Research in Veterinary Science 1992, 53, 309-314 Pathogenicity of concurrent infection of pigs with porcine respiratory coronavirus and swine influenza virus I. LANZA*, I. H. BROWN, D. J. PATON, Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, New Haw, Addlestone, Surrey KT15 3NB 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-infect- ed 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, suggest- ing a possible in vivo interference between repli- cation 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 (OToole et al 1989) but rarely show clinical signs of disease. The role of the virus in field outbreaks of res- piratory 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, partic- ularly 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 out- break, but the virus was not isolated. However, the influenza isolate on its own failed to reproduce the clinical disease when inoculated into exper- imental 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. Materials and methods Experimental animals 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. 309 310 Challenge viruses PRcv. The Stopps strain (135308) of tRCV, iso- lated 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 con- taining antibiotics. The homogenates were titrat- ed 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. L Lanza, L H. Brown, D. J. Paton TABLE 1: Groups of experimental animals Group (infection) Age (days) Number of animals Control 46 2 H3N2 46 6 PRCV 47 6 PRcv+H3N2 47 6 H1N1 61 4 PRcv+H1N1 61 4 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 allan- toic 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 embry- onated chicken eggs. After three days incubation at 34C, the allantoic fluids were