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.     

Infection with porcine respiratory coronavirus does not fully protect pigs against intestinal transmissible gastroenteritis virus

Eight nine-week-old specific-pathogen-free pigs which had been infected with the transmissible gastroenteritis virus (TGEV)-related porcine respiratory coronavirus (PRCV) and four uninfected littermates were challenged with TGEV. The previous PRCV infection failed to protect them against the enteric TGEV infection. Virus excretion in faeces was detected by an ELISA in all the pigs for three to six consecutive days after inoculation. Although little diarrhoea was observed, the infection extended through much of the small intestine of one of the previously infected pigs four days after inoculation. Challenge with TGEV caused a secondary neutralising antibody response. By using a peroxidase conjugate of a monoclonal antibody which recognises a specific antigenic site on TGEV, antibodies against TGEV could be distinguished from antibodies against PRCV in an ELISA blocking test.     

Pathogenicity of porcine respiratory coronavirus isolated in Québec.

Porcine respiratory coronavirus (PRCV) is present in many countries, including Canada, but controversy still exists concerning its pathogenicity. Eight-week-old piglets were inoculated intratracheally with a Quebec PRCV isolate (1Q90). Two contact piglets were kept with the inoculated animals. Three animals served as control. Polypnea and dyspnea were the main clinical signs observed. Diffuse bronchioloalveolar damage occurred 24 hours postinoculation. Changes compatible with bronchointerstitial pneumonia were present six days postinoculation. The inoculated virus was recovered from the respiratory tract and mesenteric lymph nodes, but not from the digestive tract, of the inoculated as well as the contact piglets. No virus was isolated from the control piglets. The development of clinical signs and histopathological changes in inoculated as well as in contact piglets and the reisolation of the inoculated virus demonstrated that PRCV can be an important respiratory pathogen.     

Evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the United States

A respiratory variant of transmissible gastroenteritis virus (TGEV), designated PRCV-Ind/89, was isolated from a swine breeding stock herd in Indiana. The virus was readily isolated from nasal swabs of pigs of different ages and induced cytopathology on primary porcine kidney cells and and on a swine testicular (ST) cell line. An 8-week-old pig infected oral/nasally with the respiratory variant and a contact pig showed no signs of respiratory or enteric disease. These pigs did not shed virus in feces but did shed the agent from the upper respiratory tract for approximately 2 weeks. Baby pigs from 2 separate litters (2 and 3 days old) also showed no clinical signs following oral/nasal inoculation with PRCV-Ind/89. In a third litter, 5 of 7 piglets (5 days old) infected either oral/nasally or by stomach tube developed a transient mild diarrhea with villous atrophy. However, virus was not isolated from rectal swabs or ileal homogenates of these piglets, and viral antigen was not detected in the ileum by fluorescent antibody staining even though the virus was easily recovered from nasal swabs and lung tissue homogenates. Swine antisera produced against PRCV-Ind/89 or enteric TGEV cross-neutralized either virus. In addition, an anti-peplomer monoclonal antibody, 4F6, that neutralizes TGEV also neutralized the PRCV-Ind/89 isolate. Radioimmunoassays with a panel of monoclonal antibodies indicated that the Indiana respiratory variant and the European PRCV are antigenically similar.     

Ultrastructural features of alveolar lesions in induced respiratory syncytial virus pneumonia of calves

Ultrastructural changes occurred in alveolar epithelium in the acute and repair stages of induced respiratory syncytial virus pneumonia induced in eight calves (calf Nos. 1-7, 3 to 6 days old and calf No. 8, 2 weeks old), using a bovine strain of respiratory syncytial virus. Five of the calves were Friesians, three were Hereford x Friesians, and all were male. Tissues from three mock-infected control calves (two Friesian, one Hereford x Friesian) were also examined. Evidence of respiratory syncytial virus infection was observed in both type I and type II pneumocytes from day 4 to day 8 after infection. Infection of type I pneumocytes frequently resulted in necrosis. The response of type II pneumocytes to respiratory syncytial virus infection varied and included hypertrophy, hyperplasia, and syncytial formation. In some infected type II pneumocytes, there were numerous irregular projections of the cell surface, associated with viral budding. Hypertrophy and hyperplasia of type II pneumocytes, epithelial syncytium formation, and irregular cytoplasmic projections from epithelial cells caused considerable thickening of respiratory membrane and occlusion of alveolar lumina. Neutrophils were frequently observed in close association with virus-infected epithelial cells, but evidence of respiratory syncytial virus infection and replication was not observed in alveolar macrophages or neutrophils. Proliferation of type II pneumocytes appeared to play a major role in maintaining the integrity of the alveolar epithelium during the acute stage of the experimental pneumonia. Increased numbers of type II pneumocytes were present on alveolar walls, particularly from 4 to 8 days after infection, and some alveoli were lined entirely by this cell type. In some areas, however, squamous epithelial cells were also involved in covering exposed alveolar basement membrane.     

Study of the potential involvement of pseudorabies virus in swine respiratory disease.

In order to investigate the potential involvement of pseudorabies virus (PRV) in swine respiratory disease, nine week old pigs were intranasally inoculated with the PRV strain 4892. Two doses of infection were used: 10(4.5) median tissue culture infectious doses (TCID50)/pig and 10(3.5) TCID50/pig, with ten pigs per group. In the group of pigs inoculated with 10(4.5) TCID50, seven out of ten pigs died within six days after inoculation. The mortality rate in the group of pigs inoculated with the lower dose was only two out of ten and, there were several pigs in this group that showed signs of respiratory distress besides some mild nervous signs. Pseudorabies virus was isolated from various tissues collected postmortem, including alveolar macrophages. Virus localization in tissues was also detected by in situ hybridization. The histopathological examination of the respiratory tract tissues revealed a pathological process that was progressing from mild pneumonia to severe suppurative bronchopneumonia. The isolation of virus from alveolar macrophages provides support to the hypothesis that replication of PRV during the course of infection produces an impairment of the defense mechanisms in the respiratory tract.     

Isolation of a cytopathic virus from weak pigs on farms with a history of swine infertility and respiratory syndrome.

Severe clinical signs of swine infertility and respiratory syndrome (SIRS) of unknown cause were observed in several Minnesota swine farms between November 1990 and March 1991. Forty-five lung samples of weak pigs were collected from 13 swine farms, and virus isolation was attempted using swine alveolar macrophage (SAM) cultures. A cytopathic virus was isolated from 19 lung samples collected from 6 different farms. Four pregnant sows were infected intranasally with a tissue suspension from which virus was isolated, and 4 6-week-old pigs and 2 contact pigs were infected intranasally with 1 of the isolates. The 4 sows farrowed 12 stillborn and 32 normal pigs. Virus was recovered from 10 of 19 pigs examined. Infected 6-week-old pigs were clinically normal except for slightly elevated rectal temperatures and mild respiratory signs. No or mild interstitial pneumonic lesions were observed in inoculated pigs, but the lesion was obvious in the 2 contact pigs. Seroconversion was observed in sows and pigs as measured by indirect fluorescent antibody (IFA). Serologic identification of the isolates was carried out by IFA using reference serum prepared from an experimentally infected sow. A cytoplasmic fluorescence was observed on the SAM monolayers infected with each of the 19 different isolates. Fluorescence was also observed when the monolayers were tested with SIRS virus ATCC VR-2332-infected sow sera. Replication of the isolates was not affected in the medium containing 5-iodo-2'-deoxyuridine but was inhibited by treatment with ether. The isolates were relatively stable at 56 C and did not agglutinate with various erythrocytes tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutralizing antibody decay and lack of contact transmission after inoculation of 3- and 4-day-old piglets with porcine respiratory coronavirus.

Ten female neonatal piglets were infected with porcine respiratory coronavirus (PRCV) to measure the decay of a specific neutralizing antibody. By 42 weeks after exposure,1 of the gilts was serologically negative (< 5) for PRCV, and by 48 weeks 2 more gilts were serologically negative. These data demonstrate that young mature gilts can be serologically negative, yet they could have been exposed to PRCV. Sentinel pigs were commingled with the PRCV-infected pigs at 8 weeks after exposure, and no virus transmission occurred.     

Pathological, ultrastructural, and immunohistochemical changes caused by Lelystad virus in experimentally induced infections of mystery swine disease (synonym: porcine epidemic abortion and respiratory syndrome (PEARS))

The pathogenicity and pathogenesis of Lelystad virus was studied in six 6-day-old SPF piglets. A third passage of the agent was propagated on porcine alveolar macrophages and intranasally inoculated into pigs. Pigs were killed at hours 24, 48, 60, and 72, and on days 6 and 8 after inoculation. From day 2 on pigs developed diffuse interstitial pneumonia with focal areas of catarrhal pneumonia, and from this day on splenic red pulp macrophages were enlarged and vacuolated. Lelystad virus was re-isolated from the lungs of infected pigs from day 2 after inoculation. Lelystad virus antigens were detected by immunohistochemical techniques in bronchiolar epithelium and alveolar cells, and in spleen cells of infected pigs from day 2 after inoculation. Ultrastructural examination of tissues by electron microscopy revealed degenerating alveolar macrophages and epithelial cells in lungs and nasal mucosa, with excessive vacuolation of the endoplasmic reticulum. Although the respiratory tract seems to be the target organ for this virus, macrophages in other organs, such as the spleen, can also be infected. This preference for macrophages may impair immunological defences.     

Differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity.

The acute stages of infection with swine influenza virus (SIV), porcine respiratory coronavirus (PRCV) and porcine reproductive-respiratory syndrome virus (PRRSV) were shown to differ in terms of clinical and lung inflammatory effects and proinflammatory cytokine profiles in bronchoalveolar lavage (BAL) fluids. Caesarian-derived colostrum-deprived pigs were inoculated intratracheally with one of the three viruses. SIV infection was followed within 1 day post inoculation (d PI) by characteristic respiratory and general signs, and excessive lung epithelial desquamation and neutrophil infiltration (38 to 56 per cent of BAL cells at 1 d PI vs 0 to 1 per cent in controls). High concentrations of bioactive interferon-alpha (IFN -alpha), tumour necrosis factor-alpha (TNF -alpha) and interleukin-1 (IL -1) coincided with peak symptoms and neutrophil infiltration. PRCV infection was asymptomatic and produced a mild bronchointerstitial pneumonitis and neutrophil infiltration (13 to 22 per cent of BAL cells at 4 d PI). IFN -alpha titres parallelled those found during SIV infection, TNF -alpha was negligible and IL -1 undetectable. PRRSV infection induced anorexia and lethargy between 3 and 5 d PI. There was marked infiltration with mononuclear cells in alveolar septa and BAL fluids between 7 and 10 d PI, while neutrophils remained at less than 11 per cent of BAL cells at any time. IL -1 was produced from three throughout 10 d PI, while IFN -alpha production was minimal and TNF -alpha undetectable. These data strongly suggest that proinflammatory cytokines can be important mediators of viral respiratory disease.     

Pathological,ultrastructural, and immunohistochemical changes caused by Lelystad virus in experimentally induced infections of mystery swine disease (synonym: Porcine epidemic abortion and respiratory syndrome (PEARS))

Summary

The pathogenicity and pathogenesis of Lelystad virus was studied in six 6‐day‐old SPF piglets. A third passage of the agent was propagated on porcine alveolar macrophages and intranasally inoculated into pigs. Pigs were killed at hours 24, 48, 60, and 72, and on days 6 and 8 after inoculation. From day 2 on pigs developed diffuse interstitial pneumonia with focal areas of catarrhal pneumonia, and from this day on splenic red pulp macrophages were enlarged and vacuolated. Lelystad virus was re‐isolated from the lungs of infected pigs from day 2 after inoculation. Lelystad virus antigens were detected by immunohistochemical techniques in bronchiolar epithelium and alveolar cells, and in spleen cells of infected pigs from day 2 after inoculation. Ultrastructural examination of tissues by electron microscopy revealed degenerating alveolar macrophages and epithelial cells in lungs and nasal mucosa, with excessive vacuolation of the endoplasmic reticulum.

Although the respiratory tract seems to be the target organ for this virus, macrophages in other organs, such as the spleen, can also be infected. This preference for macrophages may impair immunological defences.     

Studies on the pathogenesis and interspecies transmission of respiratory syncytial virus isolated from sheep

Inoculation of lambs with an ovine isolate of respiratory syncytial virus (RSV) by a combined intranasal and intratracheal route resulted in mild respiratory tract illness, with respiratory tract lesions. Lung lesions were characterized by bronchitis and bronchiolitis, hyperplasia of bronchial and bronchiolar epithelium, peribronchiolar and perivascular accumulations of lymphocytes, alveolar septal thickening, and collapse. Respiratory syncytial virus was recovered from the respiratory tract of inoculated lambs, and RSV antigen was demonstrated by immunoperoxidase staining of bronchiolar and alveolar epithelial cell in pneumonic lesions of lambs euthanatized on post-inoculation days 5 and 6. Other primary respiratory tract pathogens were not isolated. Clinical signs of respiratory tract illness or respiratory tract lesions did not develop in the in-contact control lamb. Inoculation of the ovine RSV isolate into calves and deer fawns resulted in infection in both species, and at necropsy, pneumonic lesions were present. A mild to moderate respiratory tract illness developed in the calves, but clinical disease was not seen in the fawns. Lung lesions in fawns were similar to those seen in lambs; lesions in calves were characterized by collapse, scattered areas of parenchymal necrosis, and bronchiolitis. Respiratory syncytial virus was reisolated from the lower respiratory tract of inoculated calves and fawns, and immunoperoxidase-positive epithelial cells were seen in pneumonic lesions. Other primary respiratory pathogens were not detected. Respiratory syncytial virus infection was not demonstrable in control animals that were in contact with inoculated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

用固定细胞阻断酶联免疫吸附试验鉴别诊断猪传染性胃肠炎病毒和猪呼吸道冠状病毒的感染

用固定细胞阻断酶联免疫吸附试验（ＥＬＩＳＡ）对来自丹麦猪的１８０份血清进行了猪传染性胃肠炎病毒（ＴＧＥＶ）和猪呼吸道冠状病毒（ＰＲＣＶ）感染的鉴别诊断，共检出了ＰＲＣＶ抗体阳性血清１０７份（５９．４％），ＴＧＥＶ抗体阳性血清０份。同时也检测了一些来自国内不同ＴＧＥＶ感染类型的猪场血清。该鉴别诊断方法在我国的建立和应用为从ＰＲＣＶ阳性国家进口猪的ＴＧＥ的检疫提供了一条有效途径。     

Experimental bovine respiratory syncytial virus infection in conventional calves: light microscopic lesions, microbiology, and studies on lavaged lung cells

Conventionally raised male Holstein calves, 1 month of age, were infected by intranasal and intratracheal inoculation with bovine respiratory syncytial virus. Viral antigen was identified by fluorescence microscopy most commonly in the cytoplasm of tracheal and bronchial epithelial cells 3 to 5 days after inoculation. Cytoplasmic viral antigen was identified also in nasal, nasopharyngeal, bronchiolar, and alveolar epithelial cells and in alveolar macrophages. Bronchitis and tracheitis, characterized in part by epithelial necrosis, formation of syncytial epithelial cells and epithelial hyperplasia, were the most common lesions observed histologically. Rhinitis, bronchiolitis, and interstitial pneumonia were observed less frequently. Alterations were not detected in the numbers of cells recovered by bronchoalveolar lavage after inoculation. An increase in the phagocytic rate of latex beads occurred in macrophages 5 days after inoculation. Viral-induced lesions were resolved by 30 days after inoculation. The results indicated that bovine respiratory syncytial virus inoculation of calves results in reversible alterations in airway epithelial structure and in the phagocytic function of alveolar macrophages.     

Upper respiratory infection of lactating sows with transmissible gastroenteritis virus following contact exposure to infected piglets.

Ten breeding sows were left in direct contact with their newborn piglets that had been experimentally infected with transmissible gastroenteritis (TGE) virus. All sows became infected with the virus. The sows developed fever and showed mild clinical signs of the disease for a few days. The sows excreted virus in the nasal secretion, feces, and milk during the acute febrile phase of illness. Virus was isolated from the nasal secretion of one sow as early as 20 hours after contact exposure to the infected piglets. At necropsy, the virus was more frequently isolated from the tissues of the upper respiratory tract than from small intestines; this finding indicated that the TGE coronavirus replicated in the upper respiratory tract and induced an acute respiratory infection in susceptible adult swine. Neutralizing antibody was present in the sera 8 sows after 12 to 36 days during the convalescent period. From these results, we conclude that susceptible sows in direct contact with ill piglets can become infected and by excreting virus can serve as a source of TGE virus for other susceptible pigs on the premises.     

Lesions of the Gastrointestinal Tract of Pigs Infected with Transmissible Gastroenteritis

Gross, subgross and histological lesions were studied in 103 pigs infected with transmissible gastroenteritis virus and killed at daily intervals for 14 days. Twenty-three pigs served as controls. Thirty-six pigs were given colchicine four hours prior to being killed in order to determine the mitotic activity in the gastrointestinal tract. The gross lesions consisted of dehydration, excessive milk curd in the stomach, focal hemorrhage in the submucosa of the diverticulum ventriculi of the stomach, fundic and pyloric congestion in severly dehydrated animals and thinning of the small intestinal wall. The major subgross lesion was a marked shortening of the villi in the lower duodenum, jejunum and ileum within 24 hours after exposure to the virus. Regrowth of the villi became evident on about the sixth day after infection. Histological examination of the small intestine revealed that the villus-height/crypt-depth ratio in the jejunum was reduced from 7:1 in normal pigs to less than 1:1 in infected pigs. Villous atrophy was less severe in the proximal duodenum and ileum. Cells covering the atrophic villi were flatened or cuboidal and did not have well defined brush borders. Inflammatory changes in the gastrointestinal tract were minimal at all stages of infection. Goblet cell numbers increased slightly in the recovery stage of the disease and small numbers of mononuclear cells accumulated in the lamina propria during regrowth of the villi. The number of metaphase nuclei in the small intestinal crypts of infected pigs was greater than in normal pigs.     

Immunohistochemical localization of haptoglobin in porcine lungs

The extravasation of erythrocytes into the lower respiratory tract occurs in numerous lung injuries and may lead to oxidative damages in lung tissues. Haptoglobin (Hp), the major haemoglobin-binding protein, is known to reduce lung injury associated with exposure to blood in mice. In pigs, Hp is a major acute phase protein and its serum concentrations are elevated in various infections of the respiratory tract. However, information on the porcine Hp response towards inflammatory stimuli is restricted to blood. We herein investigated the presence of Hp in lung tissues from pigs with acute and chronic bronchopneumonia via immunohistochemistry. Hp was localized in airway epithelial cells and immigrated leucocytes whereas in alveolar epithelial cells there was no distinct signal. Unaltered lungs showed less Hp-positive cells compared with lungs from pigs with acute or chronic bronchopneumonia.     

Pathogenicity of concurrent infection of pigs with porcine respiratory coronavirus and swine influenza virus

Combinations of porcine respiratory coronavirus (PRCV) and either of two swine influenza viruses (H1N1 or H3N2) were administered intranasally and by aerosol to six- to eight-week-old specific pathogen-free pigs. The clinical responses, gross respiratory lesions and growth performances of these pigs were studied and compared with those of single (PRCV, H1N1 or H3N2) and mock-infected animals. PRCV infection caused fever, growth retardation and lung lesions, but no respiratory symptoms. Infection with swine influenza viruses caused rather similar, mild symptoms of disease, with H1N1 infection being the least severe. Combined infections with influenza viruses and PRCV did not appear to enhance the pathogenicity of these viruses. Furthermore, viruses were isolated more frequently from tissues and nasal swabs taken from 'single' than 'dual' infected animals, suggesting a possible in vivo interference between replication of PRCV and swine influenza virus.