Karin Kidd-Ljunggren

Occult hepatitis B virus after acute self-limited infection persisting for 30 years without sequence variation

BACKGROUND/AIMS After acute self-limited hepatitis B virus (HBV) infection, serological loss of viral antigens and appearance of anti-HBs is generally believed to signify viral clearance. Latent and occult HBV infection appearing decades after self-limited hepatitis B has not been reported, nor has the evolutionary rate of HBV DNA over the same observation period. METHODS DNA from serum and leukocytes from 16 patients with acute self-limited hepatitis B 30 years earlier was tested by polymerase chain reaction and positive samples were sequenced. Liver tissue from four patients was also tested. Additionally, another 10 HBV strains isolated from acute HBV cases in 1969-72 were compared to 11 strains isolated from acute cases in 1998-99 in the same community. RESULTS HBV DNA was detected in liver from two patients, but not in serum or leukocytes. The HBV strains detected in liver showed complete homology, in the sequences analyzed, to the strains originally infecting these patients. Ten strains from 1998-99 were identical in pre-S and core promoter/precore regions to strains from the same community isolated 30 years earlier. CONCLUSIONS HBV can persist as an occult infection three decades after acute, apparently self-limited hepatitis B, and both the mutation and evolutionary rates of HBV DNA are low.

The hepatitis B virus X gene: analysis of functional domain variation and gene phylogeny using multiple sequences

The hepatitis B virus (HBV) X gene shares sequences with both the polymerase and precore genes, carries several regulatory signals critical to the replicative cycle, and its product has a transactivating function. In this study, the X gene sequences of 29 HBV strains from 14 different countries were characterized and compared to all corresponding databank sequences where the origin of the strain was stated. The X gene and its product are relatively well conserved. However, several rare or unique point mutations in the predicted X protein are described which further define regions on the primary sequence which may be of structural and/or functional significance. Phylogenetic analysis of the 29 X genes and their predicted proteins in this study using unrooted trees indicates that a common ancestral sequence gave rise to two main groups of X genes, represented by HBV strains found predominantly either in the Western or Eastern Hemisphere. In turn, each of these two main groups of sequences appear to have branched into two main lineages. Introduction of 33 additional DNA sequences from the databank has further verified these inferences and confirmed the groupings as previously described subgroups A to D. Whilst the split of X gene lineages into subgroups A and D seems feasible on geographical/anthropological grounds, the corresponding split of Eastern Hemisphere lineages into B and C may require an alternative hypothesis. Additionally, there was a correlation between the HBeAg/anti-HBeAg status of our patients and nucleotide identity at two positions in the core promoter, 52 and 50 bases upstream from the precore start codon. This finding, also shown recently by others, suggests that control of HBeAg secretion may involve mutations affecting transcription and not only precore/core translation.

Clinical and Serological Variation between Patients Infected with Different Hepatitis B Virus Genotypes

ABSTRACT Hepatitis B virus (HBV) has eight genotypes which have distinct geographical distributions. Studies comparing differences in the clinical outcomes of infections caused by strains with genotype-related variations in the HBV genome have largely compared genotypes B and C and genotypes A and D but not all four genotypes. The present study included 196 HBV-infected patients attending an infectious diseases outpatient clinic in Sweden. The age and geographic origin, liver function, HBeAg and anti-HBe status, and the presence or absence of HBV DNA were analyzed for each patient. HBV DNA was detected in 144 patients, and the HBV genotype and the core promoter and precore sequences were determined for the isolates from 101 of these patients. Among the patients who might be considered most likely to be nonviremic, namely, anti-HBe-positive HBV carriers with normal alanine aminotransferase (ALT) levels, 65% had detectable HBV DNA and were thus viremic. Among the viremic patients, HBeAg-positive patients were more likely to have elevated ALT levels than anti-HBe-positive patients. HBV genotypes A to F were represented in the study, and their distributions coincided accurately with the origin of the patient. A significantly higher number of genotype D-infected patients were anti-HBe positive and had elevated ALT levels (42% of genotype D-infected patients but 0% of patients infected with genotypes B and C). Genotype D strains with mutations in the core promoter and precore regions were significantly correlated with elevated ALT levels in the patients. The differences were not age related. Therefore, in this large-scale cross-sectional study, genotype D appears to be associated with more active disease.

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. J. Med. Virol. 60:107–112, 2000.

Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal.

Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5′ end of the RNA pregenome. Epsilon contains an apical stem–loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem–loop based on NOE, RDC and 1H chemical shift NMR data. The 1H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5′-CUGUGC-3′ folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.

Pro- and anti-inflammatory substances modulate expression of the leukotriene B4 receptor, BLT1, in human monocytes.

The high‐affinity leukotriene B4 (LTB4) receptor, BLT1, is a chemotactic receptor involved in inflammatory responses. In this study, we have explored the regulation of BLT1 expression in human monocytes by pro‐ and anti‐inflammatory cytokines, lipopolysaccharide (LPS), and dexamethasone. We found that proinflammatory mediators, such as interferon‐γ (IFN‐γ), tumor necrosis factor‐α, and LPS, down‐regulated expression, whereas the anti‐inflammatory cytokine, interleukin‐10, and dexamethasone up‐regulated BLT1 mRNA expression. The effect of IFN‐γ on BLT1 mRNA expression was rapidly detectable (<4 h) and concentration‐dependent (1–50 ng/ml) and seems to be exerted through a block in transcriptional activity. Alterations in mRNA expression were accompanied by changes in BLT1 surface expression, and receptor down‐modulation following IFN‐γ stimulation resulted in a diminished chemotactic response to LTB4. The regulation of BLT1 mRNA and receptor protein expression was similar to the regulation of the monocyte chemoattractant protein‐1 chemokine receptor, CC chemokine recptor 2 (CCR2). Flow cytometric analysis of fresh peripheral blood cells revealed that classical (CD14++CD16–) monocytes express high levels of BLT1 and CCR2 and that both receptors are down‐regulated on CD14+CD16+ monocytes. Apart from providing insight into the regulation of BLT1 in human monocytes, our results reveal a parallel expression and regulation of BLT1 and CCR2, which may help to understand monocyte trafficking during pathophysiological conditions.

Quantitative assessment of the antiviral potencies of 21 shRNA vectors targeting conserved, including structured, hepatitis B virus sites

BACKGROUND & AIMS RNA interference (RNAi) may offer new treatment options for chronic hepatitis B. Replicating via an RNA intermediate, hepatitis B virus (HBV) is known to be principally vulnerable to RNAi. However, beyond delivery, the relevant issues of potential off-target effects, target site conservation in circulating HBV strains, and efficacy of RNAi itself have not systematically been addressed, nor can the different existing data be quantitatively compared. The aim of this study was to provide such information. METHODS To focus on the intracellular RNAi process itself and minimise other variables affecting overall RNAi efficacy, we used a robust co-transfection system to quantitatively assess the relative potencies of 21 small-hairpin (sh) RNA vectors, targeting conserved sites throughout the HBV genome, against viral RNAs, proteins, nucleocapsids, and secreted virions under standardised conditions. RESULTS The approach enabled a distinct efficacy ranking, with the six most potent shRNAs achieving 95% reductions in virion formation, sequence-specifically and without detectable interferon induction, yet by differentially affecting different steps. Efficacy correlated poorly with predictions and was not principally abolished by target structure. Sequence comparisons suggest that truly conserved, RNAi-targetable sequences comprise less than 500 nucleotides of the circulating HBV genomes. CONCLUSIONS The HBV genome can harbour only a finite number of optimal target sites, but current predictions are poorly suited to constrain the number of possible candidates. However, the small size of the highly conserved sequence space suggests experimental identification as a viable option.

Urine from chronic hepatitis B virus carriers: Implications for infectivity

Horizontal transmission of hepatitis B virus (HBV) without apparent sexual or parenteral exposure is common in hyperendemic areas. In most cases, the route of transmission is unknown. To investigate urine as a potential source of infection, serum and urine from 56 chronic hepatitis B surface antigen (HBsAg) carriers were examined for the presence of HBV DNA using the polymerase chain reaction (PCR). Thirty‐four of the patients were anti‐hepatitis B e antigen (anti‐HBe) positive and 22 were hepatitis B e antigen (HBeAg) positive. HBV DNA was detected in serum from 46 patients (82%) and in urine from 28 patients (50%). Most HBeAg‐positive patients had HBV DNA detectable in urine (91%), whereas urine samples from anti‐HBe‐positive patients were found to contain HBV DNA to a lesser extent (24%). When comparing HBV DNA from serum and urine by an end‐point titration PCR, a titration difference averaging 103 was found between serum and urine. A significant female predominance was also noted among the positive urine samples (P < 0.05), which was not correlated to the presence of haematuria. Detection of HBV DNA may indicate active viral replication, and thereby infectivity. Because a high proportion of chronic HBV carriers were found to have HBV DNA in urine, it is suggested that irrespective of HBeAg/anti‐HBe status, urine should be regarded as a potential route of transmission and therefore be investigated further as a means of horizontal and nosocomial transmission of HBV. J. Med. Virol. 60:17–20, 2000.

The Hepatitis B Virus Pregenome: Prediction of RNA Structure and Implications for the Emergence of Deletions

The terminally redundant pregenomic RNA of human hepatitis B virus (HBV) comprises some 3,330 nucleotides and is a replicative intermediate in the production of the circular DNA genome. Deletions are known to arise in the HBV genome during the course of chronic infection and are sometimes associated with interferon therapy. These deletions are limited to small parts of the genome such as the 357-nucleotide pre-S1 region. Long RNA molecules such as the HBV pregenome have considerable structural flexibility and will undergo secondary structure shifts between energetically favourable states in a continuous and semi-random fashion. Since prediction of structure elements that are highly conserved in different forms of one RNA molecule is now feasible by computer modelling, we have analysed the whole HBV pregenome by two different RNA structure prediction algorithms and by new methods that exploit these algorithms. Significantly, the ends of pregenomic RNA were predicted to undergo both short-range and long-range interactions, which has relevance to our knowledge of the virus replicative cycle. By incorporating phylogenetic information relating to the 6 recognised genotypes of HBV, it was possible to highlight short secondary structures that may be common to all HBV strains. For example, although the pre-S1 region was predicted to undergo local folding of a loosely defined nature, most observed pre-S1 deletions mapped to all or part of an arm carrying a better-defined structure. The loss of such sequences may be mechanistically attributable to polymerase skipping during reverse transcription, and the possible advantages of such deletions are considered.

Genetic conservation within subtypes in the hepatitis B virus pre-S2 region

The antigenic determinants for the main hepatitis B virus (HBV) subtypes adw, adr, ayw and ayr lie in the S (surface) polypeptide. Two amino acid residues in particular, encoded by the S gene at codon positions 122 and 160, have been postulated to determine the different antigenic subtypes. In contrast, the 165 nucleotide pre-S2 gene encodes an immunodominant region common to all subtypes that can give rise to neutralizing antibodies. We have characterized the pre-S2 gene sequences of 29 HBV strains of the three main subtypes, adw, ayw and adr. Seven base positions showed variation that was entirely subtype-specific, with six of these variations leading to subtype-specific amino acid differences. This finding affords the possibility of using pre-S2 sequences for genetic subtyping. Two ayw strains from unrelated patients infected in the Middle East had identical pre-S2 sequences with a block of 12 nucleotides deleted. A geographical correlation with subtype observed from serological results was also apparent from phylogenetic analysis of DNA identities within the pre-S2 region. The results support the concept that the main HBV subtypes truly represent families of phylogenetically different strains.