Lars O. Magnius

Genetic Diversity of Hepatitis B Virus Strains Derived Worldwide: Genotypes, Subgenotypes, and HBsAg Subtypes

Sequences of 234 complete genomes and 631 hepatitis B surface antigen genes were used to assess the worldwide diversity of hepatitis B virus (HBV). Apart from the described two subgenotypes each for A and F, also B, C, and D divided into four subgenotypes each in the analysis of complete genomes supported by significant bootstrap values. The subgenotypes of B and C differed in their geographical distribution, with B1 dominating in Japan, B2 in China and Vietnam, B3 confined to Indonesia, and B4 confined to Vietnam, all strains specifying subtype ayw1. Subgenotype C1 was common in Japan, Korea, and China; C2 in China, South-East Asia, and Bangladesh, and C3 in the Oceania comprising strains specifying adrq–, and C4 specifying ayw3 is encountered in Aborigines from Australia. This pattern of defined geographical distribution was less evident for D1–D4, where the subgenotypes were widely spread in Europe, Africa, and Asia, possibly due to their divergence having occurred a longer time ago than for genotypes B and C, with D4 being the first split and still the dominating subgenotype of D in the Oceania. The genetic diversity of HBV and the geographical distribution of its subgenotypes provide a tool to reconstruct the evolutionary history of HBV and may help to complement genetic data in the understanding of the evolution and past migrations of man.

Genotype H: a new Amerindian genotype of hepatitis B virus revealed in Central America

The complete genomes were sequenced for ten hepatitis B virus (HBV) strains. Two of them, from Spain and Sweden, were most similar to genotype D, although encoding d specificity. Five of them were from Central America and belonged to genotype F. Two strains from Nicaragua and one from Los Angeles, USA, showed divergences of 3.1-4.1% within the small S gene from genotype F strains and were recognized previously as a divergent clade within genotype F. The complete genomes of the two genotype D strains were found to differ from published genotype D strains by 2.8-4.6%. Their S genes encoded Lys(122), Thr(127) and Lys(160), corresponding to the putative new subtype adw3 within this genotype, previously known to specify ayw2, ayw3 or, rarely, ayw4. The complete genomes of the three divergent strains diverged by 0.8-2.5% from each other, 7.2-10.2% from genotype F strains and 13.2-15.7% from other HBV strains. Since pairwise comparisons of 82 complete HBV genomes of intratypic and intertypic divergences ranged from 0.1 to 7.4% and 6.8 to 17.1%, respectively, the three sequenced strains should represent a new HBV genotype, for which the designation H is proposed. In the polymerase region, the three strains had 16 unique conserved amino acid residues not present in genotype F strains. So far, genotype H has been encountered in Nicaragua, Mexico and California. Phylogenetic analysis of the complete genomes and subgenomes of the three strains showed them clustering with genotype F but forming a separate branch supported by 100% bootstrap. Being most similar to genotype F, known to be an Amerindian genotype, genotype H has most likely split off from genotype F within the New World.

Subtypes, Genotypes and Molecular Epidemiology of the Hepatitis B Virus as Reflected by Sequence Variability of the S-Gene

The serologic heterogeneity of the hepatitis B virus (HBV) has been established from immunodiffusion experiments for a long time. Four serotypes called subtypes of the hepatitis B surface antigen (HBsAg) have been defined by two mutually exclusive determinant pairs, d/y and w/r, and a common determinant a. These subtypes are adw, ayw, adr and ayr. By subdivision of the four major subtypes in the mid-70s, nine different subtypes were identified. Sequencing of viral genomes has now become a major goal of descriptive virology, and sequence data is now used to trace routes of infection, to reconstruct the phylogenetic history of viruses and two delimit genetic subtypes. A genetic classification based on the comparison of complete genomes has defined four genomic groups of HBV, later referred upon as genotypes, which were designated with A-D. However, the interrelation of the nine subtypes to the genotypes, the possible presence of more than four human HBV genotypes as well as their global geographical prevalence remained to be determined. By sequencing the S-gene of HBV the molecular basis was assessed for the serological variations of HBsAg within the major four subtypes. Thereby, also two new genotypes of HBV designated with E and F were identified. Complete genomic sequencing of E and F strains confirmed their status as new genotypes. The F genotype was found to diverge from other HBV genomes sequenced by 14%, thus being the most divergent HBV genome so far characterized. When the worldwide molecular epidemiology of HBV based on the variability of the S-gene was defined, the E and F strains seemed to originate in aboriginal populations of Africa and the New World, respectively. They shared a unique substitution at residue 140 in the second immunodominant loop of their encoded surface antigen when compared to the vaccine strain. Future research will establish whether this substitution may predispose to a vaccine escape mutant at residue 141, that now has been reported to occur in conjunction with the 140 substitution.

COMPARISON OF THE AMINO ACID SEQUENCES OF NINE DIFFERENT SEROTYPES OF HEPATITIS B SURFACE ANTIGEN AND GENOMIC CLASSIFICATION OF THE CORRESPONDING HEPATITIS B VIRUS STRAINS

The surface (S) genes of 12 hepatitis B viruses (HBVs) encoding nine different serotypes of hepatitis B surface antigen (HBsAg) were amplified by the polymerase chain reaction and sequenced. These represented the eight strains of HBV, P1 to P8, defined at an international workshop on HBsAg subtypes in Paris in 1975, and the adrq- subtype. The S genes from additional HBV strains, one ayw4, one adw4 and one ayw1, of sub-Saharan African origin, were also sequenced. The relationship of these 12 new S gene sequences to those of the 20 published previously was investigated by constructing a phylogenetic tree, which confirmed a previous classification into four groups, designated A to D, based on 18 complete HBV genomes. When relating our sequenced S genes to these genomic groups, ayw1 of African origin and P6 (adw2) were both allocated to group A, the reference P1 (ayw1 of Vietnamese origin) was allocated to group B, P5 (ayr), P8 (adr) and adrq- were all related to group C, and P2 (ayw2) and P3 (ayw3) could both be allocated to group D. Interestingly, the S genes of w4 serotype viruses, i.e. P4 (ayw4) and P7 (adw4q-), differed by 4% or more from both previous groups and from each other, suggesting their classification into two new groups, for which the designations E and F are proposed. Genomes specifying ayw were also found in groups A and B; previously sequenced genomes specifying the ayw subtype have all been confined to group D. There were indications that the epitope for subdeterminants of w resided at amino acid positions 125 to 127. Thus, at positions 125 and 127, ayw1, ayw2 and adw2 had T and P residues, respectively, whereas M and T residues were at the corresponding positions of ayw3. Both ayw4 and adw4 had L at residue 127, and all strains expressing r, apart from P5, had an I instead of a T residue at position 126.

Genetic relatedness of hepatitis B viral strains of diverse geographical origin and natural variations in the primary structure of the surface antigen

A 681 nucleotide fragment of the hepatitis B virus (HBV) genome was sequenced that corresponded to the complete gene for hepatitis B surface antigen (HBsAg) in 80 HBsAg- and hepatitis B e antigen (HBeAg)-positive sera of diverse geographical origins. These and 42 previously published HBV sequences within the S gene were used for the construction of a dendrogram. In this comparison, each of the 122 HBsAg genes was found to be related to one or other of the six previously identified genomic groups of HBV, A to F. The HBV strains within each genomic group showed a characteristic geographical distribution. Group A genomes were represented by 23 strains mainly originating in northern Europe and sub-Saharan Africa. The group B and C genomes, represented by 17 and 28 strains respectively, were confined to populations with origins in eastern Asia and the Far East. The group D genomes, represented by 38 strains, were found worldwide, but were the predominant strains in the Mediterranean area, the Near and Middle East, and in south Asia. Group E genomes, represented by nine strains, were indigenous to western sub-Saharan Africa as far south as Angola. There were indications that the F group, made up of six strains, represented the genomic group of HBV among populations with origins in the New World. Thus, HBV has diverged into genomic groups according to the distribution of mankind in the different continents. As well as giving information on the genetic relationship of HBV strains of different geographical origin, this study also provides information on the primary structure of HBsAg in different regions of the world. Such data might prove valuable in explaining the reported failures to obtain protection with current HBV vaccines.

A Natural Intergenotypic Recombinant of Hepatitis C Virus Identified in St. Petersburg

ABSTRACT Hepatitis C virus (HCV) evolution is thought to proceed by mutations within the six genotypes. Here, we report on a viable spontaneous HCV recombinant and we show that recombination may play a role in the evolution of this virus. Previously, 149 HCV strains from St. Petersburg had been subtyped by limited sequencing within the NS5B region. In the present study, the core regions of 41 of these strains were sequenced to investigate the concordance of HCV genotyping for these two genomic regions. Two phylogenetically related HCV strains were found to belong to different subtypes, 2k and 1b, according to sequence analysis of the 5′ untranslated region (5′UTR)-core and the NS5B regions, respectively. By sequencing of the E2-p7-NS2 region, the crossover point was mapped within the NS2 region, probably between positions 3175 and 3176 (according to the numbering system for strain pj6CF). Sequencing of the 5′UTR-core regions of four other HCV strains, phylogenetically related to the above-mentioned two strains (based on analysis within the NS5B region), revealed that these four strains were also recombinants. Since a nonrecombinant 2k strain was found in St. Petersburg, the recombination may have taken place there around a decade ago. Since the frequency of this recombinant is now high enough to allow the detection of the recombinant in a fraction of the citys population, it seems to be actively spreading there. The reported recombinant is tentatively designated RF1_2k/1b, in agreement with the nomenclature used for HIV recombinants. Recombination between HCV genotypes must now be considered in the classification, laboratory diagnosis, and treatment of HCV infection.

Molecular basis of hepatitis B virus serotype variations within the four major subtypes.

Amino acid residues 101 to 180 of hepatitis B surface antigen (HBsAg) were predicted by sequencing the corresponding part of the S gene of hepatitis B virus (HBV) DNA in 46 HBsAg-positive sera, which had been subtyped by immunodiffusion with respect to d/y, w/r, w1 to w4 and q. The sequences of the nine different HBV serotypes defined by these specificities were found to be homogeneous proving that they represent consistent variations of HBV at the genomic level. Residue 127 was found to be important as were Pro, Thr and Leu for w1/w2, w3 and w4, respectively. Five residues were found to differ between ayw1 and ayw2. These were at positions 134 (Phe instead of Tyr), 143 (Thr instead of Ser), 159 (Ala instead of Gly), 161 (Tyr instead of Phe) and 168 (Val instead of Ala). However, all these residues were shared by ayw1 and adw2, implying that Arg122 was also important for w1 expression. All genomes expressing r, apart from one ayr strain, had an Ile126, which might explain the pseudo-allelism of w1 to w4 in relation to r, since this substitution might influence the w epitope. There were two regions where adw4q- and adrq- differed from all the q+ subtypes. These were located at residues 158 and 159, and at residues 177 and 178, where both the q- subtypes had amino acid substitutions in adjacent positions. The mapping of the epitopes defining these antigenic specificities will help to link information on the world-wide distribution of HBsAg subtypes to future molecular epidemiology with regard to HBV.

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.

Rapid "tea-bag" peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids applied for antigenic mapping of viral proteins.

The role of individual amino acids in binding human and macaque antibodies were determined in the human immunodeficiency virus type 1 (HIV-1) gp41, residues 594-613, and for human antibodies in the hepatitis B (HB) virus core/e antigens (HBc/eAg), residues 121-140. Decapeptides with 9 amino acids (aa) overlap were synthesised using a rapid method for simultaneous multiple peptide synthesis with 9-fluorenylmethoxycarbonyl (Fmoc) protection for the alpha-amino group of the aas. One coupling cycle including washing steps was performed within 60-90 min. The crude products were analysed by reversed-phase HPLC and PD-mass spectrometry. With the 11 decapeptides covering residues 594-613 of HIV-1 gp41, the sequences SGKLI at aa 599-603 was found to be the main recognition site for 19 human anti-HIV positive sera. Two macaques repeatedly immunized with a peptide covering aa 594-613 of gp41, preferentially recognised the sequence CTTAVPW at residues 604-610 after 1-2 months of immunisation. One macaque also recognised the sequence CSGKLI, with sera sampled greater than 10 months after start of immunisation. Out of 9 human sera from patients with chronic HB, and reactive to a peptide covering residues 121-140 of HBc/eAg, 8 were found to recognise the sequence TPPA at residues 128-131, with an individual variation within residues 125-133 in regard to N- and C-terminal ends of the recognised antigenic site. Thus, human recognition of this antigenic site overlaps the reported T- and the B-cell recognition site found in mice. We believe that this simple and rapid approach to obtain large numbers of immunologically active peptides can be useful for most laboratories interested in the immunological characterisation of proteins.

Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion

Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel‐ and cone‐like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC‐associated protein Tpr and its large coiled coil‐forming domain. RNAi‐mediated loss of Tpr allowed condensing chromatin to occur all along the NEs nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub‐compartment and delimiting heterochromatin distribution.