and 6 nucleotide sequence analysis to obtain sequences in the ns5 region viral rna was reverse transcribed and amplified by polymerase chain reaction pcr in a single reaction with previously published primers thought to be highly conserved amongst different variants of hcv enomoto et al 1990 biochemical and biophysical research communications 170 1021 1025 for some sequences a second pcr was carried out with primers 554 and 555 chan et al 1992 j gen virol 73 1131 1141 in combination with two new primers 122 sense orientation 5 u2032 ctc aac cgt cac tga gag aga cat 3 u2032 seq id no 51 and 123 anti sense 5 u2032 gct ctc agg ttc cgc tcg tcc tcc 3 u2032 seq id no 52 product dna was phosphorylated purified and cloned into the smai site of puc19 yanisch perron et al 1985 gene 33 103 107 following the procedures described in simmonds amp chan 1993 analysis of viral sequence variation by pcr in molecular virology a practical approach pp 109 138 edited by a j davidson and r m elliot oxford irl press alternatively amplified dna was purified and directly sequenced as described in simmonds et al 1990 j virol 64 5840 5850 and cha et al 1992 proc natl acad sci u s a 89 7144 7148 these methods allowed comparison of a 222 bp fragment of dna homologous to positions 7975 to 8196 in the prototype virus numbered as in choo et al 1991 proc natl acad sci u s a 88 2451 2455 the results are shown in fig1 and 2 attempts have been made to isolate dna sequences from.