Peculiarities in the designations of hepatitis B virus genes, their products, and their antigenic specificities: a potential source of misunderstandings.

The nomenclature of the hepatitis B virus (HBV) genes and their products has developed stepwise, occasionally in an erratic way, creating many misunderstandings, especially among those who do not know the structure of HBV and its genome in detail. One of the most frequent misunderstandings, even presented in leading journals, is the designation of HBV "e"-antigen as envelope or early antigen. Another problem area are the so-called "pre" regions in the HBV genome present upstream of both the core and the surface genes of HBV, inadvertently suggesting that they may be a part of corresponding precursor proteins. Misnomers and misclassifications are frequent in defining the subgenotypes and serological subtypes of HBV. Even the well-established terminology for HBV surface (HBs) or HBV core (HBc) antigen deviates from the conventional virological nomenclature for viral envelopes or capsid proteins/antigens, respectively. Another matter of undesirable variability between publications is the numbering of the nucleotides and the graphical representation of genomic maps. This editorial briefly explains how the nomenclature evolved, what it really means, and suggests how it could be adapted to today's knowledge.

Hepatitis E Virus Genotype 3 Genomes from RNA-Positive but Serologically Negative Plasma Donors Have CUG as the Start Codon for ORF3.

Hepatitis E virus (HEV) is a pathogen that causes hepatitis worldwide. Molecular studies have identified HEV RNA in blood products although its significance is not understood. This study was undertaken to characterize HEV genomes in asymptomatic plasma donors from Sweden and Germany lacking anti-HEV. Complete open reading frames (ORFs) were obtained from HEV strains in 5 out of 18 plasma donors who tested positive for HEV RNA. All strains had CUG as the start codon of ORF3, while 147 GenBank strains all had AUG as the start codon (p < 0.0001). This substitution was found in both interrelated and unrelated strains belonging to different phylogenetic clades. The HEV strains from the seronegative plasma donors had no other substitution in common, which may be why the CUG substitution seems to explain the seronegativity.

Genetic Diversity and Possible Origins of the Hepatitis B Virus in Siberian Natives.

A total of 381 hepatitis B virus (HBV) DNA sequences collected from nine groups of Siberian native populations were phylogenetically analyzed along with 179 HBV strains sampled in different urban populations of former western USSR republics and 50 strains from Central Asian republics and Mongolia. Different HBV subgenotypes predominated in various native Siberian populations. Subgenotype D1 was dominant in Altaian Kazakhs (100%), Tuvans (100%), and Teleuts (100%) of southern Siberia as well as in Dolgans and Nganasans (69%), who inhabit the polar Taimyr Peninsula. D2 was the most prevalent subgenotype in the combined group of Nenets, Komi, and Khants of the northern Yamalo-Nenets Autonomous Region (71%) and in Yakuts (36%) from northeastern Siberia. D3 was the main subgenotype in South Altaians (76%) and Buryats (40%) of southeastern Siberia, and in Chukchi (51%) of the Russian Far East. Subgenotype C2 was found in Taimyr (19%) and Chukchi (27%), while subgenotype A2 was common in Yakuts (33%). In contrast, D2 was dominant (56%) in urban populations of the former western USSR, and D1 (62%) in Central Asian republics and Mongolia. Statistical analysis demonstrated that the studied groups are epidemiologically isolated from each other and might have contracted HBV from different sources during the settlement of Siberia.

Thalassemia Patients from Baluchistan in Pakistan Are Infected with Multiple Hepatitis B or C Virus Strains.

There are an estimated 2,000 children with ß-thalassemia in the province Baluchistan of Pakistan. These children are at high risk of acquiring transfusion-transmitted infections (TTIs) due to their need of regular blood transfusions for survival. Therefore, we investigated the frequencies of TTIs among these multi-transfused patients in a region where the WHO guidelines for blood safety are not always followed. Sera from 400 children (mean age 7.7 ± 4.70 years) treated at two thalassemia centers in Baluchistan were investigated for TTIs. Eleven (2.8%) were hepatitis B surface antigen positive, and 72 (18.3%) had anti-hepatitis C virus (HCV), two of which were infected with both viruses. Only 22% of the children had been reached by the program for universal hepatitis B virus (HBV) vaccination which started in 2004. Half (51%) of the HCV infected had also been HBV infected. The HBV- and HCV-infected patients were older and had received more blood transfusions than the uninfected patients (P < 0.001). Molecular characterization of the viral strains revealed the presence of several genetically different strains in at least three HBV- and seven HCV-infected children. This is the first study to demonstrate infections with multiple HBV or HCV strains simultaneously infecting thalassemia patients. These may become the source for new emerging recombinant viruses of unknown virulence. The high prevalence of anti-HCV-positive children, and the presence of HBV infections among children who should have been vaccinated, highlights an urgent need for improvements of blood safety in this region of Pakistan.

CTL escape mutations of core protein are more frequent in strains of HBeAg negative patients with low levels of HBV DNA.

BACKGROUND: Recent studies have suggested that Cytotoxic T lymphocytes (CTL) play a key role in eliminating hepatitis B virus (HBV). OBJECTIVES: We aimed to investigate the role of mutations in different immune epitopes of hepatitis B core antigen (HBcAg) among Iranians with hepatitis B e antigen negative chronic hepatitis B (e-CHB), and asymptomatic carriers (ASCs). STUDY DESIGN: Amino acids 1-150 of HBcAg were characterized for HBV strains from 29 e-CHB patients and 48 ASCs from Iran. All patients were infected with HBV genotype D and had previously been investigated for the presence of pre-core and basic core promoter (BCP) mutants. RESULTS: Amino acid mutations of core protein were observed more frequently in HBV strains from ASCs than e-CHB patients (p=0.014). Asn(67) mutation was mutually exclusive to the combination Ile(66) and Ser(69) (P<0.001). Substitutions for Ser(21) and Thr12Ser were associated with lower serum levels of HBV DNA (p<0.001). None of the patients with mutations in HLA-A2 CTL epitope, 18-27, had serum HBV DNA more than 10(5)copies/mL (p<0.001). By multivariate analysis, high level (>10(5)copies/mL) of serum HBV DNA was inversely associated with the presence of mutations in CTL epitopes of HBc (OR: 0.11, p=0.015), while it was directly associated with the presence of promoter double T(1762)A(1764) mutations together with G(1757) (OR: 16.87, p=0.004). CONCLUSION: The inverse correlation between serum levels of HBV DNA and CTL escape mutations of the core protein in HBeAg seroconverted patients, supports the notion that selection of CTL escape mutations consolidates the persistence of HBV infection despite reducing viral fitness.

Enterovirus 94, a proposed new serotype in human enterovirus species D.

The genus Enterovirus (family Picornaviridae) contains five species with strains isolated from humans: Human enterovirus A (HEV-A), HEV-B, HEV-C, HEV-D and Poliovirus. In this study, a proposed new serotype of HEV-D was characterized. Four virus strains were isolated from sewage in Egypt and one strain from acute flaccid paralysis cases in the Democratic Republic of the Congo. The complete genome of one environmental isolate, the complete coding sequence of one clinical isolate and complete VP1 regions from the other isolates were sequenced. These isolates had 66.6-69.4% nucleotide similarity and 74.7-76.6% amino acid sequence similarity in the VP1 region with the closest enterovirus serotype, enterovirus 70 (EV70), suggesting that the isolates form a new enterovirus type, tentatively designated enterovirus 94 (EV94). Phylogenetic analyses including sequences of the 5' UTR, VP1 and 3D regions demonstrated that EV94 isolates formed a monophyletic group within the species HEV-D. No evidence of recombination was found between EV94 and the other HEV-D serotypes, EV68 and EV70. Further biological characterization showed that EV94 was acid stable and had a wide cell tropism in vitro. Attempts to prevent replication with protective antibodies to known enterovirus receptors (poliovirus receptor, vitronectin alphavbeta3 receptor and decay accelerating factor) were not successful. Seroprevalence studies in the Finnish population revealed a high prevalence of this virus over the past two decades.

Genetic characterization of hepatitis C virus strains in Estonia: fluctuations in the predominating subtype with time.

During the last decade, there has been a dramatic increase in intravenous drug use in young adults in Estonia with an increased incidence of both hepatitis B and C as a consequence. Since genetic data are limited regarding hepatitis C virus (HCV) strains in Estonia, the aim of the study was to characterize HCV strains in different risk groups to determine their relatedness to strains from other geographical regions. Three hundred fifty-three anti-HCV positive sera collected during 1994-2004 from hospitalized patients, blood donors and health care workers were used as source of HCV RNA. Two hundred nine (59%) of the sera were positive for HCV RNA by PCR directed to the 5'-UTR region. For 174 strains the HCV subtype was determined by analyses of the NS5B and/or the 5'UTR-core regions. 1b (71%) was the most common subtype followed by 3a (24%), 2c (2%), 1a (1%), and 2a (1%). The 1b and 3a strains were similar to strains from other regions of the former USSR. Within genotype 1b there were several HCV lineages. However, for 3a there seemed to be two separate introductions into Estonia. There was a relative shift from subtype 1b to 3a in 1999-2000 with a further replacement of 3a with 1b in intravenous drug users in 2001 and onwards (P < 0.05). However, both subtypes were found to co-circulate in the community independent of risk factors. One patient was infected with the 2k/1b recombinant presumed to originate from St. Petersburg being the first isolate of this recombinant recovered outside Russia.

New enteroviruses, EV-93 and EV-94, associated with acute flaccid paralysis in the Democratic Republic of the Congo.

Surveillance of acute flaccid paralysis often identifies enteroviruses not typeable by virus neutralization in cell culture. During 2000 and 2001, 186 isolates from 138 children with acute flaccid paralysis in the Democratic Republic of the Congo were sent for typing to the National Reference Centre for Enteroviruses in Lyon, France. The 5' UTR of the viral genome could be amplified by PCR for 158 isolates from 114 patients. Isolates from 89 patients were neutralizable, and contained non-polio enterovirus types. Seventeen children were infected with more than one entero- or adenovirus; another three were co-infected with both these viruses. Serological typing failed with 19 isolates from 13 (9%) patients. The VP1 region of these strains could be amplified by PCR and sequenced, which revealed that five children were infected with CV-A17, EV-70, EV-76, EV-77, or CV-A13. Two patients were doubly infected, one with CV-A24 and E-9, and another with E-27 and EV-81. Isolates from six children contained strains with divergent VP1 region. The amino acid sequences of these complete VP1 regions diverged >or=28% from published types indicating that they represented two new enterovirus types, tentatively designated EV-93 belonging to HEV-B and EV-94 within HEV-D. The latter enterovirus has in parallel been isolated from sewage in Egypt. In conclusion, there was a high frequency of "untypable" enterovirus isolates from cases with acute flaccid paralysis in the Democratic Republic of the Congo. Six of these were shown to represent two enteroviruses not previously described.

Characterization of HCV strains in an oncohematological pediatric department reveals little horizontal transmission but multiple introductions by un-screened blood products in the past.

Because several children were found infected with hepatitis C virus (HCV) at a pediatric oncohematological department in Vilnius, 474 children were tested for anti-HCV. Fifty-eight percent of 96 children treated with blood and plasma products manufactured before the introduction of anti-HCV screening of blood in Lithuania in 1994 were positive for anti-HCV versus 3.4% of those treated after 1994. The possible route of transmission for 45 of these was investigated by phylogenetic analyses within the NS5B region. Children treated before 1995 were infected with a multiplicity of strains of different subtypes, predominantly 1b found in 21 cases, 3a in 5 cases, 2 in 3 cases, 1a in 1 case, and not subtypeable genotype 1 strains in 2 cases. Children who had received blood products after 1994 were infected with only two subtypes, 1b in six and 3a in seven. Genetic analysis showed multiple introductions of HCV before 1995 and that horizontal spread between patients had occurred only to a minor extent at the department. However, two transmission chains involved children treated before 1995. Another chain involved five children treated after 1994. Since the most important risk factor for acquiring hepatitis C was blood products manufactured before the introduction of donor screening for anti-HCV, the spread between children would not have been revealed without molecular tools. These and the background strains provide the first reported sequence data on Lithuanian HCV strains. In general, these were shown to form autochthonous clades, except the 3a strains that were related to strains from the former USSR.

Molecular epidemiology of hepatitis B virus in Spain: identification of viral genotypes and prediction of antigenic subtypes by limited sequencing.

The hepatitis B virus (HBV) genotypes were studied by a line probe assay (LiPA) and by direct sequencing of a 339 nucleotide fragment from the S region of the viral genome in samples from 269 carriers living in Spain, either native to Spain (231) or immigrants from Africa, Asia, and Eastern Europe (38). The sequences were also used to predict the HBV surface antigen (HBsAg) subtype on the basis of the amino acids specified at selected positions of the HBsAg molecule. Agreement between the two genotyping methods was found in most cases (98.1%) and a HBV genotype could be assigned to all samples. The viral groups D/ayw2 (30.1%), D/ayw3 (28.6%), and A/adw2 (21.2%) were prevalent, with an additional participation of the groups D/ayw4 (4.8%), F/adw4q- (1.9%), A/ayw1 (1.9%), and D/adw3 (0.7%), all of them present among the autochthonous carriers. Strains from genotypes B and C were found exclusively among Chinese immigrants. Genotype E strains were found in immigrants from Central Africa and in one patient native of Spain. Point mutations leading to amino acid changes of residues involved in the expression of the HBsAg subtype determinants were found in 12 samples (4.5%). Some mutations would predict the putative novel genotype-subtype associations A/adw4q+, A/ayr, D/ayr, and E/ayw1, while others would suggest the loss of subtype-specific determinants. The finding of HBV strains characteristic for Africa among the autochthonous carriers confirms the emergence of African HBV strains in Spain.

Full genome analysis of hepatitis B virus genotype E strains from South-Western Africa and Madagascar reveals low genetic variability.

The eight genotypes of hepatitis B virus (HBV), A-H, have a characteristic geographical distribution. The high prevalence of genotype E within a wide expanse of Africa makes it important to characterize this genotype. Although the preS/S region of a large number of genotype E isolates has been sequenced, to date only eight complete genotype E genomes have been published. The aim of this study, therefore, was to sequence and characterize the complete genome of genotype E isolates obtained from geographically distinct regions in Africa, including Namibia, Angola, and Madagascar. The nine newly sequenced genotype E isolates were closely related with an intra-group nucleotide divergence of 1.2%, reflecting their close geographical origin. All genotype E strains have the same characteristics, an in-frame deletion of three nucleotides (one amino acid) in the 5' pre-S1, a signature pattern of amino acids in the pre-S1 region and the serological subtype ayw4. In addition, we have identified another unique attribute of genotype E strains, the introduction of another start codon Met(83) in the pre-S1 region that may result in the translation of an elongated middle hepatitis B surface protein (MHBs).

T1764G1766 core promoter double mutants are restricted to Hepatitis B virus strains with an A1757 and are common in genotype D.

To investigate the role of pre-core and basal core promoter (BCP) mutants in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (e-CHB) in Iran, Hepatitis B virus strains from 30 patients and 42 anti-HBe-positive asymptomatic carriers (ASCs) were characterized. G1896A pre-core stop mutants, detected in 77 % of e-CHB patients and 85 % of ASCs, showed no association with virus load or aminotransferase levels. Twenty per cent of e-CHB patients and 31 % of ASCs harboured T1762A1764 mutants. When this double mutation was associated with G1757, it was linked to a higher virus load in patients than when it was associated with A1757 (10(5.2+/-1.8) vs 10(3.2+/-0.8) copies ml(-1); P=0.004). Interestingly, the most common BCP mutations were T1764 and G1766, which were present in 33 % of e-CHB patients and 29 % of ASCs. These were associated with higher virus load and aminotransferase levels compared with patients lacking core promoter mutations, although this was not significant. The T1764G1766 double mutation was only present in strains with A1757 (P<0.001), which is more frequent in strains of genotype D than in those belonging to other genotypes. On the other hand, the T1762A1764 double mutation was found more frequently in association with G1757 than with A1757. The T1762A1764 double mutation forms a binding site for hepatocyte nuclear factor 1 (HNF1), which is constrained by A1757. However, the T1764G1766 double mutant may form a binding site for HNF3. Thus, position 1757 affects the emergence of promoter double mutants and would predict a relative genotypic restriction of both the T1762A1764 and the T1764G1766 double mutants.

Full-length open reading frame of a recombinant hepatitis C virus strain from St Petersburg: proposed mechanism for its formation.

The full-length ORFs for the hepatitis C virus recombinant RF1_2k/1b (N687) and the non-recombinant 1b strain N589 were sequenced. A single recombination point was found and the sizes of the genes (C, E1, E2, p7, NS2, NS3, NS4 and NS5) were according to the parental subtypes. The PKR-eIF2alpha phosphorylation site homology domain sequence of the E2 protein was identical to those of genotype 2 strains, while the IFN-alpha-sensitivity-determining region of the NS5A protein was identical to those of interferon-resistant 1b strains. For the parental strains, two hairpin structures, HS1 and HS2, were predicted for the plus-strand up- and downstream of the crossover site, which were not present in the recombinant strain. HS2 shared similarity with the motif1 hairpin of turnip crinkle virus RNA that binds to the RNA-dependent RNA polymerase and facilitates 3'-terminal extension during recombination. This study suggests that RF1_2k/1b has emerged by homologous recombination during minus-strand synthesis via template switching because of constraints imposed by the HS1 hairpin of the 3'-parental genome.

Open reading frame sequence of an Asian enterovirus 73 strain reveals that the prototype from California is recombinant.

Phylogenetic analysis within the VP1 region now enables molecular typing of enteroviruses consistent with neutralization results. Three untypable isolates, 2776/82, 57/99 and 22/00, from Korea, North India and Bangladesh, respectively, showed within this region 98.0-99.0% amino acid identities. These were less than 77% to the previous enterovirus prototypes, but 91.5-92.5% to CA55-1988, the recently identified enterovirus 73 (EV73) prototype from California. All three strains were, however, most similar to CA64-4454, an EV73 prime strain, to which they shared 96.5-98.5% identity. Seven compared EV73 strains formed two clusters in the VP1 dendrogram, one cluster with strains from South and East Asia and CA64-4454, and the other with strains from Oman and California including the prototype. When sequencing the complete open reading frame of 2776/82, its non-structural region was found to be divergent from all human enterovirus B (HEV-B) strains, including CA55-1988, indicating that one or other strain was recombinant. Boot scanning of the genomes showed a recombination point within the P2 region. Therefore, part of this was sequenced for 57/99 and 22/00 and was found similar to 2776/82, while CA55-1988 was similar to coxsackievirus B3, demonstrating that CA55-1988 was the recombinant. Since all strains of EV73 isolated so far outside California originate from Asia, where it has a broad geographical distribution, it seems that EV73 may have been introduced to California from Asia. Further analysis of EV73 strains will reveal if the recombination occurred in the USA or in Asia and will help to elucidate the origin of this virus.

A natural intergenotypic recombinant of hepatitis C virus identified in St. Petersburg.

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 city's 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.

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.

The T(1858) variant predisposing to the precore stop mutation correlates with one of two major genotype F hepatitis B virus clades.

The precore mutation G(1896)-->A occurs frequently in anti-HBe-positive carriers of HBsAg with T(1858) in the stem of the encapsidation signal. Hepatitis B virus (HBV) genotype F, considered an Amerindian genotype, subdivides into two clades and the precore mutation occurs in Central American F strains. To investigate the relationship between substitutions at position 1858 and these clades, the precore and small S genes of 48 strains of HBV genotype F were subjected to phylogenetic analyses. Isolates of one clade, formed mainly of Central American strains, all had T(1858) and Thr(45) in the S gene, whereas in the other clade, formed mainly of South American strains and one strain from Polynesia, all had C(1858) and Leu(45). The latter strain was related to strains from Venezuela and Colombia, supporting an Amerindian contribution to the Polynesian population. The position of the Polynesian strain in the phylogenetic tree indicates that the two clades have resulted from an early split, showing a high degree of genetic stability of the stem of the HBsAg encapsidation signal.

Hepatitis B virus genotype D strains from Estonia share sequence similarity with strains from Siberia and may specify ayw4.

The genotypes and subtypes of 205 HBV isolates collected during 1989-2002 in Estonia and 14 other regions of the former USSR were determined by sequencing and phylogenetic analysis of the S gene. The in Europe prevailing genotypes, A and D, were also circulating in the whole territory of the former USSR including Estonia and accounted for 18.5 and 81% of the strains, respectively. All genotype A strains specified adw2, and a single genotype C strain specified adrq+. Most genotype D strains specified ayw3 and ayw2, although, three strains from Estonia and Siberia specified ayw4. Due to unique substitutions, Ser122 and Ala127, four strains could not be classified according to the subtype. One strain specifying ayw3 encoded Leu143 and Ala145 and was possibly an immune "escape" mutant. At phylogenetic analysis 93% of the Estonian genotype D strains belonged to a cluster specifying mainly ayw3 and were more similar to isolates from Siberia and the Far-East of Russia than to isolates originating from Central Russia which belonged to another cluster of strains specifying mainly ayw2. This pattern might be explained by part of the Estonian population, has roots east of European Russia, based on linguistic evidence. Eight dominant HBV strains represented by identical S gene sequences were identified, one within genotype A and seven within genotype D, three of which included isolates from Estonia and Siberia. Some of these strains were collected over a period of at least 13 years indicating there are genetically stable variants of HBV that remain conserved over decades.

Sequential changes in hepatitis A virus genotype distribution in Estonia during 1994 to 2001.

Hepatitis A virus (HAV) isolates from a large outbreak and from non-outbreak cases in Estonia were characterized by sequencing the aminoterminal VP1 region. From January 1998 to December 1999, a total of 1084 cases of hepatitis A were reported to the Harjumaa-Tallinn and Ida-Virumaa Health Protection Services in Estonia. The attack rate was highest among males aged 15-29. Initial cases were noted to be associated with injecting drug use. IgM anti-HAV positive sera were available from 107 hospitalized outbreak cases and from 68 patients sampled during 1994 to 2001. HAV RNA was detected in 42% of sera from 1994-1996 and in 88% of sera from 1998-2001. It was possible to obtain HAV sequences from 83 outbreak and 29 background cases. The outbreak strain was represented by five different sequences, all belonging to subtype IIIA. During the outbreak, this IIIA strain also spread into the general population. All available non-outbreak isolates from 1994 to 2001 but one belonged to genotype IA and formed distinct clusters as compared to isolates from other parts of the world. One subtype IIIA isolate from 1995 was unrelated to the outbreak strain. Subtype IA had been dominating in Estonia during 1994-2001, but the outbreak strain from 1998 to 1999 was IIIA. This subtype was encountered previously in addicts in Sweden during the 1980s and in Norway at the end of the 1990s. This study supports the use of limited sequencing within the aminoterminal VP1 region for studying the molecular epidemiology of hepatitis A.