Pathogenicity of three isolates of porcine respiratory coronavirus in the USA.

The pathogenicity of three isolates of porcine respiratory coronavirus (AR310, LEPP and 1894) from the USA was assessed in specific pathogen-free pigs. Pigs inoculated with 1894 developed mild respiratory disease and pigs inoculated with AR310 and LEPP developed moderate respiratory disease from four to 10 days after they were inoculated, but all the pigs recovered fully by 14 days after inoculation. Gross and microscopic examination revealed mild (1894) to moderate (AR310 and LEPP) multifocal bronchointerstitial pneumonia from four to 10 days after inoculation. The lesions were characterised by necrotising bronchiolitis, septal infiltration with mononuclear cells, and a mixed alveolar exudate. No clinical signs or microscopic lesions were observed in control pigs that had not been inoculated.

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.

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.

A sero-epizootiological study of porcine respiratory coronavirus in Belgian swine.

A porcine respiratory coronavirus (PRCV), antigenically closely related to transmissible gastroenteritis virus (TGEV), appeared in the European swine population in 1984. The present serological study was performed to obtain insight into the epizootiology of PRCV and of TGEV. PRCV-induced neutralizing antibodies were found in 90.6 per cent of the 160 sera collected from sows at slaughter, demonstrating the enzootic appearance of PRCV in the Belgian swine population. A serological study of fattening swine on 33 farms revealed that 11 farms situated in an area with a high farm density (all farms within 4 km2) and 11 on 22 closed breeding-fattening farms situated in areas with a low farm density (only one to four farms per 12 km2) were infected with PRCV throughout the year, whereas the other 11 closed breeding-fattening farms were temporarily free of PRCV. PRCV disappeared from the farms mainly in spring and summer. All the 11 farms became reinfected in autumn or winter, indicating that PRCV is regularly reintroduced in farms in the colder seasons. There was no correlation between the herd size and the temporary disappearance of PRCV from farms. It was observed on some farms that PRCV could infect pigs shortly after weaning in the presence of declining maternal antibodies, indicating that PRCV can persist on a farm by regularly infecting newly weaned pigs. TGEV-specific antibodies were found in 7.6 per cent of the 160 sera from the slaughterhouse sows. TGEV-specific antibodies were also detected in sera from fattening swine of 5 of the above mentioned 33 farms. TGEV-outbreaks were not observed on these farms.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of natural recombination within the S1 gene of infectious bronchitis virus.

During an outbreak of severe respiratory disease, a field strain of infectious bronchitis virus (IBV), PP14, was isolated from a bird in a Texas flock that had been previously vaccinated with an attenuated Mass serotype virus. After cloning and sequencing the S1 gene from several IBV strains, it was found that the 5' end of the cDNA was 96% identical to the published sequences of Mass41 and 77% identical with Ark99. The following 402 bases which included the hypervariable regions (HVR) of the S1 gene were 94% homologous with Ark99 and only 69% with Mass41. In addition, the HVR in the 3' noncoding region of the genome, which is totally absent in Mass41, was 99% homologous with the Ark99 strain. This abrupt shift in identity of PP14 in the S1 strongly indicated that a recombination event had occurred about 98 bases from the beginning of the S1 gene between an Ark-like and a Mass-like virus. Downstream, 33 bases from the PP14 recombination junction, a second putative "cross-over" site was identified in the S1 of the SE17 strain where the 5'131 bases of the S1 gene of the Ark99 and SE17 were found to be 95% identical and the following 368 base sequence was only 78% homologous. In addition, a second shift in homology in the S1 of SE17 was identified between nucleotide 1112 and 1460 which shared 95% identity with Mass41. The putative recombination junctions which were downstream of the signal sequence and upstream of the S1 HVR may represent a "hot spot," but not an exclusive region, for exchanging genetic material between IBV strains. Genetic shifts are apparently not only common mechanisms for variation in nature, but vaccine strains may actually play a critical role in these events in the evolution of virulent strains of IBV.

Porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs.

A clinical and virological study of an outbreak of porcine epidemic diarrhoea in a combined breeding and finishing pig herd used ELISA techniques to detect porcine epidemic diarrhoea virus in faeces and antibodies in blood. No seropositive pigs were found at the start of the outbreak. The first signs of the disease were observed in fattening pigs and the infection spread rapidly to pregnant sows, farrowing sows and their suckling pigs, gilts and weaners housed in separate barns. Depression and diarrhoea in the fattening pigs and pregnant sows were the clearest clinical signs. An endemic form of the disease developed which would not normally have been recognised as epidemic diarrhoea because no typical signs were apparent. Eleven groups of seronegative replacement gilts, which were brought in monthly, became infected with the virus and most of the groups developed a profuse diarrhoea lasting a few days. The infections and diarrhoea persisted in six- to 10-week-old pigs in separate barns. The suckling pigs and young weaners appeared to be spared from the infections.

Porcine respiratory coronavirus: molecular features and virus-host interactions.

Since 1984, a previously unrecognized respiratory coronavirus, causing a mostly unapparent infection, has rapidly and massively spread within the swine population in Europe, and few years later, a virus with similar characteristics has been identified in the USA. The agent, designated PRCV, appears to be derived from the porcine enteric coronavirus TGEV. The aim of the present article is to review comprehensively the state of the knowledge about this new virus and its infection. The review includes the following topics: epizootiology, molecular characterization and antigenic features of PRCV, pathogenesis and clinical aspects, immunity and laboratory diagnosis. The authors' views concerning the impact of the emergence of PRCV on both coronavirus research and swine production are presented in the conclusion.

Polymerase chain reaction and a biotin-labeled DNA probe for detection of infectious bronchitis virus in chickens.

Polymerase chain reaction (PCR) and a biotin-labeled DNA probe were used to amplify and detect the genome of infectious bronchitis virus (IBV) from tracheal swabs taken from chickens that were experimentally inoculated with the IBV Beaudette, Arkansas, and Gray strains. The viral genome was successfully detected by PCR and confirmed by dot-hybridization assay using a biotin-labeled DNA probe on days 1, 3, 9, and 14 after exposure. Direct electron microscopy (EM) analysis was used to compare the ability of the two tests to detect IBV from the same tracheal swab samples. The EM analysis did not detect IBV in four of eight necropsy groups that were positive using PCR and the biotin-labeled DNA probe. Although histopathological lesions were observed in the tracheas, no clinical signs or specific antibody response were observed in the birds. The virus was also detected in the allantoic fluid of embryonating chicken eggs that had been inoculated with field samples suspected to be IBV. The field samples were passed four to six times in embryonating eggs, and 10 of 17 samples were positive using PCR and the biotin-labeled probe.

[Detection of porcine epidemic diarrhea virus using electron microscopy in the Czech Republic]. / Elektronove mikroskopický prukaz viru epidemické diarrhoey prasat v Ceské republice.

Coronavirus-induced porcine epidemic diarrhoea (PED) was diagnosed in two swine herds. The causal agent was demonstrated in intestinal contents by electron microscopy and identified by immunoelectron microscopy using specific immune serum to the reference strain PED-CV77. Experimental transmission to hysterectomy-derived, colostrum-deprived piglets with an intestinal contents filtrate was successful. The virus was demonstrable by electron microscopy in the intestinal contents between 12th hour and 4th day, and in small intestinal epithelial cells 18 hours after infection. Scanning electron microscopy revealed shortening and fusion of villi of small intestinal mucosa.

Characterisation of an infectious bronchitis virus isolated from vaccinated broiler breeder flocks.

Four apparently serologically closely related isolates of infectious bronchitis virus were obtained from two flocks of vaccinated broiler breeders, one mile apart, which were experiencing increased mortality and decreases in egg production. The isolates were serologically distinct from isolates previously described and capable of causing characteristic infectious bronchitis-like respiratory infection in young chicks. In one experiment, the H120 vaccine strain of the virus did not protect the trachea against challenge with the new isolates 21 days later.

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.

The prevalence of types I and II feline coronavirus infections in cats.

The types of feline coronaviruses that are prevalent throughout Japan were determined by competitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (MAb) to feline infectious peritonitis virus (FIPV) Type II and neutralizing test using Type II FIPV as challenge virus. A total of 1,079 cat serum samples were tested by indirect fluorescent antibody (IFA) assay for FIPV Type II antigen, all 42 sample from natural cases of FIP, 138 of 647 (21.3%) from cases with some chronic diseases and 57 of 390 (14.6%) from apparently non-diseased cases were positive. Of the 42 cases with FIP, 29 (69%) and 13 (31%) were found to have infection with FIPV Types I and II, respectively. Of the cases with chronic diseases, 111 (80.4%) were shown to have infection with FIPV or FECV Type I, while 14 (10.1%) with FIPV or FECV Type II. All of the 57 apparently non-diseased cases seemed to have been infected with FIPV or FECV Type I. These results indicated that feline coronavirus Type I is more high prevalent in Japan.

Coronavirus infects and causes demyelination in primate central nervous system.

Two species of primates, Owl and African green monkeys, were inoculated intracerebrally with either the neurotropic mouse hepatitis virus JHM or the putative multiple sclerosis brain coronavirus isolate SD. These viruses caused an acute to subacute panencephalitis and/or demyelination in the infected animals. The course of pathogenesis and sites of detected viral RNA and antigen was dependent both on animal species and virus strain but the results clearly showed that these viruses replicated and disseminated in the central nervous system (CNS) of these primates. This study suggests that human CNS may be susceptible to coronavirus infection.

Agents associated with neonatal diarrhoea in Ethiopian dairy calves.

Faeces samples collected from diarrhoeic dairy calves in the first 8 weeks of life were examined for the presence of 5 enteropathogens. The majority of the 108 diarrhoea cases occurred in the first 5 weeks of life and a commercial ELISA kit detected bovine enteric coronavirus (BEC) in 38.9%, serogroup A rotavirus (RV) in 16.7% and K99 (F5) fimbrial adhesin-positive Escherichia coli (K99 ETEC) in 11.1 per cent. Concurrent infections of these enteropathogens were detected in 14.8% of samples (30.8% of samples positive for these agents). No evidence of cryptosporidial infection was found using a differential staining method on faecal smears nor was salmonella excretion detected. On 2 of the 8 farms only BEC was present; the other 6 farms were positive for all 3 agents. It is concluded that BEC is the major infectious cause of neonatal calf diarrhoea in the Ethiopian dairy herds studied with RV and K99 ETEC also contributing to morbidity, either alone or as mixed infections.

Isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate.

Porcine epidemic diarrhea virus (PEDV) was isolated in Vero cell cultures from the small intestine of a piglet experimentally infected with porcine coronavirus 83P-5, that had been isolated during outbreaks of porcine acute diarrhea and passaged in piglets. The isolation of the PEDV was successful only in Vero cells maintained in the maintenance medium (MM) containing trypsin. Infected Vero cell cultures exhibited CPE characterized by cell-fusion and syncytial formation, as well as cytoplasmic fluorescence when examined by the indirect immunofluorescent test using rabbit anti-83P-5 virus serum. The isolate was adapted to serial propagation in Vero cell cultures by adding trypsin to MM. Vero cell-adapted PEDV was successfully propagated in the MA104, CPK and ESK cell lines in the presence of trypsin in MM. Vero cell-adapted PEDV had morphologic and physicochemical characteristics similar to those of other members of the coronaviridae. The isolate differed serologically from porcine transmissible gastroenteritis (TGE) and porcine hemagglutinating encephalomyelitis viruses, and no antigenic relationship between the isolate and TGE virus could be detected by the indirect immunofluorescent test. Attempts to isolate PEDV in 6 types of primary fetal pig cell cultures and 6 of 10 established cell lines resulted in the failure, probably because these cells were damaged by the action of trypsin.

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.

The V5A13.1 envelope glycoprotein deletion mutant of mouse hepatitis virus type-4 is neuroattenuated by its reduced rate of spread in the central nervous system.

Following intracerebral inoculation of adult Balb/c Byj mice, the MHV-4 strain of mouse hepatitis virus (MHV) had an LD50 of less than 0.1 PFU, whereas its monoclonal antibody resistant variant V5A13.1 had an LD50 of 10(4.2) PFU. To determine the basis for this difference in neurovirulence we have studied the acute central nervous system (CNS) infection of these two viruses by in situ hybridization. Both viruses infected the same, specific neuroanatomical areas, predominantly neurons, and spread via the cerebrospinal fluid, along neuronal pathways and between adjacent cells. The neuronal nuclei infected and the spread of virus within the brain are described. The main difference between the parental and variant viruses was the rate at which the infection spread. MHV-4 spread rapidly, destroying large numbers of neurons and the animals died within 4 days of infection. The variant virus spread to the same areas of the brain but at a slower rate. This difference in the rate of virus spread was also apparent from the brain virus titers. The slower rate of spread of the variant virus appears to allow intervention by the immune response. Consistent with this, the variant virus spread slowly in athymic nu/nu mice, but in the absence of an intact immune response, infection and destruction of neurons eventually reached the same extent as that of the parental virus and the mice died within 6 days of infection. We conclude that the V5A13.1 variant of MHV-4 is neuroattenuated by its slower rate of spread in the CNS.

Acute and late disease induced by murine coronavirus, strain JHM, in a series of recombinant inbred strains between BALB/cHeA and STS/A mice.

To examine the genetic control of acute and late disease induced by a murine coronavirus, strain JHM (JHMV), BALB/cHeA, STS/A, F1 hybrids and 13 recombinant inbred (RI) strains between BALB/cHeA and STS/A mouse strains were inoculated intracerebrally with 100 pfu of JHMV. All the BALB/cHeA mice died within 2 weeks from acute encephalitis. In contrast, STS/A mice were shown to be partially resistant, with a mortality rate of 30%, longer survival times and lower rates of viral production. The mortality rates, survival times and viral titers of F1 hybrids and the RI strains varied, suggesting involvement of multiple genes. STS/A, F1 hybrid and RI mice surviving the acute infection occasionally developed severe paraparesis about 1 month post-infection. In these mice, vacuolar degeneration, astrocytosis, the absence of perivascular cuffing and minimal demyelination were found in the central nervous system. No infectious virus could be recovered from these mice. Although the paralysis of delayed onset was limited to STS/A, F1 hybrid and eight of the 13 RI strains, the incidence varied significantly among the RI strains. These results may suggest that JHMV-induced late disease is also under multifactorial control. The pathogenesis of JHMV infection is discussed.