Arch Virol (2012) 157:1113–1121 DOI 10.1007/s00705-012-1283-1
ORIGINAL ARTICLE
Hepatitis C virus amino acid sequence diversity correlates with the outcome of combined interferon/ribavirin therapy in Chinese patients with chronic hepatitis C Yanli Zeng • Wei Li • Jingtao Li • Junjie Wang Bin Zhou • Jian Zhang • Shuwen Liu • Yingsong Wu • Jinlin Hou • Yuanping Zhou
•
Received: 2 December 2011 / Accepted: 13 February 2012 / Published online: 18 March 2012 Ó Springer-Verlag 2012
Abstract Evidence has shown that the p7, NS2 and NS3 genes affect the outcome of pegylated-IFN-a/ribavirin (PEG-IFN/RBV) combination therapy in different populations with HCV infections. Here, we test the hypothesis that diversity in the p7, NS2 and NS3 genes influences the probability of obtaining either a sustained (SVR) or nonsustained (non-SVR) viral response in Chinese patients with genotype 1b chronic hepatitis C. There were significantly more unique variations in the p7, NS2 and NS3 genes in the sequences from SVR than non-SVR patients. Inter-patient variations related to treatment outcome in NS3 were concentrated in the protease domain. There were no significant differences in the frequency of variations in the core, E1 and E2 proteins between the groups. In conclusion, increased amino acid sequence diversity in the p7, NS2 and NS3 genes is associated with an SVR to PEGIFN/RBV therapy in Chinese patients with genotype 1b chronic hepatitis C.
Y. Zeng, W. Li and J. Li contributed equally. Y. Zhou is the first corresponding author. Y. Zeng W. Li J. Li J. Wang B. Zhou J. Zhang J. Hou Y. Zhou (&) Department of Infectious Diseases, Hepatology Unit, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China e-mail:
[email protected] S. Liu (&) School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China e-mail:
[email protected] Y. Wu School of Bio-Technology, Southern Medical University, Guangzhou, China
Abbreviations ALB ALT AST CHC EVR ETVR eIF2a HGB HCC HCV HBV HIV IFN NS PEG-IFN/RBV PLT PKR PePHD RVR RBV SVR TBIL WBC 50 -UTR
Albumin Alanine aminotransferase Aspartate aminotransferase Chronic hepatitis C Early virological response End-of-treatment virological response Protein kinase eukaryotic initiation factor 2 alpha Hemoglobin Hepatocellular carcinoma Hepatitis C virus Hepatitis B virus Human immunodeficiency virus Interferon Nonstructural protein Pegylated-interferon/ribavirin Blood platelets Protein kinase protein Phosphorylation homology domain Rapid virological response Ribavirin Sustained virological response Total bilirubin White blood cell 50 Untranslated region
Introduction Hepatitis C virus (HCV) infection is a serious global health problem that affects 180 million people worldwide and 41 million people in China [1, 2]. Almost 85% of patients infected with HCV are unable to clear the virus [3] and are
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at high risk of progressive liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) [4]. HCV is a member of the family Flaviviridae, and has a 9.6-kilobase (kb) plusstrand RNA genome that encodes a long polyprotein precursor of about 3000 amino acids. This precuror is processed proteolytically by both cellular and viral proteases to at least 10 individual proteins, including four structural proteins (C, E1, E2 and p7) and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). To date, six major genotypes of HCV have been identified that differ by 31%-34% in their nucleotide sequences and by about 30% in their amino acid sequences. Each genotype is divided into multiple subtypes with 80%-85% similarity. Isolates within each subtype are also variable, with 8%-12% divergence between isolates from different patients [5–9]. The standard therapy for chronic hepatitis C (CHC) is the combination of interferon alpha (IFN-a) or pegylatedIFN-a (PEG-IFN) plus ribavirin (RBV) for 24–48 weeks. This leads to different rates of sustained virological response (SVR) in different HCV genotypes. The SVR rate is between 75%-80% of patients infected with genotypes 2 or 3 [10] but only 39.8%-40.9% of patients infected with genotype 1 [11]. Genotype 1 is the most prevalent HCV genotype in the United States, Europe and China [12, 13]. Although progress has been made in the field of clinical treatment [14, 15], the efficiency of therapy is still not satisfactory [10, 11], especially in patients infected with the ‘‘difficult-to-treat’’ genotype 1 HCV [16]. There is a high rate of significant side effects associated with treatment of these patients, and an assay to predict which patients are unlikely to respond to interferon/ribavirin would be valuable. Evidence shows that inter-patient genetic variation within the genotype 1 HCV has an influence on the treatment outcome. Although whole-genome sequencing has not identified any new specific amino acid sequences that influence outcome, previous reports have demonstrated that high levels of genetic diversity, particularly in the core, p7, NS2, NS3 and NS5A genes, is associated with both the day 28 response (rapid virological response, RVR) and the SVR [17–20]. The goal of this study was to determine whether the increased viral diversity is associated with an SVR in Chinese patients. Our results indicate that increased amino acid sequence diversity in the p7, NS2 and NS3 genes is associated with an SVR to PEG-IFN/RBV therapy in Chinese patients with genotype 1b chronic hepatitis C.
Materials and methods Patients Patients who were chronically infected with HCV genotype 1b enrolled in this study at the Department of Infectious
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Diseases, Nanfang Hospital in Guangzhou, China. All of them have completed the combination therapy of 180 lg of pegylated IFN-a-2a (Pegasys; Hoffmann-La Roche, Nutley, NJ, USA) or 1.5 lg of pegylated IFN-a-2b (Pegintron; Schering-Plough, Innishannon, County Cork, Ireland) per kilogram of body weight subcutaneously once weekly and 800-1000 mg of oral ribavirin per day for 48 weeks, with 6-months of follow-up. Patients with the following criteria were excluded: coinfection with other viruses such as hepatitis B virus (HBV) or human immunodeficiency virus (HIV), decompensated liver disease, poorly controlled diabetes, autoimmune disease or immunologically mediated disease, a history of mental disorders, angiocardiopathy, chronic nephrosis and organ transplantation. The assessment of efficacy included the early virological response (EVR), end-of-treatment virological response (ETVR) and SVR, which were defined as a negative test result for serum HCV RNA at weeks 12 and 48 of the treatment and 24 weeks after the therapy, respectively. A total of 61 patients were recruited, and serum samples were collected before the treatment. The study was performed in accordance with the ethical standards of the Declaration of Helsinki and was approved by the Ethical Committee of Nanfang Hospital in Southern Medical University. All subjects gave written, informed consent. Amplification and sequencing of a 5.2-kb HCV genome For primer design, genotype 1b HCV sequences from GenBank were aligned using the BioEdit program, and a consensus sequence was generated. Primer sequences were designed with reference to this consensus sequence, using the Primer 5 program. The primers used to amplify a 5.2-kb amplicon from the HCV genome are listed in Table 1. HCV RNA was extracted from 140 ll of serum using a QIAamp Viral RNA Kit (QIAGEN, Valencia, California, USA) according to the manufacturer’s protocol. A single 5.2-kb fragment that spanned the region between and including the core and NS3 genes was amplified as described [21]. Briefly, reverse transcription was carried out with Superscript III (Invitrogen Life Technologies, Carlsbad, CA, USA), and nested PCR was performed using high-fidelity Taq polymerase (Invitrogen Life Technologies, Carlsbad, CA, USA). Amplification was performed as follows: initial denaturation at 94°C for 2 min, then 15 cycles of 20 s at 94 °C and 6 min at 68 °C, followed by 15 cycles of 20 s at 94 °C and 6 min at 68 °C, increasing 10 s per cycle. All PCR products were purified, and consensus sequences for the 5.2-kb amplicons were obtained by direct sequencing (Invitrogen Biotechnology Co., Ltd., Shanghai, China). The sequences determined in this work have been deposited in GenBank (JQ246509-JQ246545, JQ253518JQ253541).
HCV diversity and SVR Table 1 Sequences and positions of the primers utilized for the HCV 5.2-kb amplification
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Primers
Name
Position (nt)a
Sequence (50 –30 )
RT
6093R
6093–6078
GGTTCATCCACTGCAC
Outer primers
P1
57–83
ACTGTCTTCACGCAGAAAGCGTCTAGC
P2
5283–5303
CTCCAGGTCRGCCGACATGCA
P3
69–95
CAGAAAGCGTCTAGCCATGGCGTTAGT
P4
5274–5295
CRGCCGACATGCATGYCATGAT
a
Primer position according to the numbering of HCV-con1 (GenBank accession number: AJ238799)
Inner primer
Sequence analysis Generated sequence files from patients were assembled using Vector NTI software (Invitrogen Life Technologies, Carlsbad, CA, USA), and base-calling errors were corrected following inspection of the chromatogram. The mixed-base positions, which were due to HCV quasispecies, were resolved by identifying the predominant base at each position. Sequence alignments were done with Clustal W software. Amino acid sequences were deduced from the nucleotide sequences, and all analyses were performed at the amino acid level. Shannon’s entropy between groups was calculated with Bioedit software, while the mean genetic distance was calculated using the p-distance algorithm in MEGA 5 [22]. The population consensus reference sequence was generated from 120 full-length open reading frames of genotype 1b isolates published in the HCV databases. Statistical analysis Statistical differences in the patients’ baseline parameters were determined by Student’s t-test for numerical variables and the chi-square probability test for categorical variables. The Mann-Whitney rank sum test was used to compare the number of all variations and unique variations between the SVR and the non-SVR groups, while the proportions of unique variations and the association of these amino acid variations with treatment outcomes were compared using Fisher’s exact test. The Mann-Whitney rank sum test was also used to compare the Shannon’s entropy values between the SVR and the non-SVR groups. The average genetic distances between the groups were compared using an independent samples t-test. The level of significance for statistical significance was 0.05. All statistical analysis was done using SPSS version 13.0 (SPSS Inc., USA).
Results Characteristics of the study population Of the 61 patients enrolled in this study, 35 (57.4%) achieved an SVR at week 72, while 26 (42.3%) were
referred to as non-SVR at that time. Patients with EVR and ETVR achieved SVR at a higher rate (p=0.006 and p\0.001, respectively). Patients with SVR tended to be younger than those with non-SVR (p=0.005). In addition, blood platelet (PLT) counts and albumin (ALB) were higher in SVR patients than in non-SVR patients ((p=0.008 and p=0.033, respectively). The lower platelet counts and ALB levels reflect slightly more severe liver disease, which is known to affect response to therapy, and patients with SVR tended to be younger. However, there were no significant differences between the two groups with respect to gender, alanine aminotransferase (ALT), aspartate aminotransferase (AST), HCV RNA, total bilirubin (TBIL), white blood cell (WBC), or hemoglobin (HGB) (Table 2). Amino acid sequence variation in HCV between the groups relative to an external reference sequence To determine whether there was a difference between the response groups in the frequency of amino acid sequence variations in pre-treatment HCV, we compared the number of amino acid sequence variations in the SVR and the nonSVR patients relative to a genotype 1b consensus sequence. There were no significant differences between the two groups in the total number of amino acids in any of the viral proteins that varied from the consensus sequence (Fig. 1a). Since variations that occur in both groups are unlikely to contribute to any differences in treatment response between the groups, all variations that were common to both patient groups were then eliminated. The numbers of amino acid variations that were unique to each group were compared. There were significantly more unique variations in the NS2 (p=0.004) and NS3 (p=0.036) proteins from SVR than non-SVR subjects (Fig. 1b). There were no differences in the frequency of variable amino acids in the core, E1, E2 and p7 proteins between the groups. As another measurement of amino acid sequence diversity, we compared the proportion of unique variations relative to the total number of variations in the SVR and the non-SVR groups by Fisher’s exact test. The proportion of unique variations in the p7 (p=0.03), NS2 (p=0.003) and NS3 (p=0.046) genes was significantly higher in HCV from the SVR than the non-SVR subjects (p\0.001, Table 3).
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Table 2 Baseline characteritics of all patients in the study Characteristic
SVR (n=35)
Non-SVR (n=26)
P-value
Gender (M/F)a
17/18
17/9
0.191
Age (years)b
37.71±13.58
47.81±12.79
0.005
HCV RNA (log10 copies/ml)b
5.96±0.78
6.06±1.08
0.653
ALT (U/ml)b
80.26±75.62
82.31±64.81
0.912
AST (U/ml)b
64.46±58.96
86.00±69.65
0.197
ALB (g/L)b
45.17±8.20
41.07±5.72
0.033
TBiL (lmol/L)b
14.41±6.51
18.04±10.28
0.097
WBC (G/L)b
5.36±1.62
5.73±2.56
0.489
HGB (g/L)b
139.74±23.95
138.38±24.56
0.829
b
198.74±74.21
149.27±62.12
0.008
30/5 34/1
14/12 10/16
0.006 0.000
PLT (G/L)
EVR (Yes/No)a ETVR (Yes/No)a a
Chi-square test, btwo-sample t-test
For categorical data, the number of patients in each category is shown. For continuous data, the mean ± SD are displayed M, male; F, female; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALB, albumin; WBC, white blood cells; HGB, hemoglobin; PLT, platelets; EVR, early virological response; ETVR, end-of-treatment virological response; SVR, sustained virological response; nonSVR, non-sustained virological response
Fig. 1 Variations in samples with different responses to PEG-IFN/ RBV treatment. a The total number of variations relative to a population consensus reference sequence for each gene within the 5.2-kb amplicon. b The number of unique variations relative to a
population consensus reference sequence for each gene within the 5.2-kb amplicon. PP, 5.2-kb amplicons including polyprotein from core to NS3; E2-c, E2 (Residues 617-711). Statistical significance is shown for genes with pB0.05
The serine protease domain and the helicase domain of the NS3 protein were then analysed independently. There were significantly more unique variations in the protease domain of the SVR group than that in the nonSVR group (p=0.022). However, there was no significant difference in the number of unique variations in the helicase domain between the SVR and the non-SVR groups (Fig. 2). Similar results were obtained when the proportion of unique variations in the SVR and the nonSVR groups was compared using Fisher’s exact test (Table 3).
Amino acid sequence variations between response groups without using an external reference sequence
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The previous measurements of inter-patient genome diversity can be influenced by variations in the external reference sequence, even though we used a populationwide consensus sequence instead of an arbitrary isolate. Therefore, we employed other measures of amino acid sequence diversity to confirm the differences in genetic variation between the SVR and the non-SVR groups. Figure 3 shows significantly higher genetic distances
HCV diversity and SVR
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Table 3 Comparison of the proportion of unique variations in the SVR and non-SVR sequences Proteins
Non-SVR
SVR
Unique Count
Total Exp
Count
Fisher’s exact test
Unique Exp
Count
Total Exp
Count
Exp
Core
10
11.2
55
53.8
18
16.8
79
80.2
0.675
E1
28
29.4
116
114.6
41
39.6
153
154.4
0.785
E2 P7
89 12
97.7 17.9
541 53
532.3 47.1
133 42
124.3 36.1
668 89
676.7 94.9
0.212 0.031
NS2
39
54.4
217
201.6
109
93.6
332
347.4
0.003
NS3
36
45.5
206
196.5
81
71.5
299
308.5
0.046
216
260.6
1188
1143.4
424
379.4
1620
1664.6
