Zumbika Edward, Ruan Bing, Xu Chen-Huai, Ni Qin, Hou Wei, Chen Zhi, Liu Ke-Zhou
Institute of Infectious Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, People's Republic of China.
Hepatobiliary Pancreat Dis Int. 2005 Nov;4(4):535-43.
There are 8 well-documented genotypes of hepatitis B virus (HBV) at this time point. Genotyping can be accomplished based on a partial sequence of hepatitis B virus (HBV) genome such as the pre-S or S gene. Several methods have been developed and used for HBV genotyping including direct sequencing, restriction fragment length polymorphism, line probe assay and enzyme-linked immunoassay. Recently, a novel, rapid and cost-effective genotyping method based on PCR amplification assay using type-specific primers that can identify all six major genotypes has been developed. This study was undertaken to characterise HBV genotypes and investigate the association between the prevalence of different genotypes and the severity of HBV-induced liver diseases.
Serum samples from carriers of HBV and patients with HBV-related liver diseases from Zhejiang Province were screened for viral serological markers using commercially available radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) kits. Serum HBV DNA load was determined by real-time detection PCR. A type-specific primer based the nested-PCR method was employed in the HBV genotyping. The genotype results obtained were confirmed by direct sequencing of nested PCR amplicons of the pre-S region. Ten samples of each genotype (B and C) were sequenced.
The survey on a cohort of 125 HBV carriers in and around Hangzhou City, Zhejiang Province showed the existence of HBV genotypes A (0.8%), B (48%), C (40.8%), D (0.8%), mixed B and C (9.6%) and an absence of E and F genotypes. Distribution of HBV genotypes in patients with liver diseases revealed a statistically insignificant higher prevalence of genotype B in mild chronic hepatitis (CH). Among the three genotypes B, C and mixed B/C infections 11 (73.3%), 3 (20%) and 1 (6.7%), (P<0.05), respectively in subjects with moderate CH, genotype B was significantly predominant. The infection patterns for genotypes B, C and B/C mixed in (i) liver cirrhosis (LC) 4 (23.5%), 10 (58.8%) and 3 (17.7%) and (ii) hepatocellular carcinoma (HCC) 2 (28.6%), 5 (71.4%) and 0 (0.0%) respectively revealed a marked association of C genotype with liver disease; however, the association was statistically insignificant (P>0.05). Differences in positive rate of HBeAg for the three genotypes B, 16 (30.8%), C, 27 (51.9%), and mixed B/C, 9 (17.3%) were significant (P<0.05), with genotype C showing predominance.
These findings show an interesting distribution of HBV A-D genotypes in Zhejiang Province. Furthermore, our results indicate a novel and markedly high prevalence of mixed B/C genotype infections in subjects with severe CH and LC, and a possible association of mixed B/C infections with the severity of liver diseases in this region of Mainland China.
在这一时间点，有8种记录完备的乙型肝炎病毒（HBV）基因型。基因分型可基于乙型肝炎病毒（HBV）基因组的部分序列，如前S或S基因来完成。已开发并使用了多种方法进行HBV基因分型，包括直接测序、限制性片段长度多态性分析、线性探针分析和酶联免疫吸附测定。最近，已开发出一种基于PCR扩增分析的新型、快速且经济高效的基因分型方法，该方法使用型特异性引物，可识别所有六种主要基因型。本研究旨在对HBV基因型进行特征分析，并调查不同基因型的流行率与HBV所致肝脏疾病严重程度之间的关联。
使用市售放射免疫分析（RIA）和酶联免疫吸附测定（ELISA）试剂盒，对来自浙江省的HBV携带者和HBV相关肝病患者的血清样本进行病毒血清学标志物筛查。通过实时荧光定量PCR测定血清HBV DNA载量。采用基于巢式PCR方法的型特异性引物进行HBV基因分型。通过对前S区域巢式PCR扩增产物进行直接测序，确认所获得的基因型结果。对每种基因型（B和C）的10个样本进行测序。
对浙江省杭州市及周边地区125名HBV携带者的队列研究表明，存在HBV基因型A（0.8%）、B（48%）、C（40.8%）、D（0.8%）、B和C混合型（9.6%），不存在E和F基因型。肝病患者中HBV基因型的分布显示，轻度慢性肝炎（CH）中基因型B的流行率略高，但无统计学意义。在中度CH患者中，B、C和B/C混合型感染分别为11例（73.3%）、3例（20%）和1例（6.7%），（P<0.05），基因型B显著占优势。B、C和B/C混合型在（i）肝硬化（LC）中的感染模式分别为4例（23.5%）、10例（58.8%）和3例（17.7%），（ii）肝细胞癌（HCC）中的感染模式分别为2例（28.6%）、5例（71.4%）和0例（0.0%），显示C基因型与肝脏疾病有明显关联；然而，该关联无统计学意义（P>0.05）。三种基因型B（16例，30.8%）、C（27例，51.9%）和B/C混合型（9例，17.3%）的HBeAg阳性率差异有统计学意义（P<0.05），基因型C占优势。
这些发现显示了浙江省HBV A - D基因型的有趣分布。此外，我们的结果表明，在重度CH和LC患者中，B/C混合型基因型感染的发生率很高且为新发现，并且在中国大陆该地区，B/C混合型感染可能与肝脏疾病的严重程度有关。
