In vitro replication phenotype of a novel (-1G) hepatitis B virus variant associated with HIV co-infection

The -1G mutant HBV is more prevalent in individuals co-infected with HIV/HBV than in individuals infected with HBV alone and in some cases is the dominant virus in circulation. This mutant is created by the deletion of a dGMP (-1G) from the guanine rich homopolymer sequence located at nts 2,085-2,090 (numbering from EcoRI site as position 1) in the HBV core gene. This deletion causes a frameshift generating a premature stop codon at (64) Asn in the HBV core gene (codon 93 in the precore gene), that truncates the precore protein, precursor of the secreted hepatitis B "e" antigen (HBeAg), and the core protein which forms the viral nucleocapsid. However, the replication phenotype of the -1G mutant HBV is unknown. An in vitro cell culture model in which hepatoma cells were transiently transfected with infectious cDNAs was used to show that the -1G mutant HBV is incapable of autonomous replication and, as expected, replication was restored to wild-type (wt) levels by supplying HBV core protein in trans. Although the -1G mutation had no deleterious effect on intracellular HBV-DNA levels, high levels of -1G mutant HBV relative to wt HBV reduced virus secretion and HBeAg secretion relative to empty vector controls. Importantly, the -1G mutant HBV also caused intracellular retention of truncated precore protein in the endoplasmic reticulum (ER) and Golgi apparatus. Together, these effects may be contributing to the increased pathology observed in the setting of HIV/HBV co-infection.     

Epidemiology of precore mutants of hepatitis B in the United Kingdom

A point mutation assay was used to study the codon 28 and codon 1 precore mutant status of 310 chronic hepatitis B carriers (82 HBeAg positive and 228 HBeAg negative). Fourteen of 228 (6%) of HBeAg negative carriers had high levels of serum HBV DNA. Nine of these were explained by precore variants, three by core promoter variants, and two were not explained by recognised precore changes. Nested PCR detected serum HBV DNA in 36% (82/228) of HBeAg negative carriers and 63% (52/82) of these had precore variants. Four of 82 (4%) of the HBeAg positive carriers had precore variants, all as mixed mutant/wild type populations and evidence indicated that these carriers were seroconverting. Overall 23% (52/228) of HBeAg negative carriers had both serum HBV DNA and codon 1 or 28 precore mutations. A sexual transmission event from an HBeAg negative carrier with a relatively low serum HBV DNA level (10(4)-10(6) genome copies/ml) and only core promoter mutations was observed. Despite high rates of variant carriage in the antenatal sub-group perinatal transmission was not observed. The results of direct sequencing on 45 carriers validated the point mutation assay and also showed that codon 28 mutations were only seen in carriers with the genotype CCT at codon 15. For the Caucasian population a higher prevalence of codon 28 mutations (13/25 or 52%) than expected was seen. Liver biopsy data indicated that there was no link between the presence or absence of precore mutants and the severity of liver disease.     

Genetic heterogeneity of the precore and the core promoter region of genotype C hepatitis B virus during lamivudine therapy

It has been reported that spontaneous or interferon (IFN)-induced hepatitis B e (HBe) seroconversion has usually been associated with the development of a stop codon in the precore region. However, the difference between lamivudine-induced seroconversion and spontaneous or IFN-induced seroconversion is not known. The aim of this study was to investigate the correlation between the evolution of the precore and core promoter mutations and lamivudine-induced seroconversion. Forty-five patients with chronic hepatitis B virus (HBV) infection who were treated with lamivudine for more than 1 year were enrolled. The nucleotide sequence of the precore and core promoter region was determined before and after treatment with lamivudine for 1 year. Among 29 patients who were hepatitis B e antigen (HBeAg)-positive before treatment, 12 (41.3%) lost HBeAg during the course of treatment for 1 year. Of these, eight patients (66.7%) still had precore wild type HBV after 1 year. After 1 year, reversion to precore wild type HBV was detected in 11 (64.7%) of 17 patients who had precore mutant HBV before treatment. Twelve (70.6%) of 17 patients who were persistently HBeAg-positive had precore wild type HBV before and after treatment for 1 year. Despite the loss of HBeAg, two thirds of the patients still had precore wild type HBV after the 1-year treatment. It is suggested that lamivudine-induced seroconversion differs from spontaneous or IFN-induced seroconversion in the change of nucleotides in the precore region. The reversion in the precore region may be caused by the difference of drug-susceptibility to lamivudine. The antiviral effect of lamivudine may be more effective in the precore mutant HBV than in the precore wild type HBV.     

Enzymatic amplification and sequence analysis of precore/core DNA in HBsAg-positive patients

Serum or plasma from 69 HBsAg-positive patients was tested for the presence of precore core gene specific DNA by the polymerase chain reaction (PCR). In both healthy individuals (n = 26) and chronic carriers (n = 25), there was a strong correlation between presence of circulating anti-HBe and the absence of detectable HBV genome in serum. In 18 serum samples where HBsAg was the only detectable marker, i.e., anti-HBc-negative specimens, HBV DNA could be detected in three samples. HBV strains from 21 of the 24 PCR-positive samples were sequenced over the precore/core junction. A stop codon at the end of the precore region, described by other workers, was found in strains from two blood donors, one of whom had detectable HBeAg in serum. Conversely, HBV strains from the three anti-HBc-negative patients where DNA of the HBV precore region could be amplified and who had no detectable serum HBeAg or anti-HBe did not have this stop codon. The study indicates that further investigations are required before lack of HBeAg can be correlated with evidence of mutations in the precore region.     

Prevalence and type of pre-C HBV mutants in anti-HBe positive carriers with chronic liver disease in a highly endemic area

The sequence variability in the pre-C region of the hepatitis B virus (HBV) genome in the serum of 42 anti-HBe antibody positive carriers with chronic hepatitis B was studied by PCR and direct sequencing to determine prevalence and type of HBV pre-C mutants in a highly endemic area. Except for one, all patients were infected with viruses containing mutations in the pre-C region which prevent precore and e-antigen (HBeAg) expression: 33 were infected predominantly or exclusively with variants containing a stop codon; two had a mixture of wild-type and a pre-C stop codon mutant virus; three had precore variants with mutations of the pre-C initiation codon and two of them an additional stop codon; four had a frameshift mutation; and one had two stop codons. One patient was infected with viruses which contained a mutation creating an amino acid exchange which should not prevent precore and HBeAg expression. These data demonstrate that in an endemic area a higher prevalence and even broader spectrum of pre-C HBV mutants are found than has been recognized previously in anti-HBe positive patients with chronic hepatitis B.     

Effect of mutating the two cysteines required for HBe antigenicity on hepatitis B virus DNA replication and virion secretion

Hepatitis B virus (HBV) variants with impaired expression of e antigen (HBeAg) frequently arise at the chronic stage of infection, as exemplified by precore and core promoter mutants. Since an intramolecular disulfide bond maintains the secondary structure of HBeAg, we explored effect of missense mutations of either cysteine codon. Consistent with earlier reports, substitution of each cysteine rendered HBeAg nearly undetectable. With underlying nucleotide changes at the loop of pregenome encapsidation signal, the C-7 mutants were severely impaired in pregenomic RNA packaging and hence DNA replication. Although none of the missense mutations at C61 reduced DNA replication, replacement with arginine, but not alanine, aspartic acid, phenylalanine, or serine, blocked virion secretion. Consistent with the detection of C61R genome from a patient serum, secretion block of the C61R mutant could be overcome by co-expression of wild-type core protein. In conclusion, point mutations of the C61 codon may generate viable HBeAg-negative variants.     

Hepatitis B e antigen‐negative mutations in the precore and core promoter regions in Korean patients

Most patients with hepatitis B e antigen (HBeAg)‐negative chronic hepatitis B have variants of the hepatitis B virus (HBV) that include mutations in the precore or core promoter regions of the HBV genome. The aim of this study was to investigate the patterns of precore and core promoter mutations and their relationship to HBeAg expression in Korean patients. Four hundred seventy‐five Korean patients with chronic HBV infection between February 1995 and December 2003 were enrolled in this study. There were 236 HBeAg‐positive and 239 HBeAg‐negative patients. Blood samples were tested for HBsAg, anti‐HBs, HBeAg, hepatitis B e antibody (anti‐HBe), liver function tests, and serum HBV DNA. Mutations in the precore and core promoter regions were determined by direct sequencing. In the core promoter region, the C1740, C1753, T1762/A1764, and T1766 mutations were associated with HBeAg escape (all; P < 0.05). In the precore region, a higher frequency of the C1802, A1828, T1846, A1850, C1858, T1862, and A1896 mutations was found in HBeAg‐negative patients (all; P < 0.05). In particular, the A1896 mutation was associated with high serum levels of ALT and HBV DNA in HBeAg‐negative patients (P = 0.014 and 0.026, respectively). Mutations around the Kozak sequence (nucleotides 1809–1812) were found in 6.7% of patients and were not associated with undetectable HBeAg (P = 0.13). In Korean patients, various mutations in the precore and core promoter regions were associated with HBeAg escape and amelioration of hepatic inflammation in HBeAg‐ negative patients. Only the A1896 mutation contributed to HBeAg‐negative chronic hepatitis B. J. Med. Virol. 81:594–601, 2009 © 2009 Wiley‐Liss, Inc.     

Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis

BACKGROUND. The presence of the hepatitis B e antigen (HBeAg) in serum is known to be a marker of a high degree of viral infectivity. However, fulminant hepatitis may occur in persons who are negative for HBeAg. A single point mutation has been reported to produce a stop codon in the precore region of hepatitis B virus DNA and prevent the formation of the precore protein required to make HBeAg. To determine whether a precore-mutant virus is causally related to severe liver injury, we analyzed the entire precore region in viral strains isolated from patients with fatal cases and uncomplicated cases of hepatitis B. METHODS. Serum was obtained from 9 patients with fatal hepatitis B (5 with fulminant and 4 with severe exacerbations of chronic hepatitis) and 10 patients with acute, self-limited hepatitis B. Serum samples from a sex partner implicated as the source of the virus in one case of fulminant hepatitis were also studied. The 87 nucleotides in the precore region of the hepatitis B virus were amplified by the polymerase chain reaction and then directly sequenced. RESULTS. Of the nine patients with fatal hepatitis, seven had retrievable hepatitis B DNA. In all seven there was a point mutation from G to A at nucleotide 1896 of the precore region, converting tryptophan (TGG) to a stop codon (TAG). In contrast, this mutation was not found in the 10 patients with acute, self-limited hepatitis B. The hepatitis B DNA from the implicated source contained a sequence with the stop-codon mutation that was identical to the sequence in her partner, who had fulminant hepatitis. CONCLUSIONS. The presence of a mutant viral strain is associated with and may be involved in the pathogenesis of fulminant hepatitis B and severe exacerbations of chronic hepatitis B.     

Strong association between genotype F and hepatitis B virus (HBV) e antigen-negative variants among HBV-infected argentinean blood donors

A number of reports have indicated an increased risk of cirrhosis and hepatocellular carcinoma in hepatitis B virus (HBV)-infected individuals carrying HBV e antigen (HBeAg)-negative variants. Although distinct core promoter and precore mutations distributed according to geographical locality and viral genotype have been reported, epidemiological data from South America are still scarce. The prevalences of HBV genotypes and core promoter and precore polymorphisms in 75 HBeAg-negative Argentinean blood donors were surveyed. The observed frequencies of HBV genotypes were 64.0% for genotype F, 17.3% each for genotypes A and D, and 1.3% for genotype C. Genotype F strains were widely distributed and significantly more prevalent in the northern region of the country (P < 0.001). An overall high proportion of a stop codon mutation (UAG) at precore codon 28 (66.7%) was observed. Wild-type codon 28 (UGG) was present in 29.3% of the samples, and the remaining 4.0% of samples had mixed variants. The combination of A at nucleotide (nt) 1762 and G at nt 1764 of the core promoter was found in 58.7% of the samples. The variant profiles--T at nt 1762 and A at nt 1764 or A at nt 1762 and A at nt 1764--were detected in 28.0 and 1.3% of the samples, respectively. The observed core promoter polymorphisms could not be related to the ratio of HBeAg to anti-HBeAg antibody, HBV genotype, or precore codon 28 status. Nevertheless, a clear association of genotype F and a precore stop codon mutation was found (P < 0.05). In conclusion, HBV genotype F and mutant codon 28 strains predominated and were strongly associated in a geographically broad Argentinean blood donor population.     

乙/丙型肝炎病毒双重感染患者前C区终止变异低频率

目的了解乙型肝炎病毒（ＨＢＶ）与丙型肝炎病毒（ＨＣＶ）双重感染患者前Ｃ区基因变异，及其可能的临床意义。方法用聚合酶链反应（ＰＣＲ）与限制片段长度多态性（ＲＦＬＰ）来分析２５例ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性（Ａ组）和３１例ＨＢｓＡｇ和ＨＢＶＤＮＡ阳性但抗－ＨＣＶ和ＨＣＶＲＮＡ均阴性（Ｂ组）的慢性肝病患者前Ｃ区密码２８终止变异（终２８）。结果ＨＢＶ和ＨＣＶ双重感染患者（Ａ组）血清ＨＢＶＤＮＡ第１次ＰＣＲ阳性率（１６％）明显低于单独ＨＢＶ感染组（６５％）（Ｐ＜０．００１）；前Ｃ终２８检出率（２８％）亦明显低于单独ＨＢＶ感染（６８％）（Ｐ＜０．００１）。结论提示双重感染患者ＨＢＶ前Ｃ终止变异低频率可能与ＨＢＶ低水平复制有关     

HBe antigen loss during lamivudine therapy is not caused by mutations in precore and core promoter genes in patients with chronic hepatitis B

HBe antigen (HBeAg) loss or seroconversion can occur during lamivudine therapy. The purpose of this study was to analyze nucleotide sequences in precore and core promoter regions, and examine the influence of mutations in these regions on the disappearance of HBeAg during lamivudine therapy. Serial serum samples were obtained from 51 patients (HBeAg loss in 26 patients) at commencement of therapy (baseline) and after 1 year of lamivudine therapy. Serum samples were amplified with PCR and nucleotide sequences of HBV were analyzed. At baseline, a precore stop codon mutation (A1896) was identified in 8 of 26 HBeAg loss patients and in 8 of 25 HBeAg non-loss patients. At 1 year, precore mutation was observed in 4 of 14 patients analyzed who showed HBeAg loss. At 1 year, however, a precore mutation was observed also in 3 of 9 analyzed patients who showed no HBeAg loss. Core promoter mutations were noted in 21 of 26 HBeAg loss patients and in 20 of 25 HBeAg non-loss patients. At 1 year, core promoter mutations were noted in 11 of 14 HBeAg loss patients and in 8 of 9 HBeAg non-loss patients. Our data suggested that during lamivudine therapy, core promoter and precore mutations do not influence HBeAg loss or seroconversion but may reduce the viral level upon HBeAg loss or seroconversion.     

Genetic heterogeneity in the precore region of hepatitis B virus in hepatitis B e antigen-negative chronic hepatitis B Patients: Spontaneous seroconversion and interferon-induced seroconversion

To elucidate the relationship between the clinical severity of chronic liver disease and the precore mutations in hepatitis B e antigen (HBeAg)-nega-tive hepatitis B virus (HBV) carriers, mutations were investigated in the precore region of HBV DNA in 20 chronic hepatitis B patients who sero-converted either spontaneously or after the administration of α-interferon (IFN), and 5 asymptomatic carriers. The precore mutation with a stop codon at nucleotide 1896 was found in all patients, irrespective of the histology and in all asymptomatic carriers. The second mutation at nucleotide 1899 was found in 40% of cases studied but always followed by the first mutation at nucleotide 1896. The mixed viral infection of precore mutant and wild-type HBV virus was found in 40% of seroconverted cases after IFN treatment and in sera of HBV carriers obtained within a year after the spontaneous Seroconversion. These data suggest that the precore mutants prevail over wild-type HBV in all HBeAg-negative HBV carriers within several years after the sero-conversion, but their prevalence could not confine the clinical severity of chronic liver disease. © 1995 Wiley-Liss, Inc.     

Genotype F prevails in HBV infected patients of Hispanic origin in Central America and may carry the precore stop mutant

The distribution of HBV genotypes and the presence of the precore stop mutation were investigated in HBV strains from Central America. 333 HBsAg positive sera from chronic HBsAg carriers and acute hepatitis B cases from five different countries (Costa Rica, Nicaragua, Honduras, El Salvador and Guatemala) were tested for HBV DNA by nested PCR. Genotyping by limited sequencing within the S gene was performed on 90 strains, 66 from sera with a high level of HBV DNA, and another 24 from sera positive for HBV DNA only after nested PCR. 23 of the samples were anti-HBe positive. Genotype F was found in 71 (79%), A in 13 (14%), D in 5 (6%) and C in one of the 90 sera. 18 patients with genotype F infection had anti-HBe and HBV DNA in serum. Since the three published precore sequences of genotype F strains have a C¹⁸⁵⁸, which is known to prevent the precore stop mutation from G to A at position 1896, the precore and part of the core genes were sequenced from 19 anti-HBe positive sera with HBV DNA, 17 with genotype F and 2 with genotype A. The A¹⁸⁹⁶ mutation was found in 11 of the 17 genotype F strains. All these had a T¹⁸⁵⁸, which was also present in 5 of the 6 genotype F strains with G¹⁸⁹⁶. The precore region was therefore sequenced from genotype F strains from 5 HBeAg positive sera from the five different Central American countries. These also had a T¹⁸⁵⁸, which thus is the wild type substitution in genotype F in Central America. A number of mutations were recorded between residues 57 and 68 in the core protein corresponding to a unique clustering region of the genotype F strains. The predominance of genotype F in Central American populations of Hispanic origin was not anticipated since this genotype is regarded as indigenous to the Amerindian populations of the New World. J. Med. Virol. 51:305–312, 1997. © 1997 Wiley-Liss, Inc.     

Conservation of precore and core sequences of hepatitis B virus in chronic viral carriers

Mutations in the precore region of hepatitis B virus (HBV) have been associated with failure of expression of HBV e-antigen (HBeAg), however, the prevalence of these and other mutations in HBV carriers without overt chronic liver disease remains uncertain. Homosexual or bisexual males (n = 65) with chronic HBV infection attending The Middlesex Hospital, London were studied, of whom two had clinical evidence of chronic liver disease. HBV DMA was amplified from 62 of 65 serum samples using nested and double nested polymerase chain reaction (PCR) assays. Direct sequencing of the PCR products was employed to investigate sequence variation. HBV-DNA from all available HBeAg-negative (n = 9) and selected HBeAg-positive (n = 33) sera were sequenced in the entire precore gene, the 3′ terminal portion of the X gene (aa128–154), and the 5′ terminus of the core gene (aa18–73). Sequences were highly conserved in all regions studied. Samples from two anti-HBe-seropositive patients contained mutations in the precore region. In one, a single mutation in the first amino acid resulted in a change to leucine, which would prevent translation of this region and therefore HBeAg expression. Wild type sequences were also detected in this sample. In the other sample from a patient with overt chronic liver disease, a mutation of precore amino acid 28 changed a tryptophan residue to a stop codon which would also prevent HBeAg expression. Although few such patients were studied, precore mutations may be uncommon in anti-HBe-seropositive patients without overt chronic liver disease. Possibly such mutations are not related to HBeAg to anti-HBe seroconversion, but rather they may arise in patients who remain anti-HBe seropositive for prolonged periods and they may be causally associated with the development of chronic liver disease. © 1994 Wiley-Liss, Inc.     

Analysis of carriers of hepatitis B virus from a tertiary referral hospital: Does the viral load change during the natural course of infection?

This study was designed to determine the prevalence, forms of transmission, mutational profile and viral load at baseline of hepatitis B virus (HBV) carriers in Delhi. HBV surface antigen (HBsAg)-positive patients were enrolled and evaluated clinically for liver function, serological markers for hepatitis B and HBV DNA quantitation. Tests were carried out again 1 year later and the results were compared. Liver biopsy was carried out on all carriers with active viral replication. HBV DNA-positive samples were subjected to polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP) to screen mutations in the Precore, core, and the X-gene prior to sequencing analysis. Among the 100 patients examined, HBeAg was detected in 23% and 40% were HBV DNA-positive. Of the 40 HBV DNA-positive cases, 8 had precore/core mutations, [G1896A (10%), T2066A (12.5%), T2050C (10%), and G1888A (7.5%)]. No X gene mutants were detected. Reduction in viral load was higher in HBeAg-positive patients, as compared to HBeAg-negative patients, over 1 year. At follow-up, 2/8 HBV mutants corresponded with altered liver function and morphological changes suggestive of chronic hepatitis. One patient was re-designated as DNA-negative on follow-up and had wild-type virus infection with a relatively low viral load. The predominant route for HBV transmission was determined to be parenteral. Twenty percent of the HBV carriers were infected with precore and core mutant HBV. Although the clinical and biochemical profiles of these HBV carriers remained largely stable on follow-up, there was evidence of spontaneous reduction in the mean viral load over the 1-year study period.     

Genotypic differences in the hepatitis B virus core promoter and precore sequences during seroconversion from HBeAg to anti-HBe

Hepatitis B virus (HBV) strains from anti-HBe positive patients often show specific mutations in the precore gene, the core promoter region, or both. The dynamics of seroconversion in relation to the appearance of these mutations has not been studied and compared between defined HBV genotypes. Samples from patients followed during seroconversion from HBeAg to anti-HBe were amplified by polymerase chain reaction (PCR), sequenced and genotyped. Among 16 sets of samples, 6 belonged to genotype A, 6 to genotype D, 2 to genotype B, 1 to genotype C, and 1 to genotype E. Whereas strains from genotypes B, C and E showed changes in the core promoter, precore codon 28 or both, genotype A and D strains displayed a different pattern. In 4 of 6 anti-HBe positive samples from genotype A, the precore had a wild-type sequence while the core promoter sequence showed a specific TGA mutation. In another genotype A strain a precore stop mutation was preceded by a mutation in codon 15, thus conserving base-pairing at the pregenomic RNA level in this region. In contrast, all genotype D strains showed wild-type sequences in both the core promoter and precore codon 28 in pre- and post-seroconversion samples. Thus, in 8 patients with a mean follow-up time of 17 months, wild-type sequences in both the core promoter and precore codon 28 were found after seroconversion to anti-HBe. This study also confirmed, for genotype D, that HBeAg seroconversion often occurs earlier than genomic conversion.     

Reactivation of precore variant hepatitis B virus in a child with severe aplastic anaemia

Reactivation of hepatitis B e antigen (HBeAg) negative chronic hepatitis B virus (HBV) infection due to selection of precore variant virus is an uncommon complication of previous hepatitis B infection, and virtually unrecognised in children and adolescents. A child who had received treatment with methylprednisolone and antilymphocyte globulin for severe aplastic anaemia developed high levels of detectable HBV DNA associated with hepatitis B e antibody (anti-HBe) positivity. HBV DNA was extracted, amplified and the core and precore regions sequenced from 2 samples. A mixture of wild-type and the precore variants A(1896) and A(1899) was detected in both samples, with the wild-type predominating in the second sample. Reinfection was excluded by phylogenetic analysis using Phylip and the neighbour-joining method. Precore variant Hepatitis B virus can be transmitted to children as a primary infection, and it is important that aggressive liver disease, particularly in the presence of the anti-HBe phenotype, be investigated. Further studies are needed to determine the frequency of these variants.     

Evolution of wild-type and precore mutant HBV infection after liver transplantation

Recurrence of hepatitis B virus (HBV) infection after liver transplantation is associated with varying degree of graft damage. The aim of the study was to investigate longitudinally the changes of wild-type and precore A₁₈₉₆HBV mutant viral populations after reinfection and their impact on liver graft damage. The wild-type HBV and A₁₈₉₆HBV strains were quantitated before and serially after orthotopic liver transplantation (OLT) in 14 hepatitis B surface antigen (HBsAg)-positive liver graft recipients (4 hepatitis B e antigen [HBeAg]+; 10 anti-HBe+). Before OLT, the wild-type precore HBV was present in all 4 HBeAg-positive patients and in 2/10 anti-HBe-positive patients; a mixed virus population was present in 6 patients; and A₁₈₉₆HBV mutant alone in 2 patients. After OLT, A₁₈₉₆HBV mutant appeared and gradually accumulated in 5/6 patients who had the wild-type HBV before OLT and 1 of these patients seroconverted from HBeAg to anti-HBe 52 months after transplantation. A mixed HBV population was present continuously in 6 patients before and after OLT. Of the 2 patients with A₁₈₉₆HBV only pre-OLT, the wild type appeared in one patient and the other patient retained persistently the A₁₈₉₆HBV mutant. There was no relationship between liver graft histology and the type of viral population at reinfection or at the end of follow up. Changes in the HBV population occur during follow up of recurrent hepatitis B in liver transplant recipients with frequent accumulation of precore A₁₈₉₆HBV mutants, but the type of viral population does not determine the severity of hepatitis B in the graft. J. Med. Virol. 59:5–13, 1999. © 1999 Wiley-Liss, Inc.     

Two distinct types of hepatitis B virus core promoter variants in Yemeni blood donors

Genetic variations in the basic core promoter (BCP) region of hepatitis B virus (HBV) occur during the natural history of chronic HBV infection. This study investigates the presence of basic core promoter variations in 28 asymptomatic Yemeni blood donors, correlating variations with HBeAg phenotype and viral load. The core promoter/precore and surface gene region of HBV DNA were amplified using nested PCRs and the PCR products were sequenced either directly or after cloning. HBeAg and viral load were measured when HBV DNA was detectable. Sequencing of 18 surface PCR products indicated that all were of genotype D. Two distinct types of variants were identified in the basic core promoter: substitution only (N = 14) and major deletion (N = 9). The commonest substitutions were located at nucleotide positions 1753, 1762, and 1764; 10/14 (71.4%) were associated with the precore 1896A substitution resulting in the premature stop of the precore reading frame and 6/14 (42.9%) had viral loads above 400 copies/ml. Two forms of deletion variants were found: 8 bp deletion (1763-1770) (N = 2) and a novel 12 (1746-1757) + 8 bp (1763-1770) deletion (N = 7). The deletion sequences were never associated with the precore 1896A substitution and all had viral load below 400 copies/ml with negative HBeAg phenotype. The polymorphism 1773C was found in 9/14 (64.3%) substitution sequences whereas all deletion sequences had 1773T. Two donors had mixed sequences of basic core promoter substitution and major deletions (both 8 bp and 12 + 8 bp). While the deletion variants in these two donors were similar to others found in isolation, the substitutions were of a different pattern. Further studies are required to understand the selection process behind these variants.