A coronavirus was isolated from feces of a diarrheic foal and serially propagated in human rectal adenocarcinoma (HRT-18) cells. Antigenic and genomic characterizations of the virus (isolate NC99) were based on serological comparison with other avian and mammalian coronaviruses and sequence analysis of the nucleocapsid (N) protein gene. Indirect fluorescent-antibody assay procedures and virus neutralization assays demonstrated a close antigenic relationship with bovine coronavirus (BCV) and porcine hemagglutinating encephalomyelitis virus (mammalian group 2 coronaviruses). Using previously described BCV primers, the N protein gene of isolate NC99 was amplified by a reverse transcriptase PCR (RT-PCR) procedure. The RT-PCR product was cloned into pUC19 and sequenced; the complete N protein of NC99 (446 amino acids) was then compared with published N protein sequences of other avian and mammalian coronaviruses. A high degree of identity (89.0 to 90.1%) was observed between the N protein sequence of NC99 and published sequences of BCV (Mebus and F15 strains) and human coronavirus (strain OC43); only limited identity (<25%) was observed with group 1 and group 3 coronaviruses. Based on these findings, the virus has been tentatively identified as equine coronavirus (ECV). ECV NC99 was determined to have close antigenic and/or genetic relationships with mammalian group 2 coronaviruses, thus identifying it as a member of this coronavirus antigenic group.

Truncated human coronavirus HCoV-229E spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN). Soluble hAPN neutralized the infectivity of HCoV-229E virions at 37°C, but not 4°C. Binding of hAPN may therefore trigger conformational changes in the viral spike protein at 37°C that facilitate virus entry.Human coronaviruses HCoV-229E in serogroup I and HCoV-OC43 in serogroup II are, after rhinoviruses, the second most important cause of the common cold (7, 13). The first step in HCoV-229E infection is binding of the trimeric 200-kDa viral spike glycoprotein (S) to human aminopeptidase N (hAPN, CD13) on human cells (26). The three-dimensional structures of the spike glycoproteins of the plus-strand RNAcontaining coronaviruses are not yet known. However, the structure of S is predicted to be generally similar to that of type 1 viral fusion proteins of large negative-strand RNA viruses (12,22,25). The approximately 547-amino-acid (aa)-long Nterminal S1 domain of HCoV-229E spike protein has receptorbinding activity (2), and the membrane-anchored S2 domain contains several heptad repeats (3). APNs of the normal host species are also receptors for porcine, feline, and canine coronaviruses in group I (5, 6, 15, 23). The metalloprotease APN is a 150-kDa, class II glycoprotein that is expressed as a dimer on apical membranes of polarized epithelial cells, at synaptic junctions, and on antigen-presenting cells (11,17,19,21).Soluble receptors for many viruses have been used to identify critical receptor-binding sites on the viral attachment proteins, explore the specificity of virus-receptor interactions, and characterize conformational changes in the viral attachment protein that are induced by receptor binding (10,14,18,20,28). Temperature-dependent triggering of such conformational changes in a virus attachment protein by receptors on host cell membranes leads to penetration of the viral genome into the cytoplasm of the host cell. Binding of receptor to the N-terminal domain of a type 1 viral fusion protein at 37°C can trigger a conformational change in the membrane-anchored domain, leading to fusion of the viral envelope with host cell membranes (9,10,14,27). This paper reports the expression in insect cells and characterization of a soluble recombinant hAPN protein (shAPN) and interactions of shAPN with HCoV-229E virions, the S1 domain of the viral spike glycoprotein, and truncated S1 proteins. Characterization of shAPN.A soluble form of hAPN lacking the stalk, transmembrane, and intracellular domains (anchor/ stalk-minus APN), was previously expressed in mammalian cells and shown to be enzymatically active (24). For these studies, we expressed in insect cells the 95-to 98-kDa soluble, anchor/stalk-minus protein (shAPN). The open reading frame included the BiP cleavable signal sequence (MKLCILLAVVA FVGLSGL), a linker (RS), and aa 66 to 967 of hAPN with a C-terminal V5 epitope and His tag. We purified the protein on Ni-nitrilotriacetic acid (NTA) resin...

A turkey coronavirus (TCV [NC95]) was characterized by antigenic comparison with other avian and mammalian coronaviruses using immunofluorescence (FA) and immunoperoxidase (IP) procedures. Based on FA and IP procedures, TCV (NC95) was determined to be antigenically indistinguishable from turkey enteric (bluecomb) coronavirus (TECV). In addition, TCV (NC95) and TECV were found to be closely related to infectious bronchitis virus (IBV); a one-way antigenic relationship was demonstrated. Polyclonal antibodies specific for TECV and IBV reacted strongly against TCV (NC95), as determined by FA procedures. Monoclonal antibodies (MAbs) specific for IBV matrix protein (MAb 919) reacted strongly against TCV (NC95) and TECV as determined by FA and IP procedures; an IBV peplomer protein-specific MAb (MAb 94) did not recognize the two viruses. These studies suggest an identification of TCV (NC95) as a strain of TECV, and provide evidence of a close antigenic relationship between these viruses and IBV.

The 3' end of the turkey coronavirus (TCV) genome (1740 bases) including the nucleocapsid (N) gene and 3' untranslated region (UTR) were sequenced and compared with published sequences of other avian and mammalian coronaviruses. The deduced sequence of the TCV N protein was determined to be 409 amino acids with a molecular mass of approximately 45 kDa. The TCV N protein was identical in size and had greater than 90% amino acid identity with published N protein sequences of infectious bronchitis virus (IBV); less than 21% identity was observed with N proteins of bovine coronavirus and transmissible gastroenteritis virus. The 3' UTR showed some variation among the three TCV strains examined, with two TCV strains, Minnesota and Indiana, containing 153 base segments which are not present in the NC95 strain. Nucleotide sequence identity between the 3' UTRs of TCV and IBV was greater than 78%. Similarities in both size and sequence of TCV and IBV N proteins and 3' UTRs provide additional evidence that these avian coronaviruses are closely related.

A reverse transcriptase-polymerase chain reaction (RT-PCR) procedure and two monoclonal antibody (MAb)-based immunohistochemical procedures were developed for detection of turkey coronavirus (TCV) in tissues and intestinal contents/dropping samples. The RT-PCR, MAb-based fluorescent antibody (FA), and MAb-based immunoperoxidase (IP) procedures were compared with virus isolation (VI) for detection of TCV in experimentally infected turkeys. TCV was detected in experimentally infected turkeys as early as day 1 postexposure (PE) by each of the four detection procedures. TCV was detected as late as day 35 PE by FA or IP and days 42 and 49 PE by VI and RT-PCR, respectively. With VI as a reference, sensitivity and specificity of RT-PCR were 93% and 92%, respectively; specificity of both FA and IP was 96%, and sensitivities were 69% and 61%, respectively. Each of the examined procedures was highly specific, but the RT-PCR procedure was also highly sensitive. These findings demonstrate the utility of both immunohistochemistry and RT-PCR for detection of TCV. In addition, the findings indicate that RT-PCR is a highly sensitive and specific alternative to conventional diagnostic procedures.