Isolation and propagation of a human enteric coronavirus.

Coronavirus-like particles were found by electron microscopy in stools from infants with necrotizing enterocolitis. Stool samples from these infants as well as control specimens were passaged in cultures of human fetal intestinal organs. Two samples yielded virus-like particles and these have now been passaged 14 times (HEC 14). Gradient-purified HEC 14 strains had typical coronavirus morphology on electron microscopy and contained five major proteins with molecular sizes ranging from 190 to 23 kilodaltons. Infants with necrotizing enterocolitis developed specific antibody to the viral antigens between the acute and convalescent stages of the disease, as shown by examining serum specimens by single radial hemolysis, immunoenzymatic assay, and Western immunoblotting. No cross-reactivity was shown with other coronavirus strains tested, or with the newly isolated viruses of the Breda-Berne group, responsible for calf or horse diarrhea.

Infection of the basal ganglia by a murine coronavirus.

The coronavirus, mouse hepatitis virus strain A59 (MHV-A59), causes mild encephalitis and chronic demyelination. Immunohistochemical techniques showed that MHV-A59-infected C57BL/6 mice contained dense deposits of viral antigen in the subthalamic nucleus and substantia nigra, with fewer signs of infection in other regions of the brain. The animals showed extra- and intracellular vacuolation, neuronal loss, and gliosis in the subthalamic-nigral region. Such localization is unprecedented among known viral encephalitides of humans and other species. This infection by a member of a viral class capable of causing both encephalitis and persistent infection in several species may be related to postencephalitic parkinsonism.

Vacuolar degeneration in mice infected with a coronavirus JHM-CC strain.

Vacuolar degeneration was constantly induced in the CNS of 4-week-old ICR mice by intracerebral or intranasal inoculation of JHM-CC virus, a small plaque mutant of mouse hepatitis virus (JHM). Most animals showed no symptoms or only mild hindlimb paresis. Irrespective of clinical manifestations, the virus was isolated from the CNS up to days 14 to 16. Viral antigen expression in the CNS tissue was most extensive around days 5 to 7 and became undetectable on day 14. Viral antigens were localized almost exclusively to neurons, and the temporal sequence of viral antigen distribution after intranasal inoculation clearly indicated the virus spread through the olfactory and limbic systems into the brainstem and spinal cord, and possible cell-to cell transmission of the virus within the CNS. Vacuolar changes, most conspicuous in the brainstem and spinal cord, were steadily progressive up to 4 weeks after infection, but became indistinct by 4 months. Although the distribution of vacuolar lesions largely agreed with that of viral antigen-positive cells, the severity of vacuolation did not correlate with that of inflammation. Intramyelinic splitting, periaxonal edema, and swollen neurites were major ultrastructural substrates for vacuolar changes. This model could provide a better understanding of new types of neurologic disorders associated with viral infections, including vacuolar myelopathy in AIDS.

Comparative features of a coronavirus isolated from a cheetah with feline infectious peritonitis.

A coronavirus which was isolated from a cheetah (Acinonyx jubatus) that succumbed to feline infectious peritonitis was characterized in vitro. The virus was determined to be highly cell-associated with Crandell feline kidney (CrFK) cells and was routinely maintained as a persistent infection (CrFK 83-4497). The cheetah coronavirus was compared with other members of the feline coronavirus group including the feline enteric coronavirus (FECV) 79-1683 and the feline infectious peritonitis viruses (FIPV), 79-1146, and UCD-1. The cheetah coronavirus was demonstrated to have a restricted host-cell range with limited cytopathic effect. Indirect immunofluorescence with antisera to FIPV UCD-1 revealed the concentration of viral antigens in the perinuclear region of cells infected with the cheetah coronavirus. Ultrastructural studies of the cheetah coronavirus indicated a limited number of immature viral particles within cytoplasmic vesicles and at the cell surface. This was in contrast to electron microscopy results of FECV 79-1683 and FIPV 79-1146, which had numerous mature virus particles within the cytoplasmic vesicles, as well as at the cell surface. The cheetah coronavirus was tentatively placed in the feline coronavirus family based upon its antigenic reactivity by immunofluorescence; however, the possibility that it represents a unique coronavirus of cheetahs should not be dismissed without further analyses at the host and genomic levels.

In vivo and in vitro models of demyelinating diseases. XII. Persistence and expression of corona JHM virus functions in RN2-2 Schwannoma cells during latency.

The coronavirus JHMV persistently infects rat Schwannoma cells RN2-2 at 32.5 degrees C and enters a host-imposed reversible, latent state at 39.5 degrees C. JHMV can remain up to 20 days in the latent state and about 14 days before the cultures lose the capacity to resume virus production upon return to 32.5 degrees C. Although persistently and latently infected RN2-2 cells display resistance to superinfection by a heterologous agent VSV, these cells do not release detectable soluble mediators (e.g., interferon) of the antiviral state. Nevertheless, RN2-2 cells are competent to synthesize and release interferon when treated with the appropriate inducers. These observations suggest that interferon does not play any role or may not be the major factor in the control of latency in the Schwannoma cell. Hybridization with virus-specific cDNAs shows that all viral mRNAs are present during latency and that viral mRNAs are present in the polysomes of infected cells at 39.5 degrees C. Western immunoblotting with hybridoma antibodies demonstrates that viral specific proteins are produced at the restrictive temperature. These results suggest that despite the absence of production of infectious virus at 39.5 degrees C, there is active transcription and translation into virus-specified products.

In vivo depletion of interferon-gamma leads to susceptibility of A/J mice to mouse hepatitis virus 3 infection.

The possible role of interferon-gamma (IFN-gamma) in the resistance of A/J mice to MHV3 infection was investigated. Monoclonal antibodies specific for IFN-gamma, CD4 and CD8 molecules were administered in vivo to deplete selectively the IFN-gamma synthesized or the appropriate subset of T cells. The animals were then infected with MHV3 and the course of infection was followed by studying different parameters, such as, the mortality, the virus growth in the tissues and the IFN-gamma synthesis in sera and peritoneal exudates. After MHV3 infection, a full resistance of control A/J mice was observed, in contrast to the high mortality rate observed among the depleted animals, where higher virus titers were found in different tissues. The IFN-gamma synthesis in sera and peritoneal exudates of depleted mice, after MHV3 infection, drastically decreased when compared to that detected in control mice. The data presented are consistent with the hypothesis that IFN-gamma plays an essential role in the resistance of A/J mice to MHV3 infection.

An eight-year study of the viral agents of acute gastroenteritis in humans: ultrastructural observations and seasonal distribution with a major emphasis on coronavirus-like particles.

During an 8-yr period, 862 stool specimens from patients with gastroenteritis were examined by electron microscopy after negative staining with 2% phosphotungstic acid (pH 6.5). Forty-one percent of the specimens submitted over an 8-yr period were determined to be positive for virus or viruslike particles belonging to one or more of seven morphologically distinct viral groups. Coronavirus-like particles (CVLPs) were present in 69.8% of the positive stool specimens. Membranous profiles containing "complement-type" holes (10 nm in diameter) were identified in some preparations containing CVLPs. The second most prevalent viral agent found in stool specimens was the rotavirus (17% of all positive stools). The incidence of other viruses identified in the survey were as follows: adenovirus 4.5%, picorna/parvovirus agents 2.9%, Norwalk-like agent 2.9%, astrovirus 1.9%, and calicivirus 0.5%. Unclassified small round viruses (approximately 25-30 nm in diameter) represented 0.5%. It was also determined that there was a seasonal distribution in excretion of all viruses except for CVLPs. A greater number of viruses were identified in the cooler, drier months of the year.

Evaluation of an immunogold electron microscopy technique for detecting bovine coronavirus.

A solid phase colloidal gold immunoelectron microscopy (IGEM) technique for detecting bovine coronavirus (BCV) was developed and shown to be specific. This test was compared with three other diagnostic tests using fifteen faecal samples. Bovine coronavirus was detected in 2 samples by direct electron microscopy (DEM), in 3 samples by immunosorbent electron microscopy, in 5 samples by haemadsorption-elution-haemagglutination and in 6 samples by IGEM. Ninety four faecal samples were tested by DEM and IGEM. Of 26 samples found to contain BCV by IGEM only 14 were positive by DEM. The IGEM technique is simple, efficient and less susceptible than others to non-specific reactions.

Envelope proteins of avian infectious bronchitis virus: purification and biological properties.

Immunoadsorbents, made with monoclonal antibodies, were used to purify the spike and membrane proteins of infectious bronchitis virus (IBV). The purified proteins were inoculated into rabbits to produce antisera. The rabbit anti-spike sera neutralized the infectivity of the virus whereas the anti-membrane sera did not. IBV-infected chickens produced antibodies to both the spike and membrane proteins. Both these antibodies were at their highest concentration about 9-11 days after inoculation, whereas neutralizing antibodies were present only at very low concentrations at that time. Neutralizing antibodies were at their highest concentration 21 days after inoculation. A second inoculation of virus at 42 days induced an anamnestic antibody response to the spike and membrane proteins and also for the neutralizing antibodies. The neutralizing, anti-spike and anti-membrane antibodies all reached highest concentrations 7-11 days after this inoculation. The advantages of purifying viral proteins using affinity chromatography with monoclonal antibodies are discussed.

Intestinal replication of a porcine respiratory coronavirus closely related antigenically to the enteric transmissible gastroenteritis virus.

One-week-old piglets were inoculated with the porcine respiratory coronavirus (PRCV) either intravenously or directly into the lumen of the gastrointestinal tract. Both inoculation routes resulted in the isolation of virus from the caudal small intestine. Viral replication, however, was only observed upon inoculation into the digestive tract in quantities of greater than or equal to 10(3) TCID50. Replication remained limited to a few unidentified cells located in or underneath the epithelial layer at villus- or crypt-sites. Virus was excreted in the faeces for several days but infection of the respiratory tract occurred rarely in the same pigs. The results of this study indicate that small changes in molecular structure between PRCV and transmissible gastroenteritis virus have resulted in important changes in host cell tropism.

Comparison of two strains of avian infectious bronchitis virus for their interferon induction, viral growth and development of virus-neutralizing antibody in experimentally-infected chickens.

Two field strains of avian infectious bronchitis virus were tested in specific pathogen-free chickens for interferon induction, growth and stimulation of virus-neutralizing antibody. Both strains induced interferon in lungs and trachea and neither strain induced it in kidneys. Strain Kagoshima-34, isolated from the kidneys of a chicken that had died of nephrosis/nephritis, induced interferon earlier than Strain Tottori-2, which was isolated from the trachea of a chicken with respiratory disease. Virus growth was generally more prolonged with Kagoshima-34, especially in the kidneys, but Tottori-2 persisted longer in the lungs.

A monoclonal antibody blocking ELISA to detect serotype-specific infectious bronchitis virus antibodies.

A monoclonal antibody (Mab) blocking enzyme-linked immunosorbent assay (B-ELISA) was developed and compared to a conventional indirect ELISA (I-ELISA) and a virus-neutralization (VN) test for detection of specific antibodies to avian infectious bronchitis virus (IBV) serotypes. Sera used in this study were derived from chickens experimentally inoculated with the three most prevalent IBV serotypes, Arkansas (Ark), Connecticut (Conn), and Massachusetts (Mass). Overall, there was good correlation between the results of B-ELISA and the VN test. Both detected serotype-specific antibodies in chicken sera during primary and secondary phases of the immune response. Results of both tests indicated that the antibodies produced during the primary response to IBV serotypes are strongly serotype-specific. Those produced during the secondary response react more strongly with the homologous virus, but do exhibit some level of cross-reactivity with heterologous antigens. I-ELISA detected IBV group-specific and not serotype-specific antibodies. The B-ELISA which both offers the convenience of the conventional I-ELISA and the serotype specificity of the VN test, hold excellent promise for field application in IBV diagnosis and evaluation of response to IBV vaccines.

Quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (PEDV).

The porcine epidemic coronavirus (PEDV), tentatively classified as a coronavirus, was adapted to Vero cells and a plaque test developed for infectivity titration, allowing us to test the biological and biophysical properties of the virus. Growth kinetics showed peak titers of 10(5.5) plaque-forming units ml-1 15 h after infection. Filtration experiments and electron microscopy revealed a particle diameter between 100 and 200 nm. The buoyant density of the virus was 1.18. The particle lost its infectivity on treatment with lipid solvents. Virus replication could not be inhibited by 5-iodo-2'-deoxyuridine. PEDV was moderately stable at 50 degrees C, but heat sensitivity was not altered by divalent cations. At 4 degrees C, the virus was stable between pH 5.0 and 9.0, but at 37 degrees C stability was restricted to the pH range 6.5-7.5. Viral infectivity was not impaired by ultrasonication or by multiple freezing and thawing. PEDV was not neutralized by transmissible gastroenteritis virus antiserum. On the basis of the tests carried out, PEDV is a pleomorphic, enveloped RNA virus with a particle diameter of approximately 150 nm and a buoyant density of 1.18. Infectivity depends on the presence of trypsin, and infected cells show a tendency to fuse and to form syncytia. All of these properties, as well as its physicochemical characteristics, allow PEDV to be classified as a coronavirus.

Experimental infection of pigs with the porcine respiratory coronavirus (PRCV): measure of viral excretion.

Twelve pigs were experimentally infected with a porcine respiratory coronavirus (PRCV) by the oronasal route. Viral excretion was measured daily by two means-deep nasal swabs and air samples obtained in a cyclone sampler. Clinical signs were very slight on infected pigs. Airborne virus could be recovered from day 1 to day 6 post-infection in the cyclone sampler as well as in petri dishes placed in the same loose-box. Viral titres obtained from nasal swabs were significantly correlated with those obtained from air samples. Different collection media were compared. The most efficient media for the collection of infectious viral particles contained a protective agent such as foetal calf serum.

Virus shedding and immune responses in cats inoculated with cell culture-adapted feline infectious peritonitis virus.

Eight specific pathogen-free cats were inoculated orally or parenterally with a cell culture-adapted strain of feline infectious peritonitis virus (FIPV). Faeces and oropharyngeal swabs were monitored daily for infectious virus by inoculation of feline embryo lung cells. Virus was recovered from both sites for approximately 2 weeks after inoculation, before clinical signs of disease developed. Peripheral blood lymphocytes collected from these cats were tested in an in-vitro blastogenic assay using concanavalin A (con A) and FIPV antigen. All cats showed a profound suppression of the response to con A which only recovered to pre-inoculation levels in 2 cats, one of which survived. These 2 cats also responded to FIPV antigen on the 21st day after infection, the greater response being in the survivor. The other cats, surviving 16-18 days, developed no response to FIPV antigen. Antibody titres, measured by immunofluorescence and by virus neutralization, rose rapidly to very high levels in all cats, regardless of the route of inoculation.

The sites of early viral replication in feline infectious peritonitis.

The sites of early replication of feline infectious peritonitis virus were studied following oral inoculation of specific-pathogen-free (SPF) cats with virus grown in cell cultures. Viral antigen was first detected by immunofluorescence in the tonsils and small intestine within 24 h of inoculation, and was later found in caecum, colon, mesenteric lymph nodes and liver. However, histological changes in the gut did not appear until relatively late in the course of infection. Virus was recovered from the oropharynx and the faeces from as early as the second or third day after inoculation, and shedding continued until euthanasia.

Coronavirus infection of the bovine respiratory tract.

Two viruses, morphologically resembling coronaviruses and antigenically indistinguishable from bovine enteric coronavirus, were isolated in bovine tracheal organ cultures from the lungs and trachea of young calves with respiratory disease. Intranasal and intratracheal inoculation of these viruses into neonatal calves resulted in a predominantly upper respiratory tract infection, which was associated with the development of mild respiratory symptoms.

Coronaviridae, pathogenetic and clinical aspects: an update.

A review is given about pathogenetic and clinical aspects of the well-known as well as of recently detected members of the family Coronaviridae. Special attention is paid to coronavirus infections of domestic cattle and pets, whereas avian, murine, rat and human coronaviruses are summarized briefly.

Experimental reproduction of pneumonia in gnotobiotic pigs with porcine respiratory coronavirus isolate AR310.

The pathogenicity of porcine respiratory coronavirus (PRCV) isolate AR310 was determined for gnotobiotic pigs. PRCV-AR310 was isolated from the intestines of a nursery pig from a herd with endemic transmissible gastroenteritis. The AR310 isolate was plaque purified and cell culture propagated, passed once in a gnotobiotic pig, then used as inoculum for a gnotobiotic pig pathogenicity study. Eight pigs were inoculated oronasally with 2 x 10(6) plaque-forming units of PRCV-AR310. Eight pigs served as controls and received cell culture medium. Two pigs from each group were necropsied at 3, 5, 10, and 15 days postinoculation (DPI). There was moderate multifocal to coalescing reddish tan consolidation of 60% of the lung by 10 DPI. Microscopic examination revealed a necrotizing and proliferative bronchointerstitial pneumonia characterized by necrosis, squamous metaplasia, dysplasia, proliferation of airway epithelium, mononuclear cell infiltration of alveolar septa, mild type II pneumocyte proliferation, and lymphohistiocytic alveolar exudation. The microscopic lesions were mild by 3 DPI, moderate by 5 DPI, severe by 10 DPI, and mostly resolved by 15 DPI. No lesions were observed in the intestines of these pigs. There was no clinical respiratory disease. Control pigs remained normal and had no lesions. PRCV was isolated from the lungs but not from the intestines of inoculated pigs. PRCV was not isolated from the lungs or intestines of control pigs. PRCV was also isolated from the nasal and rectal swabs of inoculated but not of control pigs.

Identification of coronaviruses by the use of indirect protein A-gold immunoelectron microscopy.

Concentration by airfuge and protein A-colloidal gold immunoelectron microscopy (PAG-IEM) offered a rapid and sensitive method for detection and identification of coronaviruses from various species. The method was applied to partially purified tissue culture-adapted or egg-adapted mammalian and avian coronaviruses and to clarified fecal samples from diarrheic calves and turkey poults for detection of enteric coronaviruses. Aggregates of virus coated with specific antibody were seen in virus samples mixed with homologous antiserum but not in control samples containing preexposure serum. At least a 10-50-fold enhancement of the sensitivity of direct EM for virus detection was obtained using protein A-colloidal gold complex as an electron-dense marker. The PAG-IEM method demonstrated low nonspecific background labeling and permitted detection of soluble and particle-associated antigen. Reciprocal cross-reactivity was detected among the subgroup of mammalian hemagglutinating coronaviruses, and antisera to 4 members of other subgroups only recognized their homologous virus.