Hepatitis B genotypes and precore/basal core promoter mutants in HBeAg-negative chronic hepatitis B

Background. Mutations in the precore stop codon (G1896A) and the basal core promoter (A1762T and G1764A) are frequently found in hepatitis B envelope antigen (HBeAg)-negative chronic hepatitis B. However, the clinical significance of these mutations remains controversial. We therefore investigated the influence of hepatitis B virus (HBV) genotypes, as well as precore/basal core promoter mutants, on the clinical and virological features of patients with HBeAg-negative chronic hepatitis B. Methods. Serum samples from 37 patients with HBeAg-negative chronic hepatitis B were collected for serological and molecular assays. The precore and basal core promoter regions were amplified by polymerase chain reaction and the amplicons were directly sequenced and analyzed. HBV geno-type was determined by polymerase chain reaction-restriction fragment length polymorphism. Results. Most of the patients had detectable serum HBV DNA, and genotypes B and C were the predominant strains. The overall prevalence of the precore stop codon mutant and basal core promoter mutant was 67% and 60%, respectively. The baseline clinical and virological features of patients with genotype B and genotype C infection were comparable. However, in the patients with precore/basal core promoter dual mutations there was a significantly lower proportion of individuals with a high detectable serum HBV DNA level (>100 pg/ml) than in the patients with either the precore stop codon mutation alone or the basal core promoter mutation alone (P = 0.04 by the logistic regression test for the trend). Conclusions. Our data suggest a high prevalence of precore stop codon and basal core promoter mutation in Taiwanese patients with HBeAg-negative chronic hepatitis B, and the influence of the basal core promoter mutation on HBV replication is modulated by the emergence of the precore stop codon mutation. Received: May 14, 2001 / Accepted: September 14, 2001     

乙型重型肝炎患者血清HBV前C/BCP变异检测及临床意义

目的探索乙型重型肝炎患者HBV前C/BCP区变异与临床病情关系。方法用荧光定量PCR法检测血清HBVDNA载量;PCR扩增前C/BCP区基因后进行序列测定。结果30例乙型重型肝炎患者中BCP变异有18例,HB-VDNA平均水平为2.71×107copies/mL,未发生BCP变异者有12例,HBVDNA平均水平为5.35×106copies/mL,两者间有显著差异。测序结果显示HBV前C区nt1896G→A终止变异和BCP变异(nt1762A→T和1764G→A)在乙型重型肝炎患者中发生率分别为43.3%(13/30)和60%(18/30),在慢性乙型肝炎患者则分别为40%(12/30)和30%(9/30),两组BCP变异(nt1762A→T和1764G→A)相比有显著差异(P=0.02)。结论BCP变异可影响病毒复制,BCP变异可能与肝病的严重程度存在着相关性。     

Hepatitis B virus precore mutants in HBeAg carriers with chronic hepatitis treated with interferon

Summary. Precore mutants of hepatitis B virus (HBV) were looked for in 18 hepatitis B e antigen (HBeAg) carriers who were treated with recombinant interferon-a (rIFN) and the results were compared with those obtained in 12 untreated carriers who underwent spontaneous HBeAb seroconversion. Molecular analysis of the HBV precore region was carried out by polymerase chain reaction (PCR) amplification and direct sequencing. Precore mutants with a stop codon at codon 28 were detectable at baseline in 19/30 carriers. However, wild-type strains predominated in the baseline sera of both treated ( n = 16) and untreated ( n = 10) patients. Sera from the remaining four patients contained predominantly or exclusively mutant virions. Following IFN treatment, there was a shift from the wild-type pattern to the mutant pattern in all patients, with the precore pattern prevailing in long-term responders (six out of nine) compared with the non-responders (none of nine). The wild-type pattern predominated among the non-responders (eight vs three), suggesting that the long-term response to IFN was associated with take-over of precore mutants. There were no relationships between any pre-treatment precore molecular pattern and disease severity or outcome of treatment. Precore mutants also took over in 10 of the 12 untreated patients (83%), who underwent spontaneous HBeAb seroconversion. Thus, a shift from wild-type to precore mutant pattern occurs in most Italian patients undergoing IFN-induced or spontaneous HBeAb seroconversion.     

慢性HBV感染者抗病毒治疗前HBV基本核心启动子及前C区突变的检测及意义

目的研究慢性乙型肝炎病毒（HBV）感染者抗病毒治疗前HBV基本核心启动子（BCP）突变和前C区（PreC）突变与HBeAg、HBV DNA水平和慢性肝病进展的关系。方法收集283例慢性HBV感染者抗病毒治疗前的血清标本,其中慢性乙型肝炎（CHB）185例,肝硬化（LC）98例。采用PCR后直接测序法检测HBV BCP和PreC区突变,同时确定基因型。结果在HBeAg阴性和HBeAg阳性CHB患者中,前C区A1896变异率分别为44.6%（37/83）和21.6%（22/102）（χ2=11.154,P=0.001）,LC患者分别为43.4%（23/53）和17.0%（8/47）（χ2=8.101,P=0.004）。在HBeAg阳性患者中,BCP T1762/A1764双突变率LC组和CHB组分别为89.4%（42/47）和70.6%（72/102）（χ2=6.310,P=0.012）。在单变量分析中,只有年龄（≥45岁）（χ2=27.861,P〈0.001）、BCP T1762/A1764双突变（χ2=8.675,P=0.003）和HBV DNA（≥105拷贝/ml）（χ2=20.499,P〈0.001）与LC进展有关。多因素Logistic回归分析（匹配年龄和性别）发现,BCP T1762/A1764双突变（OR=3.260,95%CI：1.401～7.586;wald=7.517,P=0.006）和HBV DNA（≥105拷贝/ml）（OR=4.640,95%CI：2.331～9.237;wald=19.089,P〈0.001）是LC进展的危险因素。结论前C区A1896突变与HBeAg的消失有关;年龄（≥45岁）、BCP T1762/A1764双突变和HBV DNA高载量（≥105拷贝/ml）与肝硬化进展有关。     

Variations of hepatitis B virus precore/core gene sequence in acute and fulminant hepatitis B

Variations of the hepatitis B virus (HBV) precore/core sequence has been shown to play a role in the development of active liver disease in chronic hepatitis B. Whether this is also an important viral factor in the pathogenesis of acute and fulminant hepatitis B is unknown. To determine the precore/core gene sequence in patients with acute and fulminant hepatitis B, 11 patients with fulminant hepatitis B and seven patients with acute hepatitis B were studied. The sequences of precore/core gene were determined by direct sequencing of the polymerase chain reaction amplicons generated from the HBV isolated from patients' serum. For the 11 patients with fulminant hepatitis B, the precore/core regions were successfully amplified in 10 patients. Eight patients exhibited precore stop codon mutations. In addition, nine of the 10 fulminant hepatitis B patients had frequent nucleotide substitutions with corresponding changes in the predicted amino acid sequences in the mid-core and the 5 terminus region of the core gene. In contrast, precore stop codon mutants were not detected, and variations of the HBV core gene were minimal in patients with acute hepatitis B. The association of HBV precore mutants and HBV core gene variations with fulminant hepatitis B and not acute hepatitis B suggested that these variations may be important in modulating the clinical course of HBV infection.     

重型乙型肝炎病毒C基因启动子变异

目的了解我国重型肝炎Ｃ基因调节序列变异特点。方法克隆及聚合酶链反应（ＰＣＲ）产物直接测序方法对,检测７例重型乙型肝炎患者乙型肝炎病毒（ＨＢＶ）Ｃ基因调节序列变异。结果４例存在Ｔ１７６２Ａ１７６４变异;但在Ｔ１７６２Ａ１７６４以外位点也存有诸多变异,其中１例在ｎｔ１７７６－１７７７之间有１１ｂｐ的插入变异。结论重型肝炎ＨＢＶＣ基因启动子区存在较多的变异位点,推测这些变异对Ｃ启动子的调节活性影响可能是综合性的。     

Association of hepatitis B virus subgenotypes and basal core promoter/precore region variants with the clinical features of patients with acute hepatitis

BACKGROUND: In endemic areas, including Japan, basal core promoter (BCP) and precore (PC) variants of hepatitis B virus (HBV) have been reported to be associated with the clinical outcome of acute hepatitis B patients. However, the associations of BCP/PC variants with clinical outcomes have not been observed in nonendemic areas. HBV subgenotypes, which show geographic variations in prevalence, may underlie this discrepancy in clinical outcomes. Little is known about the differences in the clinical and virological features of HBV subgenotypes and BCP/PC variants. The aim of this study was to investigate the distributions of subgenotypes and BCP/PC variants to identify clinical differences in acute hepatitis B patients. METHODS: One hundred thirty-nine patients with acute hepatitis were enrolled. Nested polymerase chain reaction was used to amplify the pre-S region of HBV for genotyping and the BCP/PC regions for variant screening. RESULTS: HBV subgenotypes A1 (n = 3), A2 (n = 28), B1 (n = 3), B2 (n = 9), C1 (n = 5), C2 (n = 84), C variant (n = 1), D2 (n = 3), and H (n = 3) were detected. BCP/PC variants were not associated with progression to chronic hepatitis. Patients infected with subgenotype C2 who progressed to fulminant hepatic failure frequently carried variants at nucleotides non-T1753 and non-T1754 and T1762, A1764, and A1896. CONCLUSIONS: BCP/PC variants would be associated with progression to fulminant hepatitis in subgenotype C2. Knowledge of HBV subgenotypes and BCP/PC variants is useful for developing strategies to treat acute hepatitis B patients.     

An outbreak of hepatitis B with high mortality in India: association with precore, basal core promoter mutants and improperly sterilized syringes

In 2009, an outbreak of hepatitis B with high mortality was observed in Sabarkantha district, Gujarat state, India with 456 cases and 89 deaths. Hospitalized patients with self-limiting disease (152, AVH)) and fulminant hepatic failure (39, FHF including 27 fatal and 12 survivals) were investigated. These were screened for diagnostic markers for hepatitis viruses, hepatitis B virus (HBV) genotyping and mutant analysis. Complete HBV genomes from 22 FHF and 17 AVH cases were sequenced. Serosurveys were carried out in the most and least affected blocks for the prevalence of HBV and identification of mutants. History of injection from a physician was associated with FHF and AVH cases. Co-infection with other hepatitis viruses or higher HBV DNA load was not responsible for mortality. Four blocks contributed to 85.7% (391/456) of the cases and 95.5% (85/89) mortality while two adjacent blocks had negligible mortality. Sequence analysis showed the presence of pre-core and basal core promoter mutants and 4 amino acid substitutions exclusively among FHF cases. None of the self-limiting patients exhibited these dual mutations. Genotype D was predominant, D1 being present in all FHF cases while D2 was most prevalent in AVH cases. Probably due to violation of accepted infection control procedures by the qualified medical practitioners, HBV prevalence was higher in the affected blocks before the outbreak. Gross and continued use of HBV contaminated (mutant and wild viruses) injection devices led to an explosive outbreak with high mortality with a striking association with pre-C/BCP mutants and D1 genotype.     

慢性重型乙型肝炎患者HBVDNA前C／BCP区突变基因分析

目的分析慢性重型乙型肝炎（慢重乙肝）患者HBVDNA前C区和基本核心启动子（前C／BCP）区突变特点与意义。方法收集87例慢重乙肝和196例慢性乙型肝炎（慢乙肝）患者血清，提取HBVDNA，用巢式PCR扩增HBVDNA前C／BCP区基因，PCR产物进行DNA测序，用NBI软件比对结果，重点分析G1896、G1862、G1899、A1762、G1764、T17536个位点突变。结果慢重乙肝组和慢乙肝组6个位点突变全阴率分别为3．4％和28．1％（P〈0．01）；慢重乙肝组在其中5个位点上的突变检出率显著高于慢乙肝组。此外，慢重乙肝组和慢乙肝组≥三联突变检出率分别为56．3％和35．2％（P〈0．01），≥四联突变检出率分别为25.3％和8．7％（P〈0．01），插入／缺失突变检出率分别为10．3％和1．0％（P〈0．01）。结论HBVDNA前c／BcP区基因突变发生频率的增加与慢乙肝发生重症化相关，结合临床资料分析突变的意义将有助于认识慢乙肝重症化的发生机制。     

新疆维吾尔族人群中HBV基因亚型与核心启动子区、前C区及核心区基因变异的关系

C基因基本核心启动子区(BCP)的T1762/A 1764双突变和前C区的A1896终止变异影响HBeAg的状态,可能与HBV的持续感染和HCC有关[1-2].我们分析了新疆维吾尔族慢性HBV感染者HBV基因亚型与C区和前C区变异的关系,旨在了解维吾尔族慢性HBV感染者的HBV共性及特性.     

Hepatitis B virus genotypes and G1896A precore mutation in 486 Spanish patients with acute and chronic HBV infection

This study aims to determine the prevalence of hepatitis B virus (HBV) genotypes (A-F) and their association with the G1896A precore mutation in 486 patients positive for HBV surface antigen. Genotypes were determined by RFLP and precore mutation by real-time PCR. Genotypes D (48.1%) and A (39.5%) were the most common, followed by F (4.1%) and B, C and E (<1%). The A to D ratio (A:D) was 1.4 in HBeAg+ chronic hepatitis B (CHB), 0.6 in HBeAg- CHB and 1.4 in HBeAg- inactive carriers. Distribution of these genotypes was different between HBeAg+ CHB and HBeAg- CHB (P = 0.02), and between HBeAg- CHB and HBeAg- inactive carriers (P = 0.009). Genotype A was the most prevalent in HBeAg+ CHB with elevated alanine aminotransferase (ALT) (68.6%) and genotype D in HBeAg+ CHB with fluctuating ALT (60.7%). There was a difference in genotype prevalence between chronic and acute infection (P = 0.03). The precore mutant correlated with high levels of HBV-DNA in genotype d HBeAg- CHB. Genotype D is not as highly prevalent in Spanish patients as would be expected in a Mediterranean area. The unequal prevalence of genotypes between acute and chronic infection suggests that genotype A is associated with a higher tendency to cause chronic infection.     

乙型肝炎病毒前C区和BCP区突变及基因型对HBeAg表达的影响

研究乙型肝炎病毒前C区和BCP区突变及基因型对HBeAg表达的影响。分别用基因芯片和基因测序的方法检验HBV DNA阳性的乙型肝炎病毒前C区和BCP区突变及基因型,应用时间分辨免疫荧光法检测HBeAg含量,按照有无HBV前C区1896、BCP区1762、1764双突变、基因型等指标进行分组分析。无前C区1896和BCP区1762/1764双突变组、单纯1762、1764双突变和1896突变组以及1896、1762、1764联合突变组之间相互比较,HBeAg含量相差显著,F=6.47,P〈0.01;B、C基因型间HBeAg含量相比,相差无显著性,t=0.1394,P〉0.05。乙型肝炎病毒前C区和BCP区突变能显著影响HBeAg量的表达,从而导致乙型肝炎病毒致病能力的变化。     

Hepatitis B virus replication and clinical outcome in carriers of HBsAg. Perspectives of treatment with DNA inhibitors

ABSTRACT— HBV-DNA was measured by the spot hybridization technique in serial serum samples obtained from 47 HBsAg carriers followed up for a mean of 4 years. The levels of HBV-DNA were compared to the conventional HBV serology and immunopathology to determine the relation of active HBV replication to the outcome of hepatitis and the suitability of Italian HBsAg carriers for treatment with DNA inhibitors. HBV-DNA was found in 26 carriers (53%) and persisted with comparable serum levels in 24 of them throughout the follow up. The occurrence rate of an unfavorable outcome as determined by histological evidence of cirrhosis was 6% versus 44% (p<0.01) in carriers with active viral infection (> 1 ng/ml of HBV-DNA) and in patients with absent or low levels of viral DNA (< 1 pg/ml), respectively. Progression of the liver disease could not be predicted on the basis of active HBV replication and was presumably related to factors other than synthesis of HBV. In many patients with inactive viral infection a primary pathogenic factor was the HBV-associated δ, an agent with a putative RNA genome against which DNA inhibitors have no rationale and possibly no effects. The majority of Italian carriers do not appear suitable for treatment with DNA inhibitors and they should be considered for a different therapy.     

Clinical significance of precore and core promoter mutations in genotype D hepatitis B-related chronic liver disease

SUMMARY: The impact of mutations in the precore and basal core promoter (BCP) regions of the hepatitis B virus on the course of chronic liver disease is not well established. We sought to examine the relationship of these characteristics to the clinical expression of liver disease in patients infected with genotype D chronic hepatitis B (CHB). BCP and precore mutations in 110 patients with genotype D1 CHB were determined and correlated with clinical phenotype. Of 110 patients, 95 (86.5%) were HBeAg-negative. Compared with HBeAg-positive subjects, HBeAg-negative patients were over a decade older and had lower viral loads (3.70 +/- 0.98 vs 5.77 +/- 0.69 log copies/ml, P < 0.001). The double mutation A1762T-G1764A was more prevalent in patients with advanced liver disease (AdLD) and was associated with higher alanine aminotransferase and viral load. After adjusting for age, there was a more than fourfold increase in the risk of AdLD with this mutation (OR = 4.4; 95% CI: 1.13-16.92, P < 0.03). Conversely, the G1757A substitution was associated with protection, being 90% less frequent among patients with AdLD (P = 0.001). The results indicate that in genotype D CHB, the presence of the A1762T-G1764A mutation was associated with more aggressive liver disease while the G1757A substitution was associated with protection from advanced disease.     

Correlation of hepatitis B virus (HBV) genotypes and mutations in basal core promoter/precore with clinical features of chronic HBV infection.

BACKGROUND/AIMS: To investigate the correlation of hepatitis B virus (HBV) genotypes and basal core promoter (BCP) and precore (PC) mutations in patients with chronic hepatitis B. METHODS: HBV genotyping, nucleotide mutation, serum HBV DNA level and serological markers were analyzed in 121 patients with chronic HBV infection using INNO-LiPA HBV genotyping, polymerase chain reaction (PCR) product-based sequencing, fluorescence quantitative PCR and enzyme-linked immunosorbent assays respectively. RESULTS: Forty (33.0%), 77 (63.6%), two (1.7%) and two (1.7%) patients had genotypes B, C, B/C and D infections respectively. Significant differences were found in serum HBV DNA levels (log10 copies/ml: 6.18 vs. 5.61, P=0.042) and mutations at nucleotide (nt) 1762/1764 (71.4% vs. 42.5%, P=0.002) between genotypes C- and B-infected patients. There were significant differences in the mean age, serum biochemical parameter levels and mutation rates in BCP/PC among hepatitis e antigen (HBeAg)-positive and -negative chronic hepatitis B (CHB) and liver cirrhosis (LC) groups. CONCLUSION: Genotypes C and B are predominant in China, and the frequent nt 1762/1764 mutation, which occurs commonly in HBeAg-negative CHB, especially in genotype C patients, may be associated with the progress of chronic HBV infection.     

Detection of HBV core promoter and precore mutations helps distinguish flares of chronic hepatitis from acute hepatitis B

Background and Aim: Acute exacerbation of chronic hepatitis B has to be distinguished from acute hepatitis, because treatment strategies differ between them. Methods: Mutations in the core promoter and precore region of hepatitis B virus (HBV) were determined in 36 patients with acute exacerbation of chronic hepatitis B, in whom alanine aminotransferase (ALT) increased above 500 IU/L, as well as the 36 patients with acute hepatitis. Results: Mutations in the core promoter (A1762T/G1764A) and precore region (G1896A) were more frequent in patients with acute exacerbation of chronic hepatitis than acute hepatitis (81% vs 19%; P < 0.0001 and 58% vs 6%; P < 0.0001, respectively). Of the 19 patients with mutations in both the core promoter and precore region, 17 (89%) had acute exacerbation of chronic hepatitis. In contrast, among the 32 patients with the wild‐type for both the core promoter and precore region, 29 (89%) developed acute hepatitis. By multivariate analysis, the double mutation in the core promoter was predictive of acute exacerbation in chronic hepatitis with the highest odds ratio at 26.4. Conclusions: In patients with hepatitis B having ALT levels >500 IU/L, mutations in the core promoter and precore region are useful in distinguishing acute exacerbation of chronic from acute HBV infection. Detection of these mutations would be useful for commencing prompt antiviral treatments on patients with acute exacerbation of chronic hepatitis for a better prognosis.     

Frequent integration of precore/core mutants of hepatitis B virus in human hepatocellular carcinoma tissues

The development of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) frequently follows persistent HBV infection and may arise in individuals who are hepatitis B e antigen (HBeAg) negative, indicating the possible presence of precore/core mutants. It is unclear whether precore/core mutants are associated with tumour development or are selected for after chromosomal integration of the wild-type viral DNA. We studied the status and sequence variation of the precore/core region of HBV in 56 patients with HBV-associated HCC and in various corresponding non-tumour tissues by Southern blot analysis, polymerase chain reaction and direct sequencing. Southern blot showed that integrated HBV DNA existed in 43 of 56 HCC tissues. Sequence analysis revealed mutations in 65% of the HCC (26/40) and 45% (14/31) of the corresponding non-tumour tissues. The mutation at nucleotide (nt) 1896, known to prevent HBeAg synthesis, was detected in 40% (16/40) of the tumours and in 35.4% (11/31) of the non-tumour tissues. Other mutations were found at nt 1899 (eight of 40 in HCC; three of 31 in non-tumour tissues), nt 1898 (seven of 40 in HCC; two of 31 in non-tumour tissues), nt 1912 (seven of 40 in HCC; none of 31 in non-tumour tissues) and nt 1886 (three of 40 in HCC; none of 31 in non-tumour tissues). To determine whether this finding merely reflected the prevalence of such mutants in this geographical region, HBV DNA from the sera of patients (also in this region) with acute and chronic hepatitis were sequenced. The nt 1896 mutant was found in 5.6% (one of 18) of patients with acute hepatitis B and in 22.8% (nine of 35) of patients with chronic hepatitis B. However, the nt 1898 mutation was not found in any of these sera. The precore/core mutant was observed with increasing frequency from acute hepatitis to chronic hepatitis, non-tumour and HCC, and this difference in frequency was significant between HCC and acute hepatitis B groups (P < 0.01), suggesting that the precore/core mutant or hepatocytes harbouring this mutant may be under immune selection and that such mutations may facilitate integration and subsequent tumour development.     

World-wide epidemiology of HBeAg-negative chronic hepatitis B and associated precore and core promoter variants

Hepatitis B is a serious disease that is endemic in many parts of the world. A significant proportion of patients with chronic hepatitis B (CHB) are infected with a variant form of the hepatitis B virus (HBV) which decreases or abolishes the production of hepatitis B e-antigen (HBeAg). The purpose of this literature review is to describe the epidemiology of HBeAg-negative CHB (e-CHB) worldwide. A literature search was conducted to identify studies pertaining to e-CHB and underlying variants (precore and core promoter). Fifty studies were included in our analysis. The median prevalence of e-CHB among patients with chronic HBV infection was 33% in the Mediterranean, 15% in Asia Pacific, and 14% in the USA and Northern Europe. The pre core stop codon variant was detected in a median of 60% (range 0–100%) of HBeAg-negative patients overall, 92% in the Mediterranean, 50% in Asia Pacific and 24% in the USA and Northern Europe. There were very few data on the prevalence of core promoter variants outside Asia where the median prevalence among HBeAg-negative patients was 77%. This literature review revealed that e-CHB is more common than previously suspected and that it is present worldwide with marked variations in the prevalence of associated HBV variants across different geographical regions. Additional research using population based samples of adequate size based on a consensus definition of e-CHB and using standardized HBV DNA assays is needed to better estimate the true prevalence of e-CHB and its associated HBV variants.     

Association of core promoter/precore mutations and viral load in e antigen-negative chronic hepatitis B patients

Apart from core promoter A1762T/G1764A and precore G1896A mutations, other hepatitis B virus (HBV) mutants are detected in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB). The aim of this study was to determine the effects of those mutants on clinical manifestation and viral loads of genotypes B and C HBV. Seventy-nine HBeAg-negative CHB patients with hepatitis flare were enrolled in this study and their HBV precore/core region were sequenced. Serial biochemical profiles and viral loads were assessed and compared. Fifty-three patients (67%) were infected by genotype B HBV and 26 (33%) were infected by genotype C HBV. The clinical manifestation and HBV viral loads were comparable between the two groups. However, genotype B was significantly associated with precore G1896A mutation (92.5%), and more mutations within nucleotide 1809-1817 were detected in patients infected by genotype B as compared with those infected by genotype C (18.9%vs 3.8%). Most of the cases had mutations at the -2, -3 or -5 position from the precore AUG initiation codon. Triple core promoter mutations T1753C/A1762T/G1764A [corrected] appeared to be linked to genotype C rather than genotype B HBV (19.2%vs 1.9%; P = 0.013). In multivariate analysis, the presence of either triple core promoter 1753/1762/1764 mutation or nucleotide 1809-1817 mutation was the only factor associated with lower HBV viral load (<70 Meq/mL) (odds ratio = 9.01; 95% CI 1.11-71.43; P = 0.04). In conclusion, minor HBV variants with mutations in the core promoter and precore region were detectable in genotypes B and C. Such HBV variants are genotype specific and related to viraemia levels.