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1、Characterization of a recombinant canine coronavirus with a distinct receptor-binding (S1) domain Andrew D. Regan a,1, Jean K. Milleta,1, Long Ping V. Tsea, Zach Chillaga, Vera D. Rinaldia, Beth N. Licitraa, Edward J. Dubovi b, Christopher D. Townc, Gary R. Whittakera,n aDepartment of Microbiology a
2、nd Immunology, Veterinary Medical Center, Cornell University, Ithaca, NY 14853, United States bAnimal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States cJ. Craig Venter Sequencing Center, Rockville, MD, United States a r t i c l e i n f o A
3、rticle history: Received 29 February 2012 Returned to author for revisions 30 March 2012 Accepted 17 April 2012 Available online 18 May 2012 Keywords: Canine coronavirus Spike protein Receptor binding domain Recombination a b s t r a c t Canine alphacoronaviruses (CCoV) exist in two serotypes, type
4、I and II, both of which can cause severe gastroenteritis. Here, we characterize a canine alphacoronavirus, designated CCoV-A76, fi rst isolated in 1976. Serological studies show that CCoV-A76 is distinct from other CCoVs, such as the prototype CCoV- 1-71. Effi cient replication of CCoV-A76 is restri
5、cted to canine cell lines, in contrast to the prototypical type II strain CCoV-1-71 that more effi ciently 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. Ge
6、nomic analysis shows that CCoV-A76 possesses a distinct spike, which is the result of a recombination between type I and type II CCoV, that occurred between the N- and C-terminal domains (NTD and C-domain) of the S1 subunit. These data suggest that CCoV-A76 represents a recombinant coronavirus form,
7、 with distinct host cell tropism. Perlman et al., 2008). Research on these viruses has greatly increased since 2003 due to the severe acute respiratory syndrome (SARS) pandemic caused by a zoonotic transmission of an animal coronavirus into the human population (EatonandWang,2007).Coronavirusesareph
8、ylogenetically divided into several genera termed alpha, beta, gamma and delta (King et al., 2011). There are two known coronavirus species that infect dogs: a newly identifi ed betacoronavirus termed canine respiratory coronavirus (CRCoV), and a more characterized alpha- coronavirus termed canine e
9、nteric coronavirus (CCoV) (Decaro and Buonavoglia, 2008). Within the CCoV alphacoronavirus species, there are two distinct serotypes (type I and type II), with both forms of CCoV transmitted via a fecaloral route (Decaro and Buonavoglia, 2008). CCoV is usually thought to cause only mild to inapparen
10、t enteritis (Pollock and Carmichael, 1983). However, in some cases CCoV infection can be fatal in young dogs, in particular when coinfections occur with canine parvovirus or other pathogens (Pratelli et al., 1999). In recent years, an increasing number of highly virulent CCoV infections have also be
11、en reported in healthy adult dogs without apparent coinfections (Buonavoglia et al., 2006; 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 to
12、 spread to other organs and are thought to be novel recombinant forms of CCoV, such as the CCoV type IIb viruses that are more closely related to transmissible gastroenteritis virus (TGEV) of pigs (Decaro and Buonavoglia, 2008; Decaro et al., 2009; Erles and Brownlie, 2009; Ntafi s et al., 2011). Th
13、e emergence of such highly virulent isolates of CCoV has signifi cantly renewed interest in this group of viruses. CCoV particles are composed of four major structural proteins termed spike (S), envelope (E), membrane (M) and nucleocapsid (N) (Hogue and Machamer, 2008). CCoV type I and CCoV type II
14、are closely related to each other except for markedly different spike proteins and the presence of an additional open reading frame (ORF3) in CCoV type I isolates (Lorusso et al., 2008). This is analogous to the situation with feline coronavirus (FCoV), which also exist as two serotypes (type I FCoV
15、 and type II FCoV) based 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 unidentifi ed genetic source (Lorusso
16、et al., 2008). Also closely related to the Contents lists available at SciVerse ScienceDirect journal homepage: Virology 0042-6822/$-see front matter Hohdatsu et al., 1998). Receptor determinants for CCoV type I remain essentially unknown. In addition to a specifi c 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;
