VIROLOGY 133,2!58-270 (1984) Restriction Site Map of African Swine Fever Virus DNA J. M. ALMENDRAL, R. BLASCO, V. LEY,’ A. BELOSO, A. TALAVERA, AND E. VINUELA2 Cmtro de Biologic Molecular (CSIC-VAM), Universidod Autbmma, Fawltud de Ciencias, Canto B&co, Madrid-.%, Spain Received July 7, 1983; accepted November 29, 1989 Treatment of African swine fever virus DNA (about 170 kbp) with the restriction endonucleases SolI, EcoRI, Kpn1, PvuI, and SmaI yielded 14,31,17,13, and 11 fragments, respectively. The order of the restriction fragments produced by each nuclease was es- tablished by identifying the crosslinked EcoRI and Sal1 terminal fragments and then finding overlapping fragments. The five restriction fragment maps were integrated into a single map by locating SalI, KpnI, PvuI, and SvzaI sites in cloned EcoRI fragments, and orienting each fragment in the overall map. INTRODUCTION African swine fever (ASF) virus is an icosahedral cytoplasmic deoxyvirus, which infects only domestic pigs and related an- imals (Hess, 1971,198l). The viral genome is a double-stranded DNA molecule with a molar mass of about 100 X 10” g mol-’ (Enjuanes et al, 197613) and terminal co- valent crosslinks (Ortin et al, 1979). The availability of cloned fragments spanning the viral genome and the knowl- edge of the order of restriction fragments in the DNA molecule are prerequisites for studies of regions coding for viral poly- peptides and for analysis of genetic het- erogeneity of the virus. For these aims Ley et al (1984) have prepared a collection of cloned ASF virus DNA restriction frag- ments, which account for about 98% of vi- ral DNA. In this paper we show a map of ASF virus DNA with about 80 sites recognized by the restriction endonucleases S&I, EcoRI, KpnI, PvuI, and SmuI, with an av- erage distance of about 2 kilobase pair (kbp) between contiguous sites. ’ Present address: Abello, S.A., Juliin Camarillo, 8, Madrid-17, Spain. * Author to whom requests for reprints should be addressed. MATERIALS AND METHODS Viruses and cells. ASF virus, adapted to grow in VERO cells (CCLSl, American Type Culture Collection), was cloned by plaque purification (Enjuanes et al, 1976a). Virus stocks were obtained from VERO cells infected at a multiplicity of infection (m.0.i.) of 0.001-0.01 plaque-forming units (PFU) per cell, in Dulbecco modified Ea- gle’s medium (DME) with 2% newborn calf serum. When the cytopathic effect was complete, cell debris was removed by low- speed centrifugation and the supernatant centrifuged for 6 hr at 8000 rpm at 4’ in a Sorvall GS3 rotor. The pellet was resus- pended in phosphate-buffered saline (PBS) and the virus suspension was stored in portions at -70”. Un$brm ltxbeli~ of ASF wires DNA with %P. VERO cells (about 4 X lo4 cells/cm2) were added to a 75-cm2 plastic flask in 10 ml of DME with nonessential amino acids and 10% calf serum. After cell attachment, 50 &i/ml of carrier-free [32P]phosphate was added to the medium. When the cul- ture reached a density of about lo5 cells/ cm’, the medium was removed and the cells were infected with plaque-purified ASF vi- rus at a m.o.i. = 10 PFU/cell, in 2 ml of medium with 2% calf serum and radioac- tive phosphate as above. After 2 hr ad- sorption, 6 ml of the last medium with 0042-6822/84 $3.00 258 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.