Quasispecies evolution in NS5A region of hepatitis C virus genotype 1b during interferon or combined interferon-ribavirin therapy

Table 1 Sequences of amplification and sequencing primers

Forward primers		Positions nt
E1	5' GAGGGGGCTGTGCAGTGGATG 3'	6057-6077
I3	5' TCCGGCTCGTGGCTAAGGGA 3'	6246-6265
S2	5' GGATTTCCACTACGTGACGGG 3'	6620-6640
S4	5' GGGTCTCCCCCCTCCTTGGCC 3'	6906-6926
S31	5' GGACTACGTCCCTCCGGTGG 3'	7241-7260
Reverse primers
AS2	5' CGTCACGTAGTGGAAATCCCC 3'	6618-6638
AS32	5' GGGACGTAGTCCGGGTCCTTC 3'	7232-7252
I4	5' GCAGCAGACGAGATCCTCAC 3'	7567-7586
E2	5' GCTGCGAGATGTTGTGGCGTA 3'	7698-7718
Cloning primers	(pMOS vector)
SeqS1	5' GGGAAAGCTTGCATGCCTGC 3'
SeqAS2	5' GACGTTGTAAAACGACGGCC 3'

Nucleotide positions are numbered according to the HCV-J sequence and the NS5A region is within positions 6246-7586.

Table 2 Clinical and virological characteristics of the 10 patients studied before and during treatment mean ± SD

Patient andtime of treatment	Sex	Age	Viral loadlog IU/mL	n	NT substitutions/mastersequence	AA substitutions/mastersequence	Genetic distance
SVR1-D0	F	48	2.00	15	4.5 ± 2.3	2.5 ± 1.4	0.0065 ± 0.0008
SVR2-D0	F	53	4.55	13	5.1 ± 3.6	3.3 ± 2.1	0.0070 ± 0.0011
SVR3-D0	M	43	4.71	8	10.3 ± 3.6	4.7 ± 1.9	0.0105 ± 0.0018
SVR4-D0	M	59	6.24	16	15.4 ± 8.0	7.5 ± 4.9	0.0172 ± 0.0022
SVR4-M3	-	-	1.61	13	3.6 ± 2.1	1.8 ± 1.0	0.0051 ± 0.0008
SVR5-D0	F	65	5.44	15	10.1 ± 6.6	4.9 ± 2.0	0.0171 ± 0.0016
SVR5-M3	-	-	2.77	17	6.4 ± 2.4	3.3 ± 1.6	0.0070 ± 0.0010
SVR6-D0	M	47	7.26	17	15.9 ± 10.5	8.5 ± 5.1	0.0192 ± 0.0022
SVR6-M3	-	-	2.18	6	4.0 ± 1.7	2.4 ± 1.3	0.0062 ± 0.0012
SVR7-D0	F	51	6.61	15	29.3 ± 8.5	7.3 ± 2.7	0.0294 ± 0.0029
SVR7-M3	-	-	5.42	15	12.6 ± 4.2	4.6 ± 2.2	0.0156 ± 0.0016
NR1-D0	M	35	5.13	17	15.2 ± 4.3	3.5 ± 1.9	0.0181 ± 0.0020
NR1-M3	-	-	5.24	9	15.3 ± 4.4	4.8 ± 1.7	0.0143 ± 0.0020
NR1-M6	-	-	2.83	4	4.5 ± 3.1	2.0 ± 3.4	0.0041 ± 0.0013
NR2-D0	F	50	5.90	17	15.6 ± 6.4	5.2 ± 2.9	0.0181 ± 0.0020
NR2-M3	-	-	5.87	12	9.5 ± 3.6	3.9 ± 1.8	0.0125 ± 0.0013
NR2-M6	-	-	5.77	10	10.3 ± 3.3	4.0 ± 2.2	0.0274 ± 0.0020
NR3-D0	F	44	5.63	14	10.9 ± 3.7	4.4 ± 2.2	0.0279 ± 0.0021
NR3-M3	-	-	5.79	11	11.1 ± 2.7	4.0 ± 2.8	0.0157 ± 0.0017
NR3-M6	-	-	5.00	12	10.8 ± 3.3	4.4 ± 2.0	0.0154 ± 0.0015

F: Female; M: Male; n: number of PCR products analyzed; D0: beginning of the treatment; M3: 3 mo of treatment; SVR: Sustained Virological Responders; NR: Non-Responders to IFN-ribavirin therapy.

Table 3 Characteristics of HCV quasispecies in pre-treatment isolates from SVR and NR patients

Region andpatient type	Geneticdistance	dS	dN	dN/dS	Num. amino acidmutations
NS5A
SVR (n = 99)	0.1027	0.3239	0.039	0.120	33.9
NR (n = 48)	0.0698	0.1216	0.025	0.206	29.8
ISDR
SVR (n = 99)	0.0834	0.3084	0.0295	0.096	1.3
NR (n = 48)	0.0765	0.2645	0.0271	0.102	1.9
PKRbd
SVR (n = 99)	0.0934	0.2934	0.0424	0.145	5.3
NR (n = 48)	0.0815	0.2694	0.0332	0.123	5.7
V3
SVR (n = 99)	0.1836	0.3264	0.1282	0.393	6.0
NR (n = 48)	0.1372	0.4482	0.0556	0.124	4.7

Values were calculated as described in methods; amino acids sequences were compared to HCV-J; n: number of PCR products analyzed; dS: frequency of synonymous substitutions per site; dN: frequency of non-synonymous substitutions per site; SV: Sustained Virological Responders; NR: Non-Responders to IFN-ribavirin therapy.

Table 4 Complexity evolution in HCV quasispecies from SVR and NR patients in ISDR, PKRbd and V3 domains

Patient	Time of treatmentdomains	D0ISDR-PKRbd-V3	M3ISDR-PKRbd-V3	M6ISDR-PKRbd-V3
SVR1	n = 15	3–4–3	-	-
SVR2	n = 13	3–4–2	-	-
SVR3	n = 8	3–5–3	-	-
SVR4	n = 16–13	3–7–3	3–4–3	-
SVR5	n = 15–17	8–8–1	4–7–1	-
SVR6	n = 17–6	8–9–3	1–2–3	-
SVR7	n = 15–17	6–6–7	6–7–7	-
NR1	n = 17–9–4	6–8–7	3–4–4	1–3–2
NR2	n = 17–12–10	7–8–2	5–8–3	3–4–6
NR3	n = 14–11–12	6–7–4	5–7–3	4–5–2

Amino acids sequences were compared to HCV-J; n: number of PCR products analyzed.

Table 5 Number of amino acids mutations observed in six different regions from NS5A protein, at baseline mean ± SD

NS5A region (aa range)	2 groups (D0)(n = 147)	SVR (D0)(n = 99)	NR (D0)(n = 48)	SVR (M3)(n = 51)	NR (M3)(n = 32)	NR (M6)(n = 26)
ISDR (2209-2248)	1.5 ± 1.6	1.3 ± 1.7	1.9 ± 1.1	0.8 ± 0.8	2.1 ± 1.0	1.8 ± 1.2
PKRbd (2209-2274)	5.4 ± 2.1	5.3 ± 2.4	5.7 ± 1.2	4.7 ± 1.6	6.1 ± 1.0	5.7 ± 1.3
V3 (2353-2379)	5.5 ± 1.4	6.0 ± 1.5	4.7 ± 0.6	6.1 ± 1.3	4.8 ± 0.7	5.1 ± 0.6
Complete NS5A (1973-2208)	32.6 ± 5.6	33.9 ± 6.0	29.8 ± 3.7	31.7 ± 5.3	30.8 ± 3.9	31.9 ± 2.9
N-terminal part of NS5A (1973-2208)	12.4 ± 3.1	12.9 ± 3.3	11.6 ± 2.4	11.7 ± 2.0	11.2 ± 2.1	12.8 ± 2.1
C-terminal part of NS5A (2209-2419)	20.1 ± 3.9	21.1 ± 4.1	18.2 ± 2.4	20.1 ± 4.2	19.6 ± 2.9	19.4 ± 3.4

n: number of PCR products analyzed; SVR: Sustained Responders; NR: Non-Responders to IFN-ribavirin therapy; D0: beginning of the treatment; M3: 3 mo of treatment; M6: 6 mo of treatment.