Characteristics of hepatitis B virus in Ghana: full length genome sequences indicate the endemicity of genotype E in West Africa

Hepatitis B virus (HBV) genotypes from A to H have distinct geographical distributions and have been shown to affect the clinical features as well as the course of the HBV infection. HBV genotype E has been found only in Africa. However, the complete genomes of this genotype, which were isolated mainly from West Africa, were available only for a few samples. In this study, five full-length genomes and seven other small S genes of HBV strains from Ghanaian blood donors were sequenced and investigated. Following phylogenetic analysis, all of the Ghanaian HBV strains were clustered closely in genotype E. All of the 12 small S genes showed the same characteristic of subtype ayw4. The complete genomes of the five Ghanaian strains showed marked similarity with each other and with the reported genotype E strains (96.7%-99.1%). Genotype E strains showed low intra-genotypic diversity (1.8%) and carried the conserved signature pattern in pre-S1 as well as in the full genome sequence. Of note, the finding of the G145R escape mutant in an unvaccinated Ghanaian blood donor might raise concern as to the ongoing nation-wide hepatitis B vaccination program in Ghana.     

Naturally occurring hepatitis B virus X deletions and insertions among Korean chronic patients

Deletions and insertions in the hepatitis B virus (HBV) X region have been associated with severe forms of liver disease, including hepatocellular carcinoma (HCC). However, the molecular epidemiologic features of this virus have been described rarely. Deletions and insertions in the X region were determined by direct sequencing in a Korean cohort of 267 patients with different clinical statuses. Deletions and insertions were observed in two sets of six patients each (2.2%, 6/267). The prevalence of deletions or insertions was significantly higher in patients with severe liver disease, HCC, or cirrhosis of the liver (7.2%, 10/132) compared to patients who were carriers or had chronic hepatitis (1.5%, 2/135) (P = 0.017). All deletions in six strains were concentrated at the C terminal end of HBx, encompassing the 113th to 154th codons. A total of four novel types of insertions (PKLL, GM, FFN, and tt) were observed in six patients. Of particular interest, all six strains with insertions were accompanied by double mutations in the basal core promoter (BCP). In conclusion, these results suggest that deletions or insertions in the X region may contribute to disease progression in Korean patients with genotype C infection.     

Molecular analysis of hepatitis B virus genomes isolated from black African patients with fulminant hepatitis B

To investigate further the possible role of mutant hepatitis B viruses in the pathogenesis of fulminant hepatitis B, the genomic sequence of hepatitis B virus isolates from 9 South African blacks with this disease, including 5 entire genomes, was analysed. Seven of the isolates were genotype A. The mutation most often reported in patients with fulminant hepatitis B, the G1896A precore stop-codon substitution, was, as expected, not present in the genotype A isolates with the exception of one in which it was accompanied by a compensatory C1858T substitution. G1896A was, however, present in the one genotype D isolate. No other precore-defective mutants were detected. The other mutation commonly found in patients with fulminant hepatitis B, the paired A1762T, G1764A substitution in the basic core promoter, was present in only one patient and G1764A in one other. The pre-surface initiation-codon mutation documented in a number of patients with fulminant hepatitis B was not found in our isolates. An 18-amino acid deletion present in the pre-surface region of one isolate has not previously been described in fulminant hepatitis B. Variations within the surface region were mainly genotype specific and not previously described. A relatively large number of mutations were present in the middle region of the core gene in those isolates without G1896A or A1762T, G1764A mutations, although the pattern was not consistent with those in published studies. Thus, as in other published series in which the entire genome of hepatitis B virus responsible for fulminant hepatitis was sequenced, we detected many mutations in different genes, but none was common to all the reported isolates.     

Comparison of full length sequences of hepatitis B virus isolates in hepatocellular carcinoma patients and asymptomatic carriers of Korea

Relatively few genomic sequences of Korean hepatitis B virus (HBV) isolates are available. Moreover, no comparative study has been made between the full-length genomes of Korean HBV isolates and clinical status. To evaluate mutations in HBV isolates obtained from chronically infected HBV patients in terms of clinical significance, we determined the genomic sequences of HBV isolates obtained from three hepatocellular carcinoma (HCC) patients (He52, He53, and He82) and from three asymptomatic carriers (He74, He100, and He127). A comparison of sequence variations showed that the HBV isolates from the three HCC patients showed higher frequencies of mutation than the isolates from the three asymptomatic carriers. Three characteristic mutation patterns were identified in the HBV isolates from the HCC patients, which distinguished the HBV isolates from the asymptomatic carriers. First, HBV isolates from the three HCC patients both had double mutations in a core promoter (T1762/A1764) and a precore mutation (A1896). Second, although these isolates belonged to genotype C, 11 amino acids deletions in the preS1 region, specific for HBV genotype D, were detected in the isolates of two HCC patients (He52 and He82). Third, mutations (I127T/N, K130M, and V131I) at three codons in the carboxy functional region of X protein were observed in isolates from all three HCC patients. Additionally, phylogenetic analysis based on the entire HBV sequences showed that all six isolates belonged to genotype C2, as do other Korean strains.     

Full-length genomic analysis of hepatitis B virus isolates in a patient progressing from hepatitis to hepatocellular carcinoma.

In hepatitis B virus (HBV)-endemic countries, the majority of hepatocellular carcinoma (HCC) arises in HBV carriers. High frequency of mutations at nucleotides 1762(A-->T) and 1764(G-->A) in the core promoter region have been described in HCC. Due to the differences in genetic backgrounds, environmental risk factors and random cellular insertion sites, it is difficult to analyze the possible roles of HBV variants detected in different HCC patients. In a follow-up cohort study, an HBsAg-positive asymptomatic carrier was diagnosed HCC within 4 years. Eleven full-length HBV isolates, three from the first serum sample obtained 4 years pre-HCC, and eight from serum sample, peri-tumor and tumor tissue post-HCC of this individual were sequenced and used to transfect HepG2 cells. When sequences were compared between pre- and post-HCC isolates, no single mutation common to all post-HCC isolates that differed from pre-HCC isolates was found. Among all 11 isolates, there were 20 predicted amino acid substitutions shared by two or more post-HCC isolates. These were located in the S(5), X(4), core(4), polymerase(4), pre-S1(2) and pre-S2(1) proteins. Possible roles of amino acid substitutions and enhanced replication efficiency in cells transfected by post-HCC isolates are discussed.     

Analysis of the full-length genome of genotype 4 hepatitis E virus isolates from patients with fulminant or acute self-limited hepatitis E

It was suggested that hepatitis E virus (HEV) genotype 4 is associated more closely with the severity of hepatitis E than genotype 3, although the virological basis remains unknown. The aim of this study was to examine whether genomic differences among genotype 4 HEVs are responsible for the development of fulminant hepatitis. Full-length sequences of genotype 4 HEVs from three patients with fulminant hepatitis and six patients with acute self-limited hepatitis were determined. The sequences were analyzed with those of 13 genotype 4 HEV isolates whose entire nucleotide sequence is known. Analysis of 22 full-length sequences (fulminant hepatitis, 5; acute hepatitis, 17) revealed that C at nt 1816 and U at nt 3148 (U3148), both of which do not change the amino acid sequences, were significantly associated with fulminant hepatitis (P = 0.0489, respectively). When partial nucleotide sequences containing nt 1816 or nt 3148 were determined in 16 additional HEV isolates of genotype 4, a closer association between U3148 and fulminant hepatitis (P = 0.0018) was observed. The comparison of 86 HEV isolates of all four genotypes showed that U3148 had a stronger association with fulminant hepatitis than other nucleotides at nt 3148 (P = 0.0006). Patients infected with HEV with U3148 had a significantly lower value of the lowest prothrombin activity (P = 0.0293). Nt 3148 is located within the RNA helicase domain, and 22-nt sequence including nt 3148 was well conserved among all genotypes. A silent substitution of U3148 in HEV may be associated with the development of fulminant hepatitis. Further studies are needed to clarify the underlying mechanism.     

Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis in relation to HBeAg and anti-HBe

To investigate the relationship between viral factors and the development of chronic hepatitis B, the entire hepatitis B virus (HBV) genome of chronic carriers at different disease stages were analyzed. Eighty genotype C HBV carriers including 12 hepatitis B e antigen (HBeAg) positive asymptomatic carriers (Group A), 49 HBeAg positive patients with chronic liver diseases (Group B) and 19 anti-HBe positive patients with chronic liver diseases (Group C) were studied. HBV nucleic acid from serum samples was sequenced directly and compared with GenBank reference sequences HBV X01587 and M12906. On phylogenetic analysis, 76 cases were genotype C2. Of the 76 genotype C2 cases, the nucleotide and amino acid substitution rates in the precore/core region were significantly higher in Groups B and C than in Group A, also in Group C than in Group B. The nucleotide substitution rates in the full genome and the core promoter region were significantly higher in Group C than in Group A, also in group C than in Group B. The nucleotide and amino acid substitution rates in the X region were significantly higher in Group C than in Group A. The amino acid substitution rate in the pre-S2 region was significantly higher in Group C than in Group B. Deletion mutations were found mainly in Groups B and C. This whole genome analysis of HBV chronic carriers suggested that the nucleotide substitutions and deletions in HBV were closely associated with the pathogenesis of chronic HBV infection.     

Long-term follow-up of hepatitis B virus and hepatitis C virus replicative levels in chronic hepatitis patients coinfected with both viruses

Dual infection with hepatitis B and C viruses is often encountered in endemic areas of both viruses. However, understanding of the clinical and virological implications is limited. The aim of this study was to investigate the role of each virus in liver injury and the interaction between the two viruses in dual infection with hepatitis B and C viruses. Three patients who had chronic infection with both hepatitis B and C viruses were examined, and a longitudinal study of both serum hepatitis B virus DNA and hepatitis C virus RNA levels over 4 years was undertaken. The results were correlated with serum alanine aminotransferase levels. Serum alanine aminotransferase values showed a relationship with hepatitis B virus replicative levels, but not with hepatitis C virus replicative levels in all 3 patients. Serial changes of replicative levels of both viruses were studied, and it was found that hepatitis C virus replicative levels were enhanced after the decline of hepatitis B virus replication in 1 of the 3 patients. In the remaining 2 patients, a transient rise of hepatitis C virus replicative levels in association with a decrease of hepatitis B virus replication was also observed during part of the follow-up period. These findings indicate that hepatitis B virus may play a dominant etiological role in liver injury, and that a suppressive action between hepatitis B and C viruses may occur in dual infection with both viruses. © 1995 Wiley-Liss, Inc.     

Association of concurrent hepatitis B surface antigen and antibody to hepatitis B surface antigen with hepatocellular carcinoma in chronic hepatitis B virus infection

Antibody to hepatitis B surface antigen (HBsAg) (anti‐HBs) can exist in patients with chronic hepatitis B virus (HBV) infection. To date, little is known about the association of concurrent HBsAg and anti‐HBs (concurrent HBsAg/ anti‐HBs) with hepatocellular carcinoma (HCC). The aim of this study was to investigate the clinical relevance of concurrent HBsAg/anti‐HBs with preS deletion mutations and HCC in chronic HBV infection. A total of 755 patients with chronic HBV infection were included consecutively at a tertiary center. Logistic regression analysis was used to identify risk factors for HCC, and serum HBV DNA was amplified, followed by direct sequencing to detect preS deletions. The prevalence of concurrent HBsAg/anti‐HBs was 6.4% (48/755) and all HBVs tested were genotype C. HCC occurred more frequently in the concurrent HBsAg/anti‐HBs group than in the HBsAg only group [22.9% (11/48) vs. 7.9% (56/707), P = 0.002]. In multivariate analyses, age >40 years [odds ratio (OR), 14.712; 95% confidence interval (CI), 4.365–49.579; P < 0.001], male gender (OR 2.431; 95% CI, 1.226–4.820; P = 0.011), decompensated cirrhosis (OR, 3.642; 95% CI, 1.788–7.421; P < 0.001) and concurrent HBsAg/anti‐HBs (OR, 4.336; 95% CI, 1.956–9.613; P < 0.001) were associated independently with HCC. In molecular analysis, preS deletion mutations were more frequent in the concurrent HBsAg/anti‐HBs and HCC groups than in the HBsAg without HCC group (42.3% and 32.5% vs. 11.3%; P = 0.002 and 0.012, respectively). In conclusion, concurrent HBsAg/anti‐HBs is associated with preS deletion mutations and may be one of the risk factors for HCC in chronic HBV infection with genotype C. J. Med. Virol. 81:1531–1538, 2009. © 2009 Wiley‐Liss, Inc.     

Acute hepatitis B virus infection in Turkey: epidemiology and genotype distribution

The aim of this study was to investigate the prevalence of hepatitis B virus (HBV) genotypes in Turkey. Epidemiological and clinical data for 158 patients with acute HBV infection from 22 medical centres in the period February 2001 to February 2002 were collected prospectively. HBV genotyping was based on analysis of restriction fragment length polymorphisms and nested PCR. There were 59 female and 99 male patients, with a mean age of 34.2 +/- 15.6 years. The most common probable transmission route was blood contact in 63 (41.1%) cases, but was unknown in 78 (49.4%) cases. The mean alanine aminotransferase level was 1718 +/- 1089 IU/L. Four of the 158 patients (2.5%) died because of fulminant hepatitis. One year after discharge, 11 (10.6%) of 103 cases were positive for hepatitis B surface antigen (HBsAg) and 80 (77.7%) were positive for anti-HBsAg. Genotype determination was unsuccessful in 11 cases because of a negative PCR; genotype D was found in the remaining 147 cases. The results suggested that acute HBV infection constitutes a significant health problem in Turkey and that genotype D is predominant.     

Characterization of hepatitis B virus genome variability in Iranian patients with chronic infection, a nationwide study

Hepatitis B virus (HBV) isolates from Iranian patients around the country were characterized. Eighty-one complete genomes from HBV isolates were sequenced and analyzed. The studied population was grouped into three categories including inactive carriers, patients with chronic hepatitis, and patients with liver cirrhosis. Molecular and phylogenetic analyses revealed that Iranian patients were infected with HBV genotype D and subgenotype D1. The most common subtype was ayw2, followed by ayw3 and ayw4. Several deletions and insertions that had no correlation with disease outcome were observed in the HBV genomes. The most frequent mutation in the major hydrophilic region (MHR) of HBV surface antigen (HBsAg) was sP120S. Almost half of the patients studied carried precore (PC) mutant variants and one-third of the studied population was infected with variants carrying basal core promoter (BCP) mutations. PC and BCP mutations were observed in older patients, especially in those with chronic liver disease. Sixty-seven patients (82.7%) were HBeAg negative, and the prevalence of precore mutant isolates (G1896A) was higher in this group than in HBeAg-positive patients. Lamivudine drug resistance mutations were detected after 1 year of treatment in about 30% of lamivudine-treated patients. In conclusion, these results demonstrate that HBV subgenotype D1 is the only subgenotype circulating in Iran, and there is no evidence of any exotic genotype in the region. The HBV PC (G1896A) mutation may play an important role in the clinical outcome of the disease by increasing the risk of progressive liver disease among Iranian patients infected with HBV.     

Concurrent hepatitis C virus and hepatitis delta virus superinfection in patients with chronic hepatitis B virus infection.

Since hepatitis C virus (HCV) and hepatitis delta virus (HDV) are transmitted by the same routes as hepatitis B virus (HBV), simultaneous or concurrent HCV and HDV infection in patients with chronic HBV infection may occur. To test this hypothesis and to examine the clinicohistological and immunopathological presentations of such multiple hepatitis virus infections, acute and/or convalescent serum specimens from 86 patients with acute HDV superinfection were tested by enzyme immunoassay for antibodies to HCV. Of the 86 patients, 18 (20.9%) were associated with HCV infection. Although patients with early mortality cannot be evaluated by the HCV markers used in this study, the results showed that the clinical and histologic features were similar except that patients with HCV infection were older than those without HCV infection (P less than 0.01). Immunopathological studies carried out within 2 months after the onset of acute HDV superinfection demonstrated that hepatitis B core antigen (HBcAg) was not detected in any patient and HDV antigen was detected in 18.2% of the patients with HCV infection whereas HBcAg and HDAg were found in 7% and 65.1%, respectively, of those without HCV coinfection (P less than 0.02). It is concluded that concurrent HCV and HDV superinfections can and do occur in patients with chronic HBV infection. In these triple viral infections, HCV may even transiently suppress HDV and HBV.     

乙型肝炎病毒基因组前-前-S和前-X区基因分布的研究

目的研究不同基因型乙型肝炎病毒(HBV)前-前-S和前-X区核苷酸和氨基酸序列分布。方法应用DNAstar软件中的MegAlign程序分析GenBank登记的35株和3株由本室测定的不同基因型HBV全序列中前-前-S和前-X区基因分布情况，并用Blast软件对GenBank中598株HBV全基因组序列进行前-前-S和前-X区的核苷酸和氨基酸相似性分析。结果除D基因型外，在各型HBV基因组中均存在前-前-S区核苷酸序列，但仅在C、F和H型HBV基因组中存在前-前-S区开放读码框架(ORF)。但前-X区核苷酸序列及其ORF仪见于C基因型HBV。在GenBank登记的598株不同基因型HBV中，与推导的前-前-S区相似氨基酸序列有36株，而与推导的前-X区相似的氨基酸序列有47株。结论前-前-S区序列存在于除D基因型以外的所有HBV基因型，而前-X区基因仅存在于C基因型HBV基因组中。     

Hepatitis B virus genotypes and spontaneous hepatitis B e antigen seroconversion in Taiwanese hepatitis B carriers

Hepatitis B virus (HBV) is classified into eight genotypes (A-H), and genotype C is associated with more aggressive liver disease compared to genotype B. However, the mechanisms responsible for the clinical differences remain unclear. To test whether genotype C patients had with lower rates of spontaneous hepatitis B ge antigen (HBeAg) seroconversion than genotype B patients, stored serum samples from 146 Taiwanese adult HBeAg-positive hepatitis B carriers followed-up for a mean of 52 months (range, 12-120 months) were tested for HBV genotype by a molecular method. Genotype C patients were significantly older than genotype B patients (mean age, 37 +/- 12 vs. 29 +/- 10 years, P < 0.001). During the follow-up period, genotype C patients had a significantly lower rate of spontaneous HBeAg seroconversion than genotype B patients (27 vs. 47%, P < 0.025). Spontaneous HBeAg seroconversion occurred one decade later in genotype C patients compared with genotype B patients. Multivariate analyses identified age < or =35 years (odds ratio: 2.08; 95% confidence interval [CI], 1.07-4.0; P < 0.05), high baseline serum alanine aminotransferase level (odds ratio: 2.34; 95%CI, 1.39-4.09; P < 0.005), and HBV genotype B (odds ratio: 1.94; 95%CI, 1.03-3.63; P < 0.05) as independent factors associated with spontaneous HBeAg seroconversion. In conclusion, genotype C patients, compared to genotype B patients, have a delayed HBeAg seroconversion in the immune clearance phase of chronic HBV infection, which may contribute to a more progressive liver disease and more refractory to antiviral therapy.     

Molecular characterization of hepatitis C virus genotype 2a from the entire sequences of four isolates

Genotype 2a hepatitis C virus (HCV) has different characteristics from genotype 1b, such as responsiveness to interferon therapy. Such type-specific characteristics appear to be due to differences in the HCV genome sequence. The complete sequences of genotype 2a HCV genome isolated from four patients with chronic hepatitis C were determined, and nucleotide and deduced amino acid sequences were compared within genotype 2a, as well as between genotype 2a and 1b. Whereas the amino acid sequence similarity of the core region was highest within genotype 1b, the NS3 and NS4B regions of exhibited greater similarity than the core region in genotype 2a. The serine protease and helicase motifs in the NS3 region were well conserved in genotype 2a to the same degree as in genotype 1b. However, the putative secondary structure of 2a isolates was significantly different from that of the 1b isolates. Analysis of amino acid similarity between genotypes 2a and 1b revealed the lowest degree of similarity in the E1 region, followed by the NS2 and NS5A region. Sequences of genotype 2a in the interferon-sensitivity determining region (ISDR) located in the NS5A region had a deletion of four amino acids compared with that of genotype 1b. When the ISDR of the genotype 2a was aligned for maximal similarity, it exhibited similarity of only 52.5-55.0% when compared with that of HCV-J, which belongs to genotype 1b. These findings for the entire sequences of genotype 2a isolates will contribute to virological studies of HCV.     

A new isolate of hepatitis B virus from the Philippines possibly representing a new subgenotype C6

Hepatitis B virus (HBV) genotypes and subgenotypes show distinct geographical prevalence. A genotyping analysis of 28 samples from asymptomatic HBV carriers from the Philippines gave a distribution of HBV genotypes as expected from a previous study: 54% B (15/28), C5 18% (5/28), 14% D (4/28), 7% A1 (2/28). In addition, 7% (2/28) of the samples showed a double infection with genotypes B and D. One of the isolates sequenced completely, ph105, did not group into one of the known subgenotypes after phylogenetic analysis. Ph105 formed a separate clade in genotype C. With a genome length of 3,215 nt. and a serological subtype adr, ph105 exhibited the main features of most genotype C strains. However, ph105 differed by 4.1–7.2% from HBV subgenotypes C1 to C5 when comparing the nucleotide sequence of whole genomes. With only 4.1% difference ph105 was most closely related to subgenotype C2. SimPlot analysis gave no indication for recombination with known HBV genotypes. Ph105 fulfils all criteria for a new subgenotype C6. J. Med. Virol. 81:983–987, 2009. © 2009 Wiley‐Liss, Inc.     

慢性乙型肝炎患者HBV耐药模式及rtA181变异准种分析

目的分析慢性乙型肝炎患者HBV逆转录酶区耐药变异情况及rtA181变异准种的分布。方法提取患者血清中HBVDNA,PCR扩增逆转录酶区域,产物测序后经软件比对分析耐药变异情况和基因型;对部分rtA181变异标本进行克隆后测序分析准种分布。结果 489例标本中,检出明确耐药变异265例,在B、C基因型中分布比例存在差异（56.6%vs43.0%,P=0.022）。拉米夫定相关耐药138例（52.1%）,以M204I、M204I/V＋L180M±L80I/V变异为主;阿德福韦相关耐药35例（13.2%）,以N236T＋A181T/V较为多见;拉米夫定＋阿德福韦相关耐药70例（26.4%）,几乎都和A181有关。rtA181准种分析发现1例位于同一病毒株的多耐药组合,且未发现单一A181T变异的病毒准种。结论 HBV耐药变异主要表现为M204和A181相关变异,耐药模式复杂;检测HBV逆转录酶区的变异有助于临床及时发现和确认耐药情况,指导临床合理进行抗病毒用药。     

Molecular characterization of hepatitis C virus genotype 4 sequences in HIV-coinfected patients from Argentina

The prevalence of hepatitis C virus genotype 4 (HCV-4) is increasing in different parts of the World but in Latin America the data are still scarce. We aimed to characterize HCV-4 isolates from 383 HIV-coinfected patients in Argentina. Sequence analyses were based on the non-structural 5B region of HCV. Results from 18 patients indicated a genetic heterogeneity that involved three genotype 4 subtypes. Sequences were ascribed to subtype 4d (67%), 4a (22%), and 4m (11%). In spite of different sources of transmission were defined among patients, no statistical association was found with the genotype 4 subtype. The scenario is also compatible with multiple importation of the epidemic and there is no evidence for transmission-specific clusters or network-like transmission of HCV-4. This HCV-4 does not represent a recent introduction in Argentina, it circulates in all transmission groups and its presence is increasing among HIV-infected patients.