Kumar Deepak, Malik Abdul, Asim Mohammad, Chakravarti Anita, Das Rakha-H, Kar Premashis
Department of Microbiology, Maulana Azad Medical College, New Delhi 110002, India.
World J Gastroenterol. 2008 Feb 7;14(5):701-8. doi: 10.3748/wjg.14.701.
To elucidate the influence of quasispecies on virological response and disease severity in patients with chronic hepatitis C.
Forty seven patients with hepatitis C [32 with chronic active hepatitis (CAH), 9 with cirrhosis, and 6 with hepatocellular carcinoma (HCC)] were screened for the presence of quasispecies by single stranded conformational polymorphism (SSCP) analysis in the hypervariable region (HVR) and non-structural 5B (NS5B) viral genes of hepatitis C virus. The 41 patients excluding those with HCC were on therapy and followed up for a year with the determination of virological response and disease severity. Virus isolated from twenty three randomly selected patients (11 non-responders and 12 showing a sustained virological response) was sequenced for the assessment of mutations.
The occurrence of quasispecies was proportionately higher in patients with HCC and cirrhosis than in those with CAH, revealing a significant correlation between the molecular evolution of quasispecies and the severity of disease in patients with hepatitis C. The occurrence of complex quasispecies has a significant association (P < 0.05) with the non-responders, and leads to persistence of infection. Significant differences (P < 0.05) in viral load (log10 IU/mL) were observed among patients infected with complex quasispecies (CQS), those infected with simple quasispecies (SQS) and those with no quasispecies (NQS), after 12 wk (CQS-5.2 +/- 2.3, SQS-3.2 +/- 1.9, NQS-2.8 +/- 2.4) and 24 wk (CQS-3.9 +/- 2.2, SQS-3.0 +/- 2.2, NQS-2.1 +/- 2.3) in the HVR region. However, a statistically significant difference (P < 0.05) was observed between the viral loads of patients infected with CQS and those infected with NQS in NS5B viral gene after 24 wk (CQS-3.9 +/- 2.2, SQS-3.0 +/- 2.2, and NQS-2.1 +/- 2.3) and 48 wk (CQS-3.1 +/- 2.7, SQS-2.3 +/- 2.4, NQS-2.0 +/- 2.3) of therapy. Disease severity was significantly associated with viral load during therapy. The strains isolated from non-responders showed close pairing on phylogeny based on the NS5B gene, but dissimilar HVR regions. This revealed the possibility of the selection of resistant strains during the evolution of quasispecies in NS5B.
Viral quasispecies may be an important predictor of virological responses to combination therapy in patients with chronic hepatitis C. Complex quasispecies and resistant strains may lead to high viral loads during therapy, with a concerted effect on disease severity.
阐明准种对慢性丙型肝炎患者病毒学应答及疾病严重程度的影响。
采用单链构象多态性（SSCP）分析，对47例丙型肝炎患者[32例慢性活动性肝炎（CAH）、9例肝硬化、6例肝细胞癌（HCC）]丙型肝炎病毒高变区（HVR）和非结构5B（NS5B）病毒基因中的准种进行筛查。排除HCC患者后的41例患者接受治疗，并随访1年，测定病毒学应答及疾病严重程度。对随机选取的23例患者（11例无应答者和12例获得持续病毒学应答者）分离出的病毒进行测序，以评估突变情况。
HCC和肝硬化患者中准种的出现比例高于CAH患者，表明丙型肝炎患者准种的分子进化与疾病严重程度之间存在显著相关性。复杂准种的出现与无应答者显著相关（P<0.05），并导致感染持续存在。治疗12周（HVR区：复杂准种组-CQS-5.2±2.3，简单准种组-SQS-3.2±1.9，无准种组-NQS-2.8±2.4）和24周（HVR区：CQS-3.9±2.2，SQS-3.0±2.2，NQS-2.1±2.3）后，感染复杂准种（CQS）、简单准种（SQS）和无准种（NQS）的患者之间病毒载量（log10 IU/mL）存在显著差异（P<0.05）。然而，治疗24周（NS5B病毒基因：CQS-3.9±2.2，SQS-3.0±2.2，NQS-2.1±2.3）和48周（NS5B病毒基因：CQS-3.1±2.7，SQS-2.3±2.4，NQS-2.0±2.3）后，感染CQS的患者与感染NQS的患者病毒载量之间存在统计学显著差异（P<0.05）。疾病严重程度与治疗期间的病毒载量显著相关。基于NS5B基因的系统发育分析显示，无应答者分离出的毒株在系统发育上紧密配对，但HVR区不同。这揭示了在NS5B准种进化过程中选择耐药毒株的可能性。
病毒准种可能是慢性丙型肝炎患者联合治疗病毒学应答的重要预测指标。复杂准种和耐药毒株可能导致治疗期间病毒载量升高，对疾病严重程度产生协同影响。
