier of the CCoV group suggesting that CCoV A76 is genetically distinct Notably CCoV A76 is genetically distinct from recently isolated highly virulent type IIb CCoV strains such as CCoV CB 05 CCoV A76 S appears to be a recombinant of a type II and a type IIa CCoV with recombination occurring between the NTD and C domain of S1 Fig 7 Further proof of a recombination event occurring at the spike gene of CCoV A76 was found by analyzing its nucleotide coding sequence Indeed in close proximity of the recombination breakpoint identified by the BOOTSCAN RESCAN methodology nucleotide 890 several stretches of nucleotides that were either identical TCTAA found twice at nucleotide 932 and at nucleotide 972 or near identical ATAAAAAT at nucleotide 862 and GTAAAATG at nucleotide 990 to characterized CCoV recombination signals TCTAA and G CTAAAAA GT Wang and Lu 2009 have been identified data not shown The divergent C domain might also suggest that a second recombination may have occurred at the vicinity of the S1 S2 boundary with an as yet uncharacterized coronavirus but evidence for this is currently lacking Overall the body of evidence in favor of a recombinant origin for the spike gene of CCoV A76 together with its distinct antigenic and tropism profiles argue in favor of setting it apart from other CCoVs as a type I II canine coronavirus Fig 4 Canine APN but not feline APN as a functional receptor for CCoV A76 Non permissive FLAG canine APN cAPN feline APN fAPN or mock transfected for 18 h The transfected FCoV 1683 C or mock infected D for 8 h Cells were stained for immunofluorescence RG4 and anti FLAG M2 antibodies for feline APN and canine APN expression respectively While it is clear that CCoV A76 has a much more highly species specific receptor binding pattern compared to typical type II CCoVs e g CCoV 1 71 or to typical type II FCoVs e g FCoV 1683 the reasons behind this are less clear The APN binding site is within the C domain of S1 and can be operationally defined as the D3 domain residues 526 676 based on TGEV nomenclature Reguera et al 2011 However individual amino acids within the D3 domain that might confer strict species specificity have not been identified BLAST analysis based on the amino acid sequence of CCoV A76 D3 domain shows that it is most similar to type II CCoV but is clearly divergent Supplementary Fig 3 It also shows some similarity to porcine coronaviruses perhaps accounting for the limited tropism seen for porcine cells The divergent C domain may impart more strict species specificity to CCoV A76 via specific interactions within the D3 domain It is also possible that an interaction between the APN binding site and the distinct CCoV type I like NTD found in CCoV A76 might account for the distinct species specificity of this virus Overall while CCoV A76 clearly has a recombinant origin it should be remembered that the recombination event has not occurred within the known APN binding domain D3 domain residues 526 676 but between the NTD and the C domain around residue 297 Fig 7 As such the recombination event itself is unlikely to be directly responsible for the distinct receptor binding characteristics of CCoV A76 which may be caused by D3 domain divergence through missense mutations or via the influence of the distinct NTD In the case of APN itself previous studies have shown that molecular determinants of species specificity lie in glycosylation BHK 21 cells were transfected with 400 ng of plasmids encoding cells were then infected at an MOI of 1 5 with CCoV A76 A CCoV 1 71 B microscopy analysis with anti coronavirus N mAb FIPV3 70 for infection and with A D Regan et al Virology 430 2012 90 99 95 sequon differences Comparing cAPN to fAPN using NetNGlyc glycan prediction software reveals that cAPN is more extensively glycosylated than fAPN with cAPN having 5 potential glycosyla tion sites on the solvent exposed domain while fAPN has only 2 In particular residues 291 293 were shown to control species specificity between human porcine and feline APN Wentworth and Holmes 2001 However this glycosylation sequon is absent for both fAPN and cAPN Analysis of the spike interacting APN Fig 5 Phylogenetic analysis of CCoV A76 spike The phylogenetic analysis was performed sequences including the transmembrane TM and C termini