Table 1.

Non-synonymous ORF mutations in ED43 recombinant viruses during culture adaptation

	ED43						H77	SNP frequencies (%)
								ED43-20m		ED43-31m	ED43-31m_opt	ED43cc*
HCV gene	NT pos	NT change		AA pos	AA change		AA pos	4.1† (4th, 13)	5.0† (10th, 6)	4.3† (2nd, 8)	5.1† (2nd, 6)	5.1† (2nd, 6)
E1	923	C	A	195	R	S	195	7.1	6,9	–	–	–
E1	1152	T	G	271	V	G	271	28.1	89.1	99.9	100	100
E2	1553	T	C	405	S	P	405	7.2	–	–
E2	1712	T	C	458	C	R	458	26.2	88.9	99.9	100	100
E2	2534	T	C	732	S	P	732	8.6	–	–	–	–
p7	2597	A	G	753	N	D	753	16.8	–	–	–	–
p7	2646	T	C	769	I	T	769	10.2	–	–	–	–
p7	2730	T	C	797	F	S	797	11.5	–	–	–	–
NS2	2819	A	G	827	T	A	827	100	100	100	100	100
NS2	2883	A	G	848	Y	C	848	6.5	88.2	99.6	100	100
NS3	3710	A	G	1124	T	A	1124	–	11,3	–	–	–
NS3	4211	A	G	1291	I	V	1291	100	100	100	100	100
NS3	4487	G	A	1383	E	K	1383	11.7	–	–	–	–
NS3	4733	A	G	1465	S	G	1465	100	100	100	100	100
NS3	4736	T	A	1466	L	M	1466	99.8	100	100	100	100
NS3	5054	T	C	1572	F	L	1572	45.8	9.8	100	100	100
NS4A	5354	G	T	1672	A	S	1672	100	100	100	100	100
NS4B	5697	C	T	1786	A	V	1786	100	100	100	100	100
NS4B	5804	A	G	1822	T	A	1822	100	100	100	100	100
NS4B	5933	A	G	1865	T	A	1865	100	100	100	100	100
NS4B	5949	G	A	1870	S	N	1870	100	100	100	100	100
NS4B	6066	G	C	1909	G	A	1909	38.0	87.8	99.9	100	100
NS5A	6257	G	A	1973	A	T	1973	25.8	88.4	–	100	100
NS5A	7017	T	C	2226	L	P	2226	7.7	–	–	–	–
NS5A	7109	G	A	2257	A	T	2261	5.3	88.4	99.9	100	100
NS5A	7126	C	A	2262	D	E	2265	17.8	–	–	–	–
NS5A	7151	A	G	2271	I	V	2274	46.7	9.6	–	–	–
NS5A	7325	A	G	2329	T	A	2332	5.4	88.4	99.5	100	100
NS5A	7436	A	G	2366	T	A	2369	45.9	9.0	–	–	–
NS5A	7503	T	C	2388	M	T	2388	11.2	7.3	–	–	–
NS5A	7578	A	G	2413	D	G	2416	100	100	100	100	100
NS5B	7975	C	A	2545	D	E	2548	100	100	100	100	100
NS5B	7979	A	G	2547	N	D	2550	11.8	–	–	–	–
NS5B	8033	A	G	2565	N	D	2568	5.9	–	–	–	–
NS5B	8131	A	T	2597	K	N	2600	95.8	99.7	99.8	100	100
NS5B	8134	A	C	2598	K	N	2601	8.6	–	–	–	–
NS5B	8365	T	G	2675	D	E	2678	100	100	100	100	100
NS5B	8718	T	C	2793	V	A	2796	6.1	88.4	100	100	100
NS5B	8730	A	G	2797	K	R	2800	100	100	100	100	100
NS5B	8756	G	A	2806	E	T	2809	100	100	100	100	100
NS5B	8757	A	C					100	100	100	100	100
NS5B	9087	C	T	2916	A	V	2919	100	100	100	100	100
NS5B	9132	A	G	2931	Q	R	2934	98.1	97.3	100	-‡	-‡
NS5B	9267	A	G	2976	D	G	2979	100	100	100	100	100
NS5B	9273	A	T	2978	Y	F	2981	100	100	100	100	100
NS5B	9281	A	G	2981	M	V	2984	100	100	100	100	100
NS5B	9284	T	C	2982	S	P	2985	26.0	90.1	100	100	100
NS5B	9289	T	G	2983	H	Q	2986	6.3	7.0	–	–	–
NS5B	9312	T	G	2991	L	R	2994	100	100	100	100	100
NS5B	9315	G	A	2992	C	Y	2995	99.3	99.1	100	100	100

Only mutations with a frequency of >5% of the viral population in NGS analysis are shown. Shaded background indicates frequencies of engineered mutations.

*Nucleotide G at position 38 in 5’UTR was changed to A.

†HCV infectivity titres at indicated passage and day (parentheses) are shown as log₁₀FFU/mL.

‡These recombinant viruses had A at position 9132 instead of G, and this nt was maintained.

AA, amino acid; NGS, next-generation sequencing; NT, nucleotide; NT pos and AA pos, nucleotide and amino acid positions; SNP, single nucleotide polymorphism.