key: cord-0886836-1smzibmu authors: Lanza, I.; Brown, I. H.; Paton, D. J. title: Pathogenicity of concurrent infection of pigs with porcine respiratory coronavirus and swine influenza virus date: 1992-11-30 journal: Research in Veterinary Science DOI: 10.1016/0034-5288(92)90131-k sha: d5b920f25eecf8114c2ff0fcd08a4dddd5cd158e doc_id: 886836 cord_uid: 1smzibmu Abstract 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. PORCINE respiratory coronavirus (PRCV) is a variant of transmissible gastroenteritis virus (TGEV) (Pensaert et al 1986) . Despite complete in vitro cross-neutralisation between these two viruses (Pensaert 1989) , PRCV, unlike classical, enteric forms of TGEV, grows poorly if at all in the gut, but grows to high titres in respiratory tissues (Cox et al 1990) . Experimentally infected pigs develop puhnonary lesions (O'Toole et al 1989) but rarely show clinical signs of disease. The role of the virus in field outbreaks of respiratory disease is uncertain, although in the majority of cases seroconversion to PRCV occurs asymptomatically. It is, however, suspected that PRCV may be a contributory cause of more severe *Present address: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Le6n, Campus de Vegazana, E-24071-Le6n, Spain respiratory disease in combination with other influences such as concurrent infections (Pensaert 1989) . Since both swine influenza viruses and PRCV are circulating in the European pig population, combined infections are likely to occur, particularly at the beginning of the fattening period, when pigs from different origins are mixed. In 1987, an H3N2 swine influenza virus was isolated from a farm in England following an outbreak of severe respiratory disease (Pritchard et a11987) . PRCV seroconversion coincided with the outbreak, but the virus was not isolated. However, the influenza isolate on its own failed to reproduce the clinical disease when inoculated into experimental pigs. The aim of this study was to establish whether or not a concurrent infection in pigs with PRCV and either of two swine influenza virus strains (H1N1 or H3N2) resulted in the development of respiratory symptoms and more severe pulmonary lesions, as compared to single' agent infections. Twenty-eight specific pathogen-free pigs, from a minimal disease herd at the Central Veterinary Laboratory, Weybridge (CVL), were used. This herd was seronegative to TGEV/PRCV and to both swine influenza strains. The animals were fed a commercial diet in predetermined quantities dependent upon age. Each group was housed separately (in 20 m 2 pens) in different blocks to avoid cross-contamination. The air from each block was exhausted through high efficiency (HEPA) sterilising filters. PRcv. The Stopps strain (135308) of t'RCV, isolated in 1986 from an outbreak of respiratory disease was used to infect a colostrum-deprived, two-day-old piglet by the intranasal route. Three days after infection, it was killed and the lungs removed, each lobe being homogenised separately in phosphate buffered saline (PBS) pH 7.2 containing antibiotics. The homogenates were titrated on primary pig kidney monolayers (PPKM). The lobes with the highest viral titre were pooled, passaged once in PPKM cells and used as inocula at a titre of 105 TCID50 m1-1. Influenza viruses. Two strains of influenza A virus were used: A/SW/Weybridge/86/H1N1 and A/SW/Weybridge/87/H3N2. Challenge inocula for each strain were prepared by intranasal dosing of colostrum-deprived, two-day-old piglets, using virus previously passaged five times in the allantoic cavity of embryonated chicken eggs. Three days after infection the piglets were killed and homogenates of each lung lobe were inoculated into the allantoic cavity of 10-day-old embryonated chicken eggs. After three days incubation at 34°C, the allantoic fluids were clarified by centrifugation and tested for haemagglutinating activity using 1 per cent chicken erythrocytes. Fluids giving the highest titres were pooled and stored at -70°C until used as inocula. The H1N1 inocula had titres of 108-5 egg infective doses (EID)50 m1-1 and the H3N2 inocula had titres of 108 EID50 mkL Twenty-eight pigs, six to eight weeks old, were split into six groups (Table 1) . Animals inoculated with a single virus received 4 ml intranasally and 1 ml aerogenically (by nebuliser). Control pigs received equal volumes of Hanks' medium by the same routes. Inocula for dual infected animals were administered intranasally (8 ml) and aerogenically (2 ml) as a mixture of the corresponding single doses. All animals were monitored twice daily for the first seven days after infection and then daily until 21 days after infection. Monitoring consisted of: clinical assessment, nasal swabbing, rectal temperature taking and observation of food intake. The animals were weighed on alternate days and were bled from the anterior vena cava on days 0, 7, 10, 14 and 21 after infection. To follow the development of lesions, one pig from each infected group was killed (intraperitoneal sodium pentobarbitone) on days 3, 6 and 10 after infection in all groups. One H1N1-and one H IN1 +pRey-infected pig were killed on day I, while one H3N2-, one PRCV-and one H3N2+PRCv-infected animal were killed on day 21. Both control pigs were killed on day 21. At post mortem examination, the following tissues were taken for virus isolation: nasal turbinates, tonsil, trachea, tracheobronchial lymph nodes and lung (apical and diaphragmatic lobes). Tissues collected at necropsy were prepared as 1.0 per cent (w/v) homogenates in PBS pH 7-2 containing 1000 iu penicillin, 1000 g streptomycin and 200 iu mycostatin (nystatin) ml-1, incubated for 30 minutes at room temperature, clarified at 1500 g for 10 minutes and the resulting supernatant stored at -70°C until tested. Nasal swabs were immediately suspended in PBS pH 7.2 containing antibiotics (as for tissues) and then treated as for tissue homogenates. PRCV isolation. Virus was isolated using PPKM grown in Hanks' medium containing 10 per cent bovine serum, 0.0375 per cent sodium bicarbonate and antibiotics (1:10 dilution of tissue/swab preparation). The maintenance medium was Earle's, containing 2 per cent bovine serum, 0.15 per cent sodium bicarbonate and antibiotics. If after seven days after infection cytopathic effect was not detected, tissue culture fluid was passed on to fresh cell cultures. Following incubation for 24 hours, the presence of virus was assessed using a TGEV direct fluorescent antibody test. Influenza isolation. Nine-to ten-day-old specific pathogen-free embryonated chicken eggs were inoculated allantoically with 0.1 ml of sample and incubated for three days at 34°C. The harvested allantoic fluids were tested for haemagglutinating activity, as already described. Table 2 shows the mean daily temperatures and the proportion of pigs with fever for each group during the first seven days after infection. All sera were inactivated at 56°C for 30 minutes. PRCV serum antibodies were detected by a TGE virus neutralisation test, using A72 (canine rectal tumour) cells and TGEV strain FS68/216 (Paton 1989). H 1 N1 or H3N2 influenza serum antibodies were detected by the haemagglutination inhibition test (Palmer et al 1975) . To further destroy non-specific inhibitors, inactivated sera were treated with 25 per cent Kaolin overnight at +4°C. After adsorption with a 10 per cent suspension of chicken erythrocytes for one hour at 37°C, the sera were tested against four haemagglutinating units of H1N1 or H3N2 virus using 1 per cent chicken erythrocytes. A regression line for weight against time was calculated for each group, from the individual weights of all pigs not killed before day 10 after infection. Liveweight gains were compared for the same animals. To find out if growth changes were significantly different between groups, an analysis of variance (ANOVA) was performed with the live weight gain figures for days 4 and 10 after infection. No clinical signs were observed and their temperature remained within the normal physiological range throughout the experiment (38-4 ° to 39.6°C). At 24 hours after infection the pigs were dull and feverish, with an average temperature of 40.2°C (range 39-7 ° to 40.8°C). Temperatures ?emained elevated for five days after infection, returning to normal thereafter. A serous nasal discharge was observed in all the animals of the group until four days after infection. PRcv-infectedgroup. Respiratory symptoms were not observed in any pig. On the first day after infection, rectal temperature ranged between 39.5°C and 40.5°C (average 39.9°C) and remained elevated for three days. PRCV+H3N2-infected group. On days 4 and 6 after infection, sneezing and coughing were observed. The average temperature of the group rose to 40.4°C (range 40.1 ° to 40-8°C) on the first days after infection. From the fourth day on, the average temperature returned to normal. One pig died on day 9 from a mesenteric torsion. H3N2 and H1Nl-infected groups. In both groups, tracheobronchial lymph nodes were moderately enlarged and a few small lung lesions were observed on days 6 and 10. PRCV-infected group. Red consolidated areas affecting mainly the diaphragmatic lobes were present on days 3 after infection. The lungs of the pigs killed on days 6 and 10 had larger pneumonic lesions scattered throughout the lungs, but most pronounced in the apical and cardiac lobes. Tracheobronchial lymph nodes were enlarged in all the pigs and were haemorrhagic in the pig killed on day 10. On day 21 after infection lung lesions had disappeared. Areas of consolidation were seen on days 3, 6 and 10 spreading from the diaphragmatic to the cardiac and apical lobes. The extent of lung tissue affected was similar or slightly greater than for the pRcv-infected pigs. Tracheobronchial lymph nodes were enlarged. There were no lesions in the pigs necropsied at 21 days. PRcv+H1Nl-infectedgroup. The lesions observed on day 3 were very small, but were more pronounced and widespread in the lungs of the pig killed on day 6, with the diaphragmatic lobes being the worst affected. The extent ofpneumonic lung tissue was similar to the corresponding pp,¢vinfected pig, although on day 10, lesions in the dual infected pig were smaller and less widespread. Tracheobronchial lymph nodes were enlarged in all the pigs. For each group, the mean daily weight changes at intervals during the first 10 days are shown in Table 3 . This table also shows the regression weight-time line for each group. Since a shallow slope indicates slow growth, this indicated poorest growth in the pRcv-infected group. In all of the infected groups the growth rate slowed down, as compared to the controls, during the first two days after infection. H3N2, PRCV and H 1N 1-infected animals were the most affected. Concurrent infection did not have a synergistic effect in either of the dual-infected groups, where the liveweight gain was higher than in singleinfected animals during the first two days. Although H1N1 and PRcv+H1Nl-infected animals had a lower liveweight gain than the controls in the first four days (PRcv+H1Nl-infected animals lost weight between two and four days), the slopes of the regression lines over the full 10 day period were similar to the control group. The ANOVA showed significant differences in the growth rate between the control group and all the infected groups during the first four days after infection, although differences were not significant at 10 days. Differences between single and dual-infected groups were not significant at any stage. PRCV. All pigs given PRCV shed this virus in nasal swabs until seven to nine days after infection. In tissues, Pp, cv persisted slightly longer in pigs given this virus only (Tables 4 and 5) . Apical lobe + + --+ + + + +-Diaphragmatic lobe + + --+ + + + -- Influenza virus H1N1 and H3N2. The mean duration of excretion of influenza virus, as detected by nasal swabs (Table 4) , was shorter in dual than in single-infected pigs. Three days after infection, H3N2 virus was detected in five of six tissue samples taken from an H3N2-infected pig, whereas only two samples were positive from the corresponding l'RcV+H3N2-infected animal (Table 6 ). H1N1 influenza virus was isolated less frequently from tissues than H3N2. Concurrent infection reduced the rate of isolation, since at three days after infection the virus was detected in three out of six tissues taken from an H1N1infected pig, but not at all from the equivalent eRcv+H1Nl-infected animal. In each infection group there was seroconversion against the virus or viruses inoculated (Table 7) , while the control group remained seronegative against all three viruses throughout the experiment. The titres of influenza antibodies reached a peak at 10 days while the highest PRCV antibody titres were detected at 21 days. The antibody titres were very variable in the different pigs and there were no significant differences between thd single and the dual infected animals The animals infected with PRCV alone developed lung lesions and fever, and suffered growth retardation, as previously described (O'Toole et al 1989 , Cox et al 1990 , Vannier 1990 ). However, respiratory symptoms were not observed, in contrast to the findings of Duret et al (1988) and Van Nieuwstadt and Pol (1989) . The prolonged shedding of PRCV from infected animals, together with airborne transmission, probably helps to explain the rapid spread of PRCV across Europe. Both influenza strains used in this study had been isolated from outbreaks of respiratory disease , Pritchard et al 1987 . However, animals infected with them showed little illness, although fever and decreased growth rate were observed. The H3N2 infected pigs were the worst affected and did show mild respiratory symptoms (nasal discharge). Similar results have been described previously for the H3N2 strain (Wibberley et al 1988) . Serial passage in embry-onated eggs might have reduced the pathogenicity of these viruses for pigs. Field observations have suggested the possible association of swine influenza virus with porcine respiratory coronavirus in outbreaks of respiratory disease. However, the clinical picture observed in these experimentally induced dual infections was quite similar to that seen when PRCV was given on 'its own. Body temperatures were slightly higher after dual than after single infections and PRCV+H3N2-infected pigs were the only ones to develop coughing and sneezing. However, no significant differences in growth rates were detected between the single and dualinfected animals. Lesions observed in dual-infected pigs were generally similar to, or in some instances less severe than those found in PRCVonly-infected animals. Overall, no significant enhancement of the pathogenicity of PRCV by the simultaneous infection with either of two different influenza viruses could be detected. Under field conditions, infections with different viruses are unlikely to be absolutely simultaneous, but it is not possible to predict what if any difference this would make to the severity of disease. Other routes of infection, such as the intratracheal might also have been explored, although intranasal and aerosol routes were selected as being closest to those occurring naturally. H1N1 and H3N2 viruses were recovered from some tissues and, or, nasal swabs for longer after single than after dual infection. It is thus possible that PRCV may have actually interfered with the replication of influenza virus in vivo. An analogous situation in chickens has been described in vitro for the effect of infectious bronchitis virus on the growth of Newcastle disease virus (Buxton and Fraser 1977) . Although coinfection with influenza virus did not seem to increase the pathogenicity of PRCV, potentiation by other respiratory pathogens of the pig cannot be ruled out. For instance, Marois et al (1989) considered that concurrent infection with TGEV increased the severity and extent of the lesions produced by Mycoplasma hyopneumoniae in the lungs of experimentally infected pigs. Further research is needed to see if bacterial respiratory pathogens, such as Pasteurella rnuI-tocida and Actinobacillus pleuropneumoniae, can enhance the pathogenicity of viruses such as PRCV. Sites of replication of a porcine respiratory coronavirus related to transmissible gastroenteritis virus Isolement, identification et pouvoir pathogene chez le porc d'un coronavirus apparent6 au virus de la gastroenterit6 transmissible Enzootic pneumonia in feeder pigs: association with transmissible gastroenteritis virus infection Pathogenicity of experimental infection with 'pneumotropic' porcine coronavirus Centre for Diseases Control PATON, D. (1989) Transmissible gastroenteritis. In Manual of recommended diagnostic techniques and requirements for biological products for lists A and B diseases Isolation of a porcine respiratory, non enteric coronavirus related to transmissible gastroenteritis Porcine influenza outbreak in East Anglia due to influenza A virus (H3N2) Outbreaks of swine influenza in pigs in England in 1986 Disorders induced by the experimental infection with the porcine respiratory coronavirus Isolation of a "roE-related respiratory coronavirus causing fatal pneumonia in pigs Characterization of an influenza A (H3N2) virus isolated from pigs in England in 1987 The authors are grateful to the Central Veterinary Laboratory animal care staff, to Dr S. Done for performing the necropsies, to Mrs A. Reynolds for excellent technical assistance, and to many other staff in the virology department. I. L. was the recipient of a fellowship from the Spanish Ministry of Education and Science.