Journal of General Virology (1992), 73, 3285-3288. Printed in Great Britain 3285 Differential in vitro inhibition of feline enteric coronavirus and feline infectious peritonitis virus by actinomycin D Evelyn L. Lewis, 1 D. A. Harbour, 1. J. E. Beringer 2 and J. Grinsted 3 1Department of Veterinary Medicine, Langford House, Bristol BS18 7DU, 2Department of Botany and Zoology, Woodland Road, Bristol BS8 1 UG and 3Department of Pathology and Microbiology, Medical School, University of Bristol, University Walk, Bristol BS8 1TD, U.K. The growth of feline enteric coronavirus strain 79-1683 in whole feline embryo cells was inhibited by the presence of 1 tg/ml of actinomycin D in the culture fluid. No virus-specific mRNAs could be detected in such cultures and yields of infectious virus were depressed by 99%. By contrast, the antigenically related feline infectious peritonitis virus strain 79-1146 was unaffected by the presence of actinomycin D, indicating a fundamental difference between the two feline coronavirus strains in their requirements for host-encoded function(s). Feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV) are two coronaviruses which, although antigenically similar (Sanchez et al., 1990), show markedly different tissue tropisms and virulence. FECV causes a mild or inapparent enteritis in kittens whereas FIPV induces a fatal, immune-mediated peritonitis (Pedersen et al., 1981; Lutz et al., 1986; Evermann et al., 1991). The critical event in the pathogenesis of FIPV-mediated disease appears to be the ability of the virus to infect macrophages productively (Stoddart bovine coronavirus, Keck et al. (1988); rat coronavirus, Parker et al. (1970); mouse hepatitis virus (MHV), Keck et al. (1989), Spaan et al. (1981) and Malluci (1965); infectious bronchitis virus (IBV), Lomniczi human coronavirus (HCV) 229E, Schreiber et at. (1989). However FECV appeared to be strongly inhibited since no viral intracel- lular RNAs were detected. We therefore studied this phenomenon in more detail. Monolayers of WFE cells grown in 25 cm 2 flasks were inoculated with 10 p.f.u./cell of either FECV 79-1683 or FIPV 79-1146. After 1 h incubation at 37 C, growth medium with or without 1 tg/ml actinomycin D was added and the cultures were reincubated at 37 C. Mock-infected WFE cells were processed simultaneously. After 6 h, total cellular RNA was prepared by extraction with guanidinium isothio- cyanate, and pelleted through a caesium chloride step gradient (Maniatis et al., 1982). Viral intracellular RNAs were visualized by Northern blot hybridizations using a cDNA probe representing the 3 end of the FIPV genome. RNA preparations were electrophoresed in formaldehyde-formamide 1% agarose gels at 70 V for approximately 4 h, transferred to nitrocellulose filters (Meinkoth Vennema, 1991). However, in the presence of actinomycin D, no viral intracellular RNAs were seen in FECV-infected cells, whereas in FIPV-infected cultures viral intracellular RNA synthesis 0001-1047 1992 SGM 3286 Short communication (a) 1 2 3 4 21.2- 5.5- 2-15- (b) 1 2 3 4 (s) (M) (N) :=7 =?= = Fig. 1. Northern blot hybridizations of FIPV probe, pB12 (represent- ing the 3 end of the genome), to electrophoretically separated intracellular RNAs from FECV-, FIPV- or mock-infected WFE cells with (Act D +) or without (Act D-) actinomycin D (l Dg/ml). Ribosomal RNA size markers in kb are indicated on the left of each filter. Feline coronavirus RNAs are labelled according to the recommended nomenclature (Cavanagh et al., 1990) on the right-hand side of each filter. (a) Lanes 1 and 2, mock-infected Act D + and Act D- WFE cells, respectively. Lane 3, FECV 79-1683-infected and Act D + WFE cells. Lane 4, FECV 79-1683-infected and Act D- WFE cells. (b) Lanes 1 and 2, mock-infected ActD and ActD- WFE cells, respectively. Lane 3, FIPV 79-1146-infected and Act D WFE cells. Lane 4, FIPV 79-1146- infected and ActD- WFE cells. was comparable to that in cells without the drug (Fig. 1). Further monolayers of WFE cells or feline embryo lung (FEL) fibroblasts (OReilly et al., 1979) were infected with FECV 79-1683 or FIPV 79-1146 at an m.o.i, of 5 p.f.u./cell. After 1 h for adsorption at 37 C, 10 I I I I I (a) ! (b) / I I I I I 52 4 6 8 10 12 4 6 8 10 12 Time after infection (h) Fig. 2. Feline coronavirus yields from confluent monolayers of WFE cells with (0) or without () 1 g/ml actinomycin D. (a) WFE cells (passage 25) infected with FECV 79-1683 (passage 10). (b) WFE cells (passage 28) infected with FIPV 79-1146 (passage 9). growth medium Eagles MEM supplemented with 10 foetal calf serum (FCS) with or without I tag/ml actinomycin D was added. Virus was harvested at intervals by subjecting replicate cultures to three cycles of freezing and thawing. Cell debris was pelleted by centrifugation at 2000 g for 10 min and the supernatant fluid was titrated for infectious virus by plaque assay as follows. Confluent monolaye