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.

Modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses, both tissue-culture-origin (TCO) and chicken-embryo-origin (CEO), were passaged 20 times in specific-pathogen-free chickens. After serial bird-to-bird passage, increased virulence was observed for CEO virus but not TCO virus. Increased mortality and increased severity and duration of respiratory disease were observed in chickens inoculated with chicken-passaged CEO viruses; only mild respiratory disease (no mortality) occurred in chickens inoculated with chicken-passaged TCO viruses. These findings suggest that ML ILT vaccine viruses may increase in virulence after bird-to-bird passage.

Domestic houseflies (Musca domestica Linnaeaus) were examined for their ability to harbor and transmit turkey coronavirus (TCV). Laboratory-reared flies were experimentally exposed to TCV by allowing flies to imbibe an inoculum comprised of turkey embryo-propagated virus (NC95 strain). TCV was detected in dissected crops from exposed flies for up to 9 hr postexposure; no virus was detected in crops of sham-exposed flies. TCV was not detected in dissected intestinal tissues collected from exposed or sham-exposed flies at any time postexposure. The potential of the housefly to directly transmit TCV to live turkey poults was examined by placing 7-day-old turkey poults in contact with TCV-exposed houseflies 3 hr after flies consumed TCV inoculum. TCV infection was detected in turkeys placed in contact with TCV-exposed flies at densities as low as one fly/bird (TCV antigens detected at 3 days post fly contact in tissues of 3/12 turkeys); however, increased rates of infection were observed with higher fly densities (TCV antigens detected in 9/12 turkeys after contact with 10 flies/bird). This study demonstrates the potential of the housefly to serve as a mechanical vector of TCV.

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.