Viruses Five CAV isolates, BL-1, BL-2, BL-3, BL-4, and BL-5, isolated recently at University Putra Malaysia (UPM); three CAV isolates, SMSC-1, SMSC-2 and 3-1, isolated at Veterinary Research Institute (VRI), Malaysia, and an European CAV isolate Cux-1, kindly provided by Dr. K.A. Schat, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA, were used for the study. Cells and cell culture MDCC-MSB1 cells, a cell line derived from a Marek's disease lymphoma, were kindly provided by the Director, VRI, Ipoh, Perak, Malaysia. The cell culture was maintained following the methods described by Chowdhury et al. (2002). For each isolate, two ml of the stock virus were inoculated into 2 x 106 cells in a 75-cm2 flask containing 18 ml RPMI 1640 media. One flask was also maintained as an uninoculated control. The infected and control cells were harvested 48-72 hours after infection. DNA extraction DNA was extracted from uninfected and CAV infected MDCC-MSB1cells harvested at 48-72 hours post infection following the procedures described by Chowdhury et al. (2002) with minor modification and stored at -200C until use. Amplification of DNA fragments by polymerase chain reaction (PCR) The following DNA fragments were amplified by PCR from the whole genome of CAV for analysis by restriction endonulease enzymes. The nucleotide (nt) positions used here are based on Cux-1 sequence (Noteborn et al., 1991). (i) Fragment A: 1518 bp fragment (nt 2317-1515). Two oligonucleotide primers flanking fragment A were as follows. Forward primer (CAV5): 5'- ATC GAA TTC CGA GTG GTT ACT ATT CC -3' (nt 2317-23) and the reverse primer (CAV6): 5'- GAA GGA TCC CTC ATT CTT AGT GGC -3' (nt 1515-1492) (Soiné et al., 1993). (ii) Fragment B: 926 bp fragment (nt 1463-69). The primers flanking fragment B were as follows. Forward primer (CAV 9): 5'- GAC ACA TTG AAA CCC GCT TT -3' (nt 1463-1482) and the reverse primer (CAV 10): 5'- GCG ATT CGT CCA TCT TGA CT -3' (nt 69-50) (Todd et al., 1996). (iii) Fragment C:675 bp fragment (nt 844-1519). The primers flanking fragment C were as follows. Forward primer (CAV 13): 5'- GAC TGT AAG ATG GCA AGA CGA GCT C -3' (nt 844-868) and the reverse primer (CAV 14): 5'- GGC TGA AGG ATC CCT CAT TC -3' (nt 1519-1500) (Todd et al., 1992). (iv) Fragment D: 552 bp fragment (nt 2074-306). The primers designed based on Cux-1 sequence (Noteborn et al., 1991), were used for amplifying fragment D. Forward primer (CAV15): 5'- GTA ATG AAG AGC GAT GCA TGG GC- 3' (nt 2074-2096) and the reverse primer (CAV6b): 5'- CCA TTT TCG AAA CGT CAC TTT CGC- 3' (nt 306-283). The fragment A and fragment B covers the whole CAV genome. Fragment C falls within the fragment A at the 3'-end, and fragment D includes part of fragment B (at 3'-end) and part of fragment A (at 5'-end). All oligonucleotide primers were synthesized by Operon Technologies, Inc., USA. For amplification of all of the above fragments, PCR's were carried out in 50 'l reaction mixture containing distilled water, PCR buffer (1x), MgCl2 (2.5 mM), dNTP mixture (0.3 mM each), forward and reverse primers (30 pmole each), Taq DNA polymerase (2.0 unit) and DNA template (0.2 to 0.5 'g). The thermal cycling profiles were as follows: initial 5 min incubation at 94oC, followed by 40 cycles of 94oC for 1 min, 60oC for 1.5 min, 72oC for 2 min; a final incubation at 72oC for 10 min and cooling at 4oC. The PCR products were run on 1 to 1.5% agarose gel electrophoresis. After staining with ethidium bromide, the specific bands were excised and purified by Geneclean kit (BIO 101, Inc., USA) following the supplied instructions. Analysis of amplified DNA fragments by restriction endonuclease enzymes For all nine isolates of CAV, the fragment A (1518 bp) was digested with one restriction enzyme, Eco130I (StyI); the fragment B (926 bp) with three enzymes, Eco130I (StyI), HpaII and MboI separately; the fragment C (675 bp) with three enzymes BsuRI (HaeIII), HinfI, and HpaII; and the fragment D (552 bp) with one enzyme, EcoRI. All enzymatic digestions were performed separately in a 20 l reaction mixture containing buffer (1x) for individual enzyme as recommended by the manufacturer (MBI Fermentas, Lithuania), enzyme (15-25 units), distilled water (if any) and the purified DNA fragment. The reaction mixture was incubated at 370C for 10-14 hours. Agarose gel electrophoresis After incubation, the resulting digested products, in case of fragment A/StyI were run in 2% agarose gel electrophoresis and in case of fragment D/EcoRI were run in 2.5% agarose gel electrophoresis, and the gels were stained in ethidium bromide (1 µg ml1). For fragment A/StyI, 1 kb DNA ladder (MBI Fermentas, Lithuania) and for fragment D/EcoRI, 50 bp DNA ladder, ready to use (MBI Fermentas, Lithuania) was used as molecular size markers. The separated DNA fragments in the gel were visualised by using a UV transilluminator and photographs were taken. Sodium dodecyl sulphate polyacrylamide gel electrophesis (SDS-PAGE) The fragment B digested products with each of StyI, HpaII and MboI, and fragment C digested products with each of HaeIII, HinfI, and HpaII were separated by SDSPAGE. For the experiment, 12% separating or resolving gel and 4% stacking gel were prepared and electrophoresis was conducted at 44-48 V in a Mini-Protein® II Electrophoresis Cell (BIO-RAD, USA) following the instructions of the manufacturer. The run was maintained up to 1.5 cm from the bottom level, which took about 4 hours. Fragments generated by digesting the ØX174 DNA (replicative form) with HaeIII were used as size markers. After electrophoresis, the gel was stained with ethidium bromide (1µg ml-1). The separated DNA bands in the gel were visualised by the UV transilluminator and photographs were taken.