Complete genome sequence and phylogenetic analysis of hepatitis B virus isolated from Turkish patients with chronic HBV infection

Hepatitis viruses are the leading causes of chronic liver disease resulting in chronic hepatitis, cirrhosis, and hepatocellular carcinoma in the world and also in Turkey. Although Turkey has an intermediate rate of hepatitis B virus (HBV) infection with a prevalence reported as 5%, a complete HBV genome sequence has not been published. In this study, the molecular characterization and phylogenetic analysis are described of 11 complete HBV genomes isolated from 11 naïve patients (5 male, 6 female; ages: 18–54 years old, median 35 years old) with chronic HBV infection. Of 11 patients, 7 and 4 were HBeAg positive/anti‐HBe negative and HBeAg negative/anti‐HBe positive, respectively. All patients had no co‐infection with HCV, HDV, or HIV. HBV DNA was extracted from the sera of the patients. The complete genome was amplified by PCR and cloned into a TA vector. The PCR products were sequenced directly and the complete HBV genome sequences were determined. Ten HBV genomes were 3182 base pairs in length. There was a 183 bp deletion (between nucleotides 2987–3169) in pre‐S region in one HBeAg positive patient. There were two pre‐core stop codons (G1896A) in two HBeAg negative and three core promoter dual mutations (T1762/A1764) in one HBeAg positive and two HBeAg negative patients' HBV genomes. Phylogenetic analysis of all complete genomes yielded that all Turkish sequences were clustered in genotype D branch (ten in subgenotype D1 and one in subgenotype D2). The analysis of S gene amino acid sequences revealed that surface gene subtypes of one and ten HBV strains were subtype ayw3 and ayw2, respectively. This study indicates that Turkish patients with chronic hepatitis B infection show very little genotypic heterogeneity. Genotype D of HBV DNA and subtype ayw2 of surface gene represent almost the whole Turkish patient population infected with HBV. J. Med. Virol. 76:476–481, 2005. © 2005 Wiley‐Liss, Inc.     

Complete genome sequence and phylogenetic analysis of hepatitis B virus (HBV) isolated from Mongolian patients with chronic HBV infection

Although there is a report of a high rate of hepatitis B virus (HBV) infection in Mongolia, the entire nucleotide sequence of HBV circulating among Mongolian patients has not been reported. To obtain the complete nucleotide sequence of the Mongolian HBV, viral DNA was extracted from sera of patients with HBV infection. Six Mongolian HBV strains were amplified by PCR. Complete genomic sequences were determined for two Mongolian HBV isolates, MBT181 and MMU36. The entire genome of Mongolian HBV isolates was 3,182 bp long and genetic distance between Mongolian HBV isolates was 3.2%. Precore stop codon resulting from a guanine to adenine mutation at nucleotide 1,896 was detected in MBT181 strain. Based on phylogenetic analysis, the six Mongolian isolates were classified as genotype D.     

Distribution of hepatitis B virus (HBV) genotypes among HBV carriers in the Cote d'Ivoire: complete genome sequence and phylogenetic relatedness of HBV genotype E

The characteristics of hepatitis B virus (HBV) genotype E are not well known because only a few studies have been carried out by complete genome analysis. The aim of this study was to elucidate the distribution of HBV genotypes in Cote d'Ivoire, and to clarify the genotype-related characteristics of genotype E. The distribution of HBV genotypes among 48 HBV carriers in Cote d'Ivoire was determined using serological and genetic methods. The characteristics of genotype E were evaluated by complete genome sequences, and further investigations of small S gene, basic core promoter (BCP) mutation, and precore mutation were undertaken. HBV genotype distribution among the 48 carriers was 6.3% for genotype A, 6.3% for genotype D, and 87.4% for genotype E. Complete genomes of two genotype E strains were sequenced, and found to have 98.2% to 99.2% homology at the nucleotide level when compared with genotype E strains reported previously. In 24 genotype E carriers, the precore mutation was detected in 75% of the patients without HBeAg, in contrast to only 25% of the patients with HBeAg (P < 0.05). All 24 strains have T at nucleotide 1858 in the precore region. In contrast, BCP double mutation was detected in 17% of the patients with HBeAg, and 33% of the patients without HBeAg. These results indicated as the following: (1) genotypes A, D, and E of HBV exist in Cote d'Ivoire and genotype E is the most prevalent; (2) genotype E spread with low genetic diversity over the complete genome in West Africa; (3) HBV precore and/or BCP double variants were common among the patients with genotype E infections.     

Genotype,serotype, and phylogenetic characterization of the complete genome sequence of hepatitis B virus isolates from Malawian chronic carriers of the virus

Hepatitis B virus (HBV) genotypes have distinct geographical distribution. HBV sequences among hepatitis B carriers in Malawi have not been evaluated thus far. HBsAg serotype and genotype of HBV was determined in 20 serum samples from Malawian chronic HBV carriers, and two complete genomes and 13 entire pre-S2/S genes were sequenced directly. Genotype A HBV isolates were found in all of the samples, and serotype with adw2 and ayw2 were detected in three and 17 samples, respectively. In phylogenetic analyses, two complete genomes were classified into a subgroup A' that was described previously in South African isolates of the virus, and were separated from HBV isolates in Western countries with nucleotide differences ranging from 4.1-6.2%. The separation of subgroup A' was also evident in the tree topology of the entire pre-S1/S2, X and precore/core region, but not evident in the small-S region. The nucleotide divergences in subgroup A' were higher than those among genotype A without subgroup A' in the complete genomes as well as each of four open reading frames. All of the 13 pre-S2/S sequences were classified into the subgroup A', and clustered with known HBV isolates with ayw2 in carriers from South Africa and Zimbabwe. Three amino acids in the pre-S2/S gene were characteristic of subgroup A' with ayw2. In conclusion, unique HBV isolates of subgroup A' with ayw2 are prevalent in Malawi, and subgroup A' with a relatively higher nucleotide diversity may be a HBV isolate characteristic of the indigenous population of some African countries.     

Hepatitis B virus (HBV) genotyping in Belgian patients with chronic HBV infection

Several studies have demonstrated that pathogenic and therapeutic differences exist among hepatitis B virus (HBV) genotypes. Therefore, this study established the prevalence of different HBV genotypes in 128 Belgian patients with chronic HBV infection. The prevalences of genotypes A and D, and mixed genotypes A and D, were 53%, 37% and 8%, respectively, for a group of blood donors, and 54%, 31% and 9%, respectively, for a group of patients from the gastroenterology units. The results indicated that genotypes A and D are the predominant genotypes in Belgian patients with chronic HBV infection.     

Specific deletions in the hepatitis B virus core open reading frame in patients with chronic active hepatitis B

The polymerase chain reaction (PCR) and DNA sequencing were used to examine genomic variation in the pre-core/core open reading frame of the hepatitis B virus (HBV) in chronically infected patients. Gel electrophoresis of amplification products showed the presence of shortened forms of the core gene in addition to the full length product. These shortened forms were seen only in patients with chronic active hepatitis (CAH) seropositive for HBeAg and not in patients with chronic persistent hepatitis (CPH) or HBeAg minus CAH. Cloning and DNA sequencing revealed the presence of a number of overlapping deletions within the core gene, the majority being in-frame, which were clustered within aa 81-114 of the core gene product. These deletions were found in patients with CAH from different racial and geographical backgrounds, whereas PCR analysis of the surface and ×open reading frames showed no shortened forms suggesting deletions to be specific to the core gene in these patients. Because the product of the core gene-the HBV core antigen–is believed to be the major target for T-cell-mediated liver damage, it seems likely that the products of core genes carrying deletions will alter immune recognition and may be of importance in the progression of inflammatory liver damage.     

Complete genomic sequence and phylogenetic relatedness of hepatitis B virus isolates from Iran

Hepatitis B virus (HBV) is one of the main etiological agents of acute and chronic liver disease that is still a major public health problem in the world. Numerous HBV isolates have grouped into eight genotypes, A to H, based on the complete genome sequence. To date, no study has been carried out on the complete HBV genome sequence in Iran. The objective of this study was to investigate the complete genome sequence organization and phylogenetic analysis of the five HBV strains, which obtained from Iranian chronic infected patients. Results showed that Iranian strains were closely related to each other, with 97-100% nucleotide similarity. Phylogenetic analysis based on the complete genome sequences and the precore/core gene sequences revealed that all strains were of genotype D, sub-genotype D1 with bootstrap value 100 and 99%, respectively. The S gene encoded Arg122, Pro127, and Lys160 corresponding to subtype ayw2. Iranian HBV isolates had closely related with Turkish HBV strains. All strains had a nucleotide length of 3,182 base pair (bp) except IR-P4 strain, with a 3,185 bp in length and with a unique Phe89 insertion in the X gene. The intragenotypic divergence of the complete genome sequence of Iranian strains was 1.8% and the intergenotypic in genotype D was 3.8% and with the other genotypes was 7.9-15.4%. In conclusion, this study revealed that the HBV genotype D, sub-genotype D1, subtype ayw2 dominates in the Iranian infected patients. A single Phe89 insertion in the X gene of the one Iranian strain with an unforeseen length of 3185 bp was identified.     

Elimination of hepatitis delta virus infection after loss of hepatitis B surface antigen in patients with chronic delta hepatitis

The aim of this study was to evaluate whether patients with chronic hepatitis delta virus (HDV) infection treated with alpha interferon and subsequent loss of hepatitis B surface antigen (HBsAg) eliminate HDV. HDV RNA was detected in 26 of 28 patients with chronic delta hepatitis using the polymerase chain reaction. Seventeen patients in whom HDV RNA was detected were treated with alpha interferon; in 65%, HDV RNA remained detectable during treatment or reappeared after stopping therapy whereas in three patients HDV RNA remained absent (17.5%). HDV RNA became and remained undetectable in serum and liver of two of these three patients who lost HBsAg from serum and in one patient who was intermittently HBsAg negative during therapy. After loss of HBsAg, hepatitis B virus (HBV) DNA was still detectable in the liver, but not HBV RNA, indicating absent or very low HBV replication. Three patients were lost to follow up (17.5%). Two nontreated patients with chronic HDV infection also lost HBsAg during follow up; HDV RNA also became undetectable in their serum. Thus, HDV replication does not persist after the loss of HBsAg. Clearance of HBsAg may be a useful guide to when therapy can be stopped. © 1994 Wiley-Liss, Inc.     

Complete genomic sequence and phylogenetic relatedness of hepatitis B virus isolates in Cambodia

Although it is known that Cambodia is one of the high endemic area of hepatitis B virus (HBV) infection, molecular characterization of HBV circulating in this country has not been reported. In this study, pre-S gene of HBV from 12 Cambodian patients was sequenced. Phylogenetic analysis based on the pre-S gene sequence revealed that 8 out of 12 isolates (66.7%) belonged to HBV/C1 and remaining four (33.3%) were HBV/B4. Furthermore, complete genomic sequences were also determined for three Cambodian HBV isolates. They all comprised of 3,215 bp long and two of them belonged to subgenotype B4, which had recombination event with genotype C in the precore/core gene confirmed by SimPlot and BootScanning analyses. Our results showed that both HBV strains belonged to subgenotypes B4 and C1, which are circulating in this country. This is the first report on molecular characterization of the HBV prevalent in Cambodia.     

The role of the hepatitis B virus infection in patients with chronic hepatitis in argentina

Over a seven-year period, we monitored 221 patients with chronic hepatitis from two medical centers. By using the counterelectrophoresis (CEP) test to detect the presence of HBsAg and anti-HBc, or both, we established that 87.7% of them had hepatitis B infection. Serum specimens originally found negative for HBsAg by CEP were further tested by reversed passive hemagglutination (RPH), and those originally found negative for anti-HBc by CEP were further tested by radioimmunoassay (RIA). Five patients were anti-HBc-positive and HBsAg-negative. No sex predominance was observed, but HBsAg incidence increased with increasing age. The HBeAg antigen was detected in 46.8% of the 161 cases tested for it; the most frequent subtype found was adw (63.7%). The present findings indicate that HBV infection largely contributes to the development of chronic hepatitis in Argentinian patients.     

Preponderance of hepatitis B virus genotype B contributes to a better prognosis of chronic HBV infection in Okinawa,Japan

The present study was designed to examine the distribution of hepatitis B virus (HBV) genotypes among patients at various stages of chronic liver disease type B in Okinawa Prefecture, Japan, where the prevalence of hepatitis B surface antigen is the highest in Japan despite the lowest mortality rate from primary liver cancer. Serum samples from 227 HBV carriers were determined for HBV genotype by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Five of 227 sera were negative for HBV DNA by nested PCR and were excluded from the genotype analysis. Genotype B was predominant in asymptomatic carriers (45/67, 67%), whereas genotype C was predominant in chronic liver disease: 49% (50/103) in patients with chronic hepatitis, 63% (20/32) in patients with cirrhosis, and 60% (12/20) in patients with hepatocellular carcinoma. The distribution of genotype B decreased with increasing liver disease severity. However, this tendency was seen among patients aged less than 50 years old, whereas the prevalence of genotype B was similar among carriers with various liver diseases who were older than age 50. In conclusion, HBV genotype B was prevalent and less frequent among patients with advanced liver disease, particularly in patients aged less than 50 years. These findings suggest that the preponderance of genotype B is responsible for the low mortality rate of primary liver cancer associated with HBV seen in Okinawa Prefecture, despite having the highest HBV carrier rate in Japanese.     

Complete nucleotide sequences of hepatitis B virus genomes associated with epidemic fulminant hepatitis

Pre-core/core mutants are frequently observed in patients with fulminant hepatitis. To investigate the extent of molecular characteristics of hepatitis B virus (HBV) genomes implicated in the development of fulminant hepatitis, full-length HBV genomes were sequenced directly from sera of two patients with epidemic fatal fulminant hepatitis, after amplification by the polymerase chain reaction. These two genomes, of 3215 nucleotides, were 99.6% identical, indicating that a common source of HBV potentially caused fulminant hepatitis. Thirty unique nucleotide mutations were commonly found in the two entire HBV genomes. Three were located in the stem-loop structure, changing this element to a more stable structure. Twenty-five unique amino acid substitutions were found in each open reading frame, except for the X and pre-surface 2 genes. One was located in the pre-surface 1 gene; two were in the surface gene; three were in the pre-core gene, including codons 28 (tryptophan to stop codon) and 29 (glycine to aspartic acid); eight were in the core gene; and 11 were in the polymerase gene. The pre-core mutations at codons 28 and 29 were common to the two HBV strains reported previously in patients with epidemic fulminant hepatitis. Thus, HBV genomes associated with epidemic fatal fulminant hepatitis have numerous unique mutations, located mainly in the polymerase gene, as well as the pre-core/core gene, including mutations in the stem-loop structure of the pregenome encapsidation signal sequence. These mutations may be associated with the development of fulminant hepatitis. © 1996 Wiley-Liss, Inc.     

Hepatitis B virus infection of peripheral blood mononuclear cells is common in acute and chronic hepatitis

Hepatitis B virus (HBV) infection of peripheral blood mononuclear cells (PBMCs) has been observed in all stages of liver disease. Thus far all information about the physical state of HBV in mononuclear blood cells comes from Southern blot analysis and in situ hybridization. In this study we focused our attention on the presence of HBV DNA sequences in PBMCs of 30 patients with acute type B hepatitis and 6 patients with chronic active hepatitis by utilizing both Southern blot analysis and the polymerase chain reaction (PCR). Southern blot analysis showed no HBV DNA sequences in PBMCs of the acute hepatitis patients, although the sensitivity of our method enabled us to detect as little as 1 pg of cloned HBV insert. As far as the chronic hepatitis patients are concerned Southern blot analysis revealed the presence of HBV DNA sequences in 5 out of 6 patients but intermittently at successive follow-up times. On the other hand we were able to demonstrate the presence of HBV related sequences in 14 out of 30 acute hepatitis patients (5 HBeAg positive, 9 antiHBe positive) and in all 6 chronic hepatitis patients by PCR. Our results indicate that the involvement of PBMCs with HBV during acute HBV infection occurs at a very low level, often below the detection limit of the Southern blot technique.     

Characteristics of lamivudine-resistant hepatitis B virus (HBV) strains with and without breakthrough hepatitis in patients with chronic hepatitis B evaluated by serial HBV full-genome sequences

Lamivudine therapy often causes breakthrough of hepatitis B virus (HBV) DNA and breakthrough hepatitis. The aim of this study was to determine the viral factors that relate to HBV-DNA breakthrough with and without breakthrough hepatitis. Among 82 patients with chronic hepatitis B (CHB) who received lamivudine at a dose of 100 mg daily for more than 24 months, 23 patients had HBV-DNA breakthrough induced by a lamivudine-resistant mutant. Of these 23 patients, 16 had breakthrough hepatitis and 7 had only HBV-DNA breakthrough. Serial HBV-DNA full-genome sequences during therapy were examined in 10 (7 had breakthrough hepatitis and 3 did not) of these 23 patients by direct sequencing. Mutations in the S region were examined by cloning in representative patients. There were no significant differences in the baseline clinical backgrounds and virus marker between patients with and without breakthrough hepatitis. The HBV amino acid substitutions at breakthrough hepatitis were identical to those at HBV-DNA breakthrough. Cloning analysis revealed that monoclonal mutational strain appeared at breakthrough and no such mutations existed at baseline. Regarding HBV amino acid substitutions in the polymerase region, S region, X region, and precore-core region with breakthrough compared to baseline, there was no significant differences of the numbers of amino acid substitution between breakthrough hepatitis and non-breakthrough hepatitis. There were no common amino acid changes in patients with breakthrough hepatitis. Although monoclonal lamivudine-resistant strain emerged at HBV-DNA breakthrough in patients with CHB, there were no common amino acid changes, suggesting viral factor may have insignificant role in breakthrough hepatitis.     

HBV and HAV infection in chronic hepatitis in argentina

Sera of 1 55 chronic hepatitis (CH) patients in Argentina were tested for the presence of HBsAg, anti-HBs, anti-HBc, and anti-HAV. Our purpose was to define the role that both virus A and B might play in the etiology and pathogenesis of this condition. The patients were divided into two groups: group I (57) HBsAg-negative; group II (98) HBsAg-positive. The control group consisted of 1,209 healthy blood donors from Banco Central de Sangre de Rosario; 286/ 1,209 (24%) had viral markers for HBV. In group I, 38/57 (67%) had anti-HBs and/or anti-HBc, but none had anti-HBs alone. Group II showed a higher percentage of males (P < 0.05). We found similar incidence of anti-HAV among group I, group II, and the control group.     

Diagnosis and clinical impact of occult hepatitis B infection in patients with biopsy proven chronic hepatitis C: a multicenter study

Occult hepatitis B virus (HBV) infection in patients with chronic hepatitis C has been found associated with severe liver damage, low response to interferon treatment and increased risk of developing HCC. However, doubts remain on its clinical impact and the sensitivity and specificity of its detection. HBV-DNA was sought by PCR in plasma, peripheral blood mononuclear cells (PBMCs) and liver compartments of 89 patients with biopsy proven chronic hepatitis C, using sets of primers for core ("c"), surface ("s"), and x ("x") regions of HBV genome. Occult HBV infection was defined by the presence of HBV-DNA in at least two different PCRs in at least one compartment. Occult HBV infection was detected in 37 (41.6%) of the 89 patients investigated. It was more frequent (80.8%) in 26 anti- HBs negative/anti-HBc positive patients than in 18 anti-HBs/anti-HBc positive (61.1%, P < 0.01) and 45 anti-HBs/anti-HBc negative (11.1%, P < 0.0001), and more frequently in liver (91.9%) than in PBMCs (62.2%) and plasma (32.4%). No association was found between occult HBV infection and the degree of liver necroinflammation and fibrosis. However, considering the 52 patients without occult HBV infection, 51.4% of 35 patients with genotype 1 and 5.9% of 17 with genotype non-1 showed severe fibrosis (P = 0.003); patients with occult HBV infection did not show such difference. Instead of seeking occult HBV infection in patients with chronic hepatitis C, both anti-HBs negative/anti-HBc positive and anti-HBs positive/anti-HBc positive, in plasma alone, more reliable information can also be obtained from the liver tissue and PBMCs.     

Sequence analysis of pre-S/surface and pre-core/core promoter genes of hepatitis B virus in chronic hepatitis C patients with occult HBV infection

Although occult hepatitis B virus (HBV) infection in individuals without detectable hepatitis B surface antigen (HBsAg) may occur and has been reported to be common in patients with chronic hepatitis C, the related molecular mechanisms remain unknown. With the polymerase chain reaction, serum HBV DNA was sought in 100 HBsAg-negative patients with chronic hepatitis C virus (HCV)-infection. In those with occult HBV infection, possible genomic variability of HBV was evaluated by amplification and direct sequencing of pre-S, surface, and pre-core/core promoter genes. In total, 10 of the 100 patients (10%) had detectable serum HBV DNA, documenting an occult HBV infection. A deletion mutant in the pre-S gene was found in one patient and mutations of the a determinant of HBsAg were observed in 2. In addition, a novel core promoter mutant (a dinucleotide substitution: T-to-C at nucleotide 1,802 and T-to-G at nucleotide 1,803, T1802C/T1803G) was found frequently in patients with occult HBV infection as compared to sex- and age-matched HBsAg-positive patients (80 vs. 10%, P < 0.001). In conclusion, the data suggest occult HBV infection is not uncommon in chronic hepatitis C patients in Taiwan, and a novel core promoter mutant may be associated with the absence of circulating HBsAg in these patients.