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Antigenic and genomic relationships among tissue culture-adapted turkey enteric coronavirus (TCV) isolates, three strains of avian infectious bronchitis virus (IBV), and mammalian coronaviruses were investigated. Immunoblotting and immunoprecipitation experiments using polyclonal antisera showed that the four major structural proteins of TCV cross-reacted with the four homologous proteins of bovine enteric coronavirus (BCV), the N and M proteins of mouse hepatitis virus serotype 3, and the N protein of IBV. Close antigenic relationships between TCV and BCV were also established by seroneutralization and hemagglutination-inhibition. Of 49 monoclonal antibodies produced against either TCV or BCV, 11 differentiated the two viruses. Five of these monoclonal antibodies had neutralizing activities and were directed to either the peplomeric S (gp200-gp100) or hemagglutinin HE (gp140-gp65) glycoproteins. BCV cDNA probes tested on purified viral preparations and coronavirus-positive (by electron microscopy) fecal samples from diarrheic turkey poults confirmed the relatedness of TCV and BCV. The two viruses produced distinct cytopathic changes in HRT-18 cells in the presence of trypsin, whereas only TCV isolates were able to reproduce the clinical symptoms in turkey poults. Their matrix (M) proteins undergo different glycosylation processes.

Isolation of hemorrhagic enteritis virus (HEV) from spleens of infected turkeys in the MDTC-RP19 lymphoblastoid cell line was compared with detection of HEV antigen in the same spleens using the agar gel precipitation (AGP) test. A concordance of 80% was found between the two assays. Virus isolation had a sensitivity of 84% and a specificity of 88% compared with the AGP test. RP19 cells were also susceptible to infection with several other avian adenoviruses, but such infection was easily differentiated from that of HEV by a fluorescent-antibody (FA) test. Turkeys required 10(2) tissue-culture-infectious doses (TCID) to develop HE-specific lesions and 10(5) TCID to be killed. On the other hand, as little as 10 TCID of apathogenic HEV protected the poults against challenge with virulent HEV. The enzyme-linked immunosorbent assay (ELISA) for detection of HEV antibody was improved by using virus-infected RP19 cells as antigen. The ELISA appears to be more sensitive than the serum-neutralization test.

An enzyme-linked immunosorbent assay (ELISA) that detects antibodies specific for type II avian adenoviruses is described. Freon-treated extracts from spleens of hemorrhagic enteritis (HEV)-infected poults were a better source of antigen than the cell-culture-propagated virus. Antigen-coated microtiter wells treated with bovine serum albumin reduced nonspecific adsorption of immunoglobulins. The ELISA readily detected antibodies in hemorrhagic-enteritis- and marble-spleen-disease-infected field flocks of turkeys and pheasants. Antibody was also detected 1 week after vaccination of 2-week-old poults with cell-culture-prepared hemorrhagic enteritis vaccine. The assay was specific and more sensitive than agar-gel-precipitation and virus-neutralization tests. HEV-related antigens in infected spleens were also detected indirectly in a blocking ELISA by removal of HEV antibodies (Ab) from stock (Ab+) serum through antigen-antibody complex formation. Extracts from HEV-infected poults reduced specific antibody reactivities in HEV-antigen-coated wells, whereas extracts from HEV-challenged poults previously vaccinated with attenuated virus did not remove HEV antibodies.

Dissociated spleen lymphocytes from hemorrhagic enteritis virus (HEV)-free chickens, pheasants, and turkeys were infected with HEV in suspension cultures. Viral antigens were detected in cells of all three donor species by immunofluorescence. The age of chicken from which cells were obtained and the temperature of incubation influenced the development of viral antigens in cultured cells, which could be observed as early as 24 hr postinfection. This is the first time that HEV has been shown to infect cells in vitro.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies in turkey serum to hemorrhagic enteritis virus. The ELISA antigen was extracted from turkey spleens and partially purified with fluorocarbon. Antibodies were demonstrated in serum samples of breeding and meat flocks that had been naturally exposed to infection. These samples were also examined in parallel by agar-gel precipitin (AGP); most of the sera were AGP-positive. ELISA, however, was more sensitive in detecting antibodies in day-old sera that were AGP-negative. The passively acquired antibodies were no longer detected by 4 weeks of age. A brisk but short-lived secondary response was detected by ELISA in the sera of turkeys immunized with beta-propiolactone-inactivated extract of infected spleens.

A candidate breeder flock of turkeys was studied during and after an outbreak of rhinotracheitis. Laboratory studies revealed the presence of three pathogens during the acute phase of the disease. These agents were hemorrhagic enteritis virus (HEV), paramyxovirus type 2 (PMV2), and chlamydia. Twenty-five turkeys in the flock were individually identified, and blood samples from these birds were collected for serological studies each week for 21 weeks. The serological results revealed high titers for HEV and chlamydia but very low titers for PMV2.

Two methods for purifying the virus of hemorrhagic enteritis from infected turkey spleens are described. One procedure utilized precipitation with polyethylene glycol, and the other consisted of trichlorotrifluoroethane extraction. Both procedures included sucrose-cesium chloride gradient centrifugation in the final purification step. The buoyant density of the viral fraction was 1.34 g/cm3, typical for adenoviral particles, and the size and morphologic characteristics of the virions observed by transmission electron microscopy suggested that the purified virus belongs to the family Adenoviridae. The biologic activity of the purified virus was titrated by inoculating 10-fold dilutions of the viral suspension into turkey poults. Mortality and hemorrhagic diarrhea proved to be inconsistent parameters of infection, and the degree of splenomegaly was proportional to the virus dose. The body/spleen ratio was the parameter selected for measuring viral activity, and the body/spleen ratio 50% was adopted as the unit for the titration of the virus. By using the same system it was demonstrated that the infectivity of the virus could be neutralized with antiserum produced in turkeys.

The efficacy of oral vaccination for hemorrhagic enteritis of turkeys was assessed by comparing flocks raised on the same premises, under the same management, with and without vaccination. The immunizing virus, a strain of marble spleen disease virus of pheasants, was administered via the drinking water. Vaccinated and unvaccinated turkeys differed significantly in feed conversion rates and spleen weights after challenge.

The effect of maternal antibody (MAB) to hemorrhagic enteritis (HE) on the response of turkeys to infection with virulent and avirulent strains of HE virus (HEV) was examined. The influence of age at exposure and treatment with HEV antibody on development of clinical HE also was studied. MAB protected poults from clinical HE for up to 6 weeks of age. MAB also interfered with vaccination against the disease for at least 5 weeks after hatching, as indicated by absence of HEV antigen in spleens and by poor seroconversion at 6 days and at 3 weeks post-vaccination, respectively. The incidence of clinical HE in MAB-negative poults was significantly higher in poults inoculated with virus at 15 days of age or older than in poults inoculated at 1-13 days of age. Further, MAB-negative poults embryonally inoculated with virulent or avirulent strains of HEV did not develop disease; these poults developed antibody and resisted challenge with virulent virus at 6 weeks of age. Poults treated with HE antibody within 1 hour of challenge or at 1, 3, or 5 weeks before challenge with virulent virus were protected against lesions and mortality induced by HEV. These results suggest that MAB may influence susceptibility of turkeys to infection with HEV for at least 5 to 6 weeks after hatching, unlike the case with most other viral infections of poultry. The results confirm that early age resistance to clinical HE is independent of MAB and suggest that such resistance persists for up to 13 days of age. The data also suggest that turkeys lacking MAB can be immunized against HE by embryo vaccination.

The Minnesota strain of turkey enteric coronavirus (TCV) was propagated in HRT-18 cells, a cell line derived from human rectum adenocarcinoma. A productive non-cytopathic infection was established, without a previous adaptation, in these cells as shown by the specific hemagglutinating activity in cell culture supernatants. A post-embedding immunochemical technique, using specific antiserum directed against the original egg-adapted virus and colloidal-gold-labelled protein A as the electron-dense marker, was used for the identification of the virus and related antigens in the cells by electron microscopy. Budding of typical coronavirus particles, through intracytoplasmic membranes and accumulation of complete virus within cytoplasmic vesicles or the lumen of rough endoplasmic reticulum, were the main features of the viral morphogenesis. Late in infection, numerous progeny viral particles were shown at the outer surface of infected cells, but budding could not be demonstrated at this level. Two different types of surface projections were observed on the extracellular particles of this avian coronavirus. These morphological characteristics have been thus far described only for mammalian hemagglutinating coronaviruses.

The interaction between a panel of ten monoclonal antibodies and hemorrhagic enteritis virus, a group II avian adenovirus, was determined. The monoclonal antibodies reacted with all nine isolates of group II avian adenoviruses, but not with any of five types of group I avian adenoviruses. All ten monoclonal antibodies recognized antigenic determinants on the hexon protein of hemorrhagic enteritis virus when analyzed by immunoprecipitation and immunoblotting. They reacted only with the native hexon protein and not with protein denatured by sodium dodecyl sulfate or guanidine-HCl/urea treatment combined with reduction and carboxymethylation. Based on the results of competitive binding assays, the panel of monoclonal antibodies could be subdivided into two groups, which recognized different antigenic domains of the hemorrhagic enteritis virus hexon protein. The monoclonal antibodies in group 1 neutralized hemorrhagic enteritis virus infectivity while the monoclonal antibodies of group 2 did not. Group 1 consisted of eight monoclonal antibodies which could be further subdivided into subgroups 1A, 1B, 1C and 1D. The subdivision of the monoclonal antibodies was based on the degree of blocking in the competitive binding assays and differences in their ability to induce enhancement. In general, the monoclonal antibodies had a higher avidity for the virulent isolate of hemorrhagic enteritis virus than for the avirulent hemorrhagic enteritis virus isolate.

A double-antibody ELISA for the detection of coronaviruses in intestinal contents from turkey poults with diarrhea was developed. Antibodies were raised in rabbits and guinea pigs against a Minnesota isolate of turkey enteric coronavirus (TCV) propagated in embryonating turkey eggs and were purified by density-gradient centrifugation. The specificity of antisera was confirmed by hemagglutination-inhibition and immunoelectron microscopy. Absorption of anti-TCV hyperimmune sera with egg extracts or egg ovalbumin and the use of different dilution and blocking buffers influenced the sensitivity and specificity of the ELISA. Reciprocal cross-reactivity was detected among turkey, chicken, bovine, and murine coronaviruses. Antisera to the transmissible gastroenteritis virus of swine, the rabbit enteric coronavirus, or the human coronavirus strain 299E failed to react with TCV. The TCV cross-reacted only moderately with the avian infectious bronchitis virus and the hemagglutinating encephalomyelitis virus of swine. Investigations with samples from 47 commercial turkey flocks in Quebec with episodes of transmissible enteritis revealed that the ELISA was more sensitive than was electron microscopy for detection of TCV.

Coronaviruses were observed by electron microscopy in the intestinal contents of turkeys in Quebec flocks where repeated outbreaks of enteritis occurred. Three isolates could be serially propagated in turkey embryos inoculated by the amniotic route with clarified intestinal contents. Purification and concentration of viral particles contained in intestinal contents of infected embryos were achieved by precipitation with polyethylene glycol and ultracentrifugation on sucrose density gradients. Three particle types were demonstrated: intact virions with a density of 1.18 to 1.20 g/ml and incomplete particles with densities of 1.14 and 1.24 g/ml. Hemagglutination of rabbit and guinea pig erythrocytes was demonstrated with the intact viral particles; the hemagglutinin was not dependent on incubation temperature. All the isolates were antigenically related, as shown by hemagglutination-inhibition. The turkey coronaviruses did not cross-react with antisera against coronaviruses of avian infectious bronchitis, porcine transmissible enteritis, bovine neonatal calf diarrhea, or mouse hepatitis. One of the Quebec isolates was shown to induce syncytia formation on its third passage in primary chicken-embryo kidney cell cultures. Electron-microscopic examination of infected cell-culture fluids revealed characteristics coronavirus particles identical to those found in intestinal contents of infected turkeys.

Avian influenza and hemorrhagic enteritis viral preparations were immunoelectrophoresed in a phosphate-buffered system. Excellent separation and resolution of viral proteins were achieved. Reasons are given why this method might be preferred over the conventional method employing a veronal (barbital)-buffered system.

Enzyme-linked immunosorbent assays (ELISAs) were developed to quantitate hemorrhagic enteritis virus (HEV) antibodies in turkey sera and HEV antigens in tissue extracts. These assays were more sensitive than the commonly used agar-gel precipitin tests in detecting antigen and antibody. The antibody-ELISA was used to monitor the presence and decline of passive antibodies in turkey poults and the seroconversion of turkeys infected with HEV. The antigen-ELISA was carried out using a monoclonal antibody; this test was used to quantitate HEV antigen in experimentally infected turkeys in a time-sequence experiment. Both ELISAs measured a strong antigenic relationship between an avirulent strain (HEV-A) and a virulent strain (HEV-V).

Twenty-nine hybridoma cells lines, producing monoclonal antibodies (MAbs) to the Minnesota strain of turkey enteric coronavirus (TCV), have been established by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of the egg-adapted or tissue culture-adapted virus. The hybridomas produced mainly IgG2a or IgG1 antibodies. Western immunoblotting experiments with purified virus, and immunoprecipitation tests with [35S]methionine-labelled infected cell extracts, allowed assessment of the polypeptide specificity of the MAbs. Sixteen hybridomas secreted antibodies directed to the peplomeric protein (E2, gp200/gp100) and putative intracellular precursors of apparent Mr 170K to 180K and 90K. Four hybridomas produced antibodies that selectively reacted with a glycoprotein with an Mr of 140K (E3). This polypeptide species corresponded to the major structural component of small granular projections, located near the base of the larger bulbous peplomers, and was found to be responsible for haemagglutination. The major neutralization-mediating determinants were found to be carried by both E2 and E3 glycoproteins. Eight hybridomas produced MAbs directed to the major nucleocapsid protein (N, 52K), and only one MAb reacted with a low Mr structural glycoprotein (24K), corresponding to the matrix (E1) protein. By indirect immunofluorescence, MAbs of different specificity also revealed distinct patterns of distribution of the viral antigens within the cells. The location on the virion of the antigenic determinants recognized by MAbs of different specificity was determined by the use of an immunogold electron microscopy technique. Comparison of nine TCV Quebec strains, using MAbs directed to peplomer and haemagglutinin proteins of the prototype Minnesota strain, confirmed their close antigenic relationship, but also revealed the occurrence of at least two distinct antigenic groups.

The incidence of hemorrhagic enteritis (HE) infection in California turkeys was studied by testing 2220 turkey blood samples from 173 flocks for HE virus (HEV) antibody by the enzyme-linked immunosorbent assay (ELISA). Maternal antibody was detected at 1 day of age in all flocks tested, and it vanished after 3 weeks. Acquired HEV antibody appeared at 8 to 10 weeks, and 100% of the meat and breeder turkey flocks were positive after 11 weeks of age. HEV infection occurred earlier in the meat flocks than in the breeder flocks, and it also occurred earlier during summer than during the fall and winter months.