Emekdaş Gürol, Tezcan Seda, Aslan Gönül, Serin Mehmet Sami, Sezgin Orhan, Uçbilek Enver, Ulger Mahmut, Altıntaş Engin, Döğen Aylin
Mersin University Faculty of Medicine, Department of Medical Microbiology, Mersin, Turkey.
Mikrobiyol Bul. 2012 Jul;46(3):432-45.
Currently, ten genotypes (A-J) of hepatitis B virus (HBV) are identified based on the nucleic acid sequence heterogeneity, and these genotypes have been shown to have distinct geographic distribution. Reports of previous studies indicated that the genotype D is the predominant type among hepatitis B patients in different regions of Turkey, however there is no data for HBV genotypes to date from Mersin region. The aim of this study was to investigate the HBV genotypes by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in chronic hepatitis B patients in Mersin province (located in the Mediterranean region of Turkey). A total of 54 serum samples were obtained from the chronic hepatitis B patients (33 male, 21 female; mean age: 40.05 years) followed-up at Gastroenterology Clinic of Mersin University Hospital. Patients had detectable HBV-DNA levels in their serum samples, and they were under antiviral therapy for at least one year. Genotyping of HBV was performed by RFLP analysis with the use of AvaII and MboI restriction enzymes after amplification of pre-S gene region by PCR. Confirmation of selected 18 cases was carried out with direct DNA sequencing. The genotypes were determined by phylogenetic comparison with 43 reference NCBI (National Center for Biotechnology Information) HBV sequences. Genotype determination was not successful in seven cases; since three of them were negative in preS-PCR, three of them yielded non-specific bands, and one of them exhibited a deleted PCR product, at the 300 bp level that was shorter than expected. Four different restriction patterns were determined in PCR-RFLP analysis of the remaining 47 samples. One of these patterns which was AvaII [-]/MboI [306/89/51], was clearly discriminated in 72.3% (34/47) of the samples as genotype D. Genotype discrimination of three patterns could not be done properly and these patterns were AvaII [- ]/MboI [357/306/89/51] (7/47, 14.9%), AvaII [300/146]/MboI [306/89/51] (5/47, 10.7%), and AvaII [- ]/MboI [357/89/---] (1/47, 2.1%). Phylogenetic comparison of HBV sequences demonstrated that all patterns in our cases were clustered in NCBI genotype D sequences. Patterns of AvaII [300/146]/MboI [306/89/51] and AvaII [-]/ MboI [357/89/---] and deleted sample were recognized as pre-S gene variants of HBV isolates. Our data indicated that the predominant HBV type was genotype D as commonly seen in Turkey and other Mediterranean countries. The results of this study also showed that the genotype uniformity and pre-S gene variants within the HBV isolates could be crucial in terms of understanding the molecular epidemiology of HBV circulating in the Mediterranean region of Turkey.
目前，根据核酸序列异质性已鉴定出10种乙型肝炎病毒（HBV）基因型（A - J），这些基因型具有不同的地理分布。既往研究报告表明，基因型D是土耳其不同地区乙肝患者中的主要类型，然而，梅尔辛地区迄今尚无HBV基因型的数据。本研究的目的是通过聚合酶链反应 - 限制性片段长度多态性（PCR - RFLP）方法，对梅尔辛省（位于土耳其地中海地区）的慢性乙型肝炎患者进行HBV基因型研究。共从梅尔辛大学医院胃肠病科门诊随访的慢性乙型肝炎患者中获取了54份血清样本（男性33例，女性21例；平均年龄：40.05岁）。患者血清样本中可检测到HBV - DNA水平，且他们接受抗病毒治疗至少一年。通过PCR扩增前S基因区域后，使用AvaII和MboI限制性内切酶进行RFLP分析对HBV进行基因分型。对选定的18例进行直接DNA测序以进行确认。通过与43条美国国立生物技术信息中心（NCBI）的HBV参考序列进行系统发育比较来确定基因型。7例中基因型测定未成功；其中3例前S - PCR为阴性，3例产生非特异性条带，1例在300 bp水平出现比预期短的缺失PCR产物。对其余47份样本进行PCR - RFLP分析确定了四种不同的限制性图谱。其中一种图谱AvaII [-]/MboI [306/89/51]，在72.3%（34/47）的样本中被明确鉴定为基因型D。三种图谱的基因型鉴别无法准确完成，这些图谱分别是AvaII [- ]/MboI [357/306/89/51]（7/47，14.9%）、AvaII [300/146]/MboI [306/89/51]（5/47，10.7%）和AvaII [- ]/MboI [357/89/---]（1/47，2.1%）。HBV序列的系统发育比较表明，我们病例中的所有图谱均聚类于NCBI基因型D序列中。AvaII [300/146]/MboI [306/89/51]和AvaII [-]/ MboI [357/89/---]图谱以及缺失样本被认为是HBV分离株的前S基因变体。我们的数据表明，HBV的主要类型是基因型D，这在土耳其和其他地中海国家很常见。本研究结果还表明，HBV分离株中的基因型一致性和前S基因变体对于理解在土耳其地中海地区传播的HBV分子流行病学可能至关重要。
