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Sera from cats with naturally acquired and experimentally induced feline immunodeficiency virus (FIV) infections were tested by immunoblot analysis, radioimmunoprecipitation assay (RIPA), and a complex trapping/blocking ELISA. In sequentially obtained samples from experimentally inoculated cats, antibodies against the envelope protein gp120 and the core protein p15 were the first to appear, as indicated by results of RIPA, using lysates of FIV-infected lymphocytes. Antibodies could be detected as early as 2 weeks after infection, followed by a response against p24, p43, and p50. By immunoblot analysis, p24 and p15 were the first proteins detectable between postinoculation weeks 3 and 5; an anti-envelope response was never found by use of this assay, but was found by RIPA. Using the latter test, most sera of naturally infected cats were found to recognize the major core protein p24 in addition to 1 or more minor core proteins. All 40 sera tested precipitated the envelope protein; 3 reacted exclusively with it. A complex trapping/blocking ELISA was developed to quantitate the anti-p24 response. Sera from healthy FIV-infected cats were shown to have higher anti-p24 titer than did those from diseased cats.

An epidemiologic survey was performed to determine the incidence of torovirus infections in 2 disease entities of cattle: diarrhea of replacement calves up to 2 months old, and winter dysentery of adult cattle. Samples were obtained from 187 diarrheal and 115 healthy calves from 15 farms, as well as 149 diarrheal and 67 healthy cows from 27 farms with or without winter dysentery. Enzyme-linked immunosorbent assays for detection of torovirus, rotavirus, and coronavirus antigen in feces, and of torovirus and coronavirus antibodies in serum were used to monitor infections in these groups. Torovirus was detected in 9 of the 15 farms in the study, and in 6% of calves with diarrhea, which was significantly higher than in healthy calves (2%). Seroconversion to torovirus was found significantly more often after winter dysentery episodes than on farms without a disease history; coronavirus seroconversion was less common.

Enzyme-linked immunosorbent assays were established to detect Breda virus antigen in feces and homologous antibodies of the IgG1, IgM, and IgA isotypes in serum. With the aid of solid-phase immune-electron microscopy, torovirions in fecal material were observed. The course of natural infection was studied in 10 sentinel calves that had been obtained from different farms, and housed together at 1 week of age. They were separated from other cattle until the age of 10 months. Up to the age of 4 months, all calves regularly excreted Breda virus in the feces. Irrespective of the existence of IgG1 isotype maternal antibodies, all calves had early IgM responses in serum, but lack of IgA seroconversion. In 7 calves, antibody titer decreased below detection, whereas 3 calves had an isotype switch, resulting in persistent IgG1 titer. After introduction into the dairy herd at 10 months of age, all calves had diarrhea, and shedding of Breda virus was observed in 8 of them. Seroconversion for all antibody isotypes was observed, indicating lack of mucosal memory. In contrast, coronavirus infection in the presence of maternal antibodies led to isotype switch in all calves but one, and a memory response was observed after introduction into the dairy herd.

Feline sera from 13 countries were assayed for coronavirus antibody, using a heterologous indirect immunofluorescence test. Significantly higher percentages of antibody carriers were obtained during testing randomly collected sera from mature males (greater than 1 year old) than in testing females of the same age. Antibodies were infrequently found in immature cats (less than 6 months old); at 1 year of age or older, a plateau was reached and little change in the percentage of seroconverted animals was observed. Differences were not detected between purebred cats vs mixed-breed cats or household vs stray cats. In animals showing clinical signs of feline infection peritonitis (FIP), antibodies were encountered with higher frequency than in clinically healthy cats. Significant differences in antibody incidence were found between countries, with a range between less than 10% and greater than 50% of seropositive individuals. Antibodies were detected in sera from an isolated cat population (Marion Island) and from wild-caught cheetahs (Acinonyx jubatus). The antibody specificity for FIP virus was confirmed by neutralization tests. The antibody pattern in randomly collected solitary cats, in catteries, and cats with clinical FIP showed characteristic differences in titer and incidence. The implications of these results for the epizootiology of FIP are discussed.

Immune complexes purified from sera and ascitic fluids of cats after inoculation with feline infectious peritonitis (FIP) virus contained proteins and proteolytic fragments of the peplomer, nucleocapsid, and envelope polypeptides; in addition, host proteins were demonstrated in the immune complexes. Free (uncomplexed) antibodies against the 3 classes of virion polypeptides were detected and quantitated; the weakest and latest response was directed against the peplomer protein. Immunofluorescence titers showed the best correlation with the antibody response directed against the envelope polypeptides. Differences in reactivity were not found between sera and ascitic fluids from the same animals and between seropositive healthy cats and cats which had died of FIP. Humoral antibody and hypergammaglobulinemia showed a linear correlation, but the wide variation in antiviral titers at a given concentration of gamma-globulin indicated that additional (autoimmune) reactions occur during the pathogenesis of FIP.

The serotypes 1 and 2 of Breda virus from feces of experimentally infected gnotobiotic calves were studied with respect to their sedimentation and density properties in sucrose gradients and their structural polypeptides; Berne virus, the proposed prototype of the new family Toroviridae, was included for comparison. After Breda-1 virus had been stored at 4 C for a prolonged period, it showed a heterogeneous sedimentation behavior (480 to 520 Svedberg units [S]) and density (1.18 to 1.21 g/ml) indicative of its poor state of preservation. In contrast, freshly prepared Breda-2 virus sedimented at 350 S and showed a buoyant density of 1.18 g/ml; these values compare well with those of Berne virus (400 S and 1.16 g/ml, respectively). Efficient purification of the Breda viruses could be achieved by a 2-step method, involving pelleting by ultracentrifugation followed by isokinetic and isopyknic sucrose gradient centrifugation. Radioiodinated purified virus showed polypeptides with apparent molecular weights of 105,000, 85,000 37,000, and 20,000; another labeled protein of 65,000 D is of doubtful virus specificity. Mouse immune serum raised against Breda-2 virus recognized the polypeptides of the homologous virus and the 2 highest molecular weight proteins of Breda 1 virus in radioimmune precipitation. The same serum inhibited hemagglutination of the heterologous serotype to a low, but significant, degree and efficiently neutralized the infectivity of Berne virus. These observations are taken as indications that the 105,000- and 85,000-D polypeptides represent surface structures of torovirions, probably peplomeric proteins.

Seven hybridomas that secreted monoclonal antibodies (MAB) against the peplomer protein and one that secreted MAB against the nucleocapsid protein of Berne virus (proposed family Toroviridae) were isolated. All MAB directed against the peplomer protein neutralized virus infectivity and, with the exception of MAB 6A7, inhibited each other's binding in competition assays. Neutralization of Berne virus infectivity was potentiated when some MAB were used in pairs. The antibodies have been used to localize toroviral proteins in infected cells; use of antipeplomer MAB 6B10 yielded a diffuse intracytoplasmic immunofluorescence, whereas the antinucleocapsid MAB 1F1 detected antigen in the intra- and perinuclear compartments. By use of radioimmune precipitation, protein A of Staphylococcus aureus was found to bind directly to the nucleocapsid polypeptide, without the requirement for specific antibody. Using fluorescein isothiocyanate-conjugated protein A, the intranuclear accumulation of the nucleoprotein of Berne virus was confirmed by results of immunofluorescence.

Pig epizootic diarrhea virus cannot be grown in cell culture; for its characterization, intestinal perfusate material from a pig infected with the strain CV777 had to be used. In isopyknic sucrose gradients, a peak of virus-specific ELISA activity was detected at a density of 1.17 g/ml. Using immunoprecipitation of radioiodinated-purified virus material followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 3 proteins of low molecular weight (20,000 to 32,000 daltons [D]) were found; after blotting nitrocellulose and glycoprotein identification with concanavalin A and horseradish peroxidase, 1 of the proteins (23,000 D) gave a signal. Another protein of 58,000 D was encountered, which was the only protein binding an RNA probe. Finally, a protein of 85,000 D was visible, associated with minor bands of about 110,000 and 135,000 D in most experiments. Using the concanavalin A-blotting technique, the same bands were visualized. The demonstration of a polydisperse cluster of proteins from 20,000 to 32,000 D (of which at least 1 is glycosylated), of glycosylated proteins from 85,000 to 135,000 D, and of an RNA-binding protein of 58,000 D is taken as structural evidence that pig epizootic diarrhea virus should be classified with the Coronaviridae, irrespective of the apparent lack of an antigenic relationship with other members of that family.

Humoral changes were studied in 6 specific-pathogen-free kittens during experimental infection with feline infectious peritonitis virus. Although the incubation period and the duration of the disease differed widely, a similar pattern of rectal temperatures, serum complement values, circulating immune complexes, and antibody titers was found for all kittens during the last 15 to 20 days of life. Antibody formation started 8 to 13 days before death and was accompanied by the appearance of circulating immune complexes with subsequent increased concentrations of complement followed by complement depletion. The data are discussed as evidence for an immune complex pathogenesis of feline infectious peritonitis.