Genetic analysis of HAV strains recovered from patients with acute hepatitis from Southern Italy

Southern Italy is an endemic area for HAV infection contributing to the majority of Italian hepatitis A cases. Using molecular analysis, HAV strains have been classified in distinct genotypes and subgenotypes. To characterize HAV wild-type strains circulating in Southern Italy, sequence analysis of VP3-VP1 and VP1/2A junction regions of HAV isolates recovered from 25 patients with acute hepatitis during 2000 and 2001 was carried out. HAV isolates showed a degree of identity, after pairwise comparison with one another, ranging from 91.9-100% in the VP3-VP1 junction region and 89.9-100% in the VP1/2A junction region. All strains belonged to genotype I, with 84% (21/25) of samples clustering in subgenotype IA and 16% (4/25) in subgenotype IB. Cocirculation of subgenotypes IA and IB was observed among isolates from 2000, whereas all strains from 2001 were subgenotype IA. In addition, the subgenotype IA strains formed different clusters, one of which was related closely to some Cuban strains, showing a percent similarity of 98.8% in the 168-base pair segment encompassing the VP1/2A junction and the same amino acid substitution. The latter finding suggests that this subgenotype variant circulates also in the Mediterranean area. The results of the phylogenetic analysis confirm the genetic heterogeneity among HAV strains in Western Europe.     

Molecular epidemiology of hepatitis A in St. Petersburg, Russia, 1997-2003

The molecular epidemiology of hepatitis A virus (HAV) strains circulating in the St. Petersburg and Karelia regions was studied during 1997-2003. Hepatitis A virus RNA was isolated from both clinical samples (stools or sera) and environmental samples (sewage water). RT-PCR was carried out using different primer pairs from the VP1/2A and VP1 genomic regions, the variable parts of the HAV genome. PCR products were sequenced and 306 nucleotides from the VP1/2A and 332 nucleotides from the VP1 region were used for phylogenetic analysis. The results show that the IA subtype was the most common during the follow-up period: >90% of the isolated HAV strains belonged to that subtype. The HAV strains found in intravenous drug users belonged to subtypes IA and IIIA. Only one out of a total of 88 sequenced strains was of the IB subtype. The subtypes IB and IIIA were found only in 2001-2003, which suggests that new strains were introduced into the endemic situation. The results indicate the usefulness of molecular epidemiological methods in studying changes in the circulating HAV strains and in tracing transmission routes.     

Detection of hepatitis A virus genotype IB variants in clams from Maputo Bay, Mozambique

Clams provide an important source of food and income for the population of Maputo, Mozambique, where conditions of poor water supply and inadequate sanitation favor endemic infection with hepatitis A virus (HAV). To determine the role of bivalves in an endemic area, clams gathered from Maputo Bay were bought from market and examined for HAV. Four batches, total 150 clams, were sampled over the year. RNA extracted from individual digestive glands was assayed by nested RT-PCR and sequencing of HAV 5' noncoding region (5' NCR). Specific HAV signals were detected in one batch, 23 of 34 clams (67%) testing positive. Phylogenetic analyses of VP3/VP1, VP1/P2A, and 5' NCR determined clustering of clam strains as genotype I, subtype B. In addition to identifying HAV IB strains with predicted conserved amino acid sequence, IB variants exhibiting novel amino acid substitutions at the VP1/P2A junction were detected. HAV strains from clams showed 93%-99% homology with wild-type IB strains from South African outbreaks and from a panel of HAV IgM positive Swedish patients. DNA from enteric human adenovirus 40/41 was found in a limited number of clams from two batches, 6/34 (17%) and 4/35 (11%). Detection of HAV subgenotype IB in bivalves provided indirect evidence of the strains circulating in a densely populated coastal region where HAV is presumed to be hyperendemic. The results suggest that clams may be an important source of HAV in Maputo region, and indicate the need for further molecular study of strains circulating in the indigenous population.     

Genetic diversity of hepatitis A virus in Siberia

The nucleotide sequences of a region of VP1/2A genes of a large group of hepatitis A virus (HAV) isolates circulating in Siberia (the Altai Territory, the Irkutsk and Novosibirsk Regions) were determined. Comparison of these sequences with those of prototype HAV of genotypes IA, IB, and IIA revealed their high similarity to prototype genotype IA strains. The above domains were shown to contain the types of viruses, which were close to both the European subtypes of HAV genotypes IA (78.3%) and the Far Eastern subtypes of this genotype (21.7%). The similar comparison of the derived amino acid sequences suggests that VP1 and 2A contains the amino acid substitutions that are typical of this geographical region.     

Hepatitis A in Tunisia: phylogenetic analysis of hepatitis A virus from 2001 to 2004

Tunisia is a highly endemic area for hepatitis A virus (HAV) infection. In the present study, the phylogenetic characterization of the VP1 gene (882 nucleotides) and of the VP1/2A junction (336 nucleotides) of Tunisian strains were examined. One hundred strains isolated from patient with anti-HAV IgM from 2001 to 2004 were amplified by RT-PCR, sequenced at the VP1 and at the VP1/2A junction and aligned with the published sequences to establish phylogenetic analysis. All Tunisian strains belong to genotype I with a greater presence of sub-genotype IA (98%) originate from most of Tunisian regions and 2% of sub-genotype IB. In addition, sub-genotype IA and IB strains formed 25 different clusters. Genetically similar strains were also identified between 2001 and 2004 isolated from the southern and the central part of Tunisia, suggesting that an indigenous strain has been circulating in the Tunisia. The genetic profile of the VP1 region showed that Tun159-02 and Tun40-03 clustered respectively in the IB and IA sub-genotype, however, analysis of VP1/2A junction revealed in contrast that Tun159-02 and Tun40-03 clustered respectively in IA and IB. This is the first report to identify sub-genotype IA in Tunisia and provides new data on the genetic relatedness of HAV from Tunisia and the distribution of sub-genotype IA in this part of the world.     

First full-length genomic sequence of a hepatitis A virus isolated in Argentina shows recombination between subgenotypes IA and IB

A hepatitis A virus (HAV) recovered in Argentina from a stool sample of a sick child in the year 2006 (HAV-Arg/06) was entirely sequenced. Phylogenetic analysis included the HAV-Arg/06 sequence in subgenotype IA, either considering the usual VP1-2A variable junction fragment or the full length nucleotide sequence. Interestingly, a recombination event with subgenotype IB, involving a portion of the 2C-3A nonstructural proteins coding region (nucleotides 4961-5140) was detected using specific software. Only subgenotype IA strains have been detected in Argentina or Uruguay, whereas subgenotype IA and IB strains have been reported to circulate in Brazil. Although recombination has been given an important role in the evolution of picornaviruses, there have been only a few reports of its involvement in the evolution of HAV, probably due to the limited number of complete HAV sequences available. This study constitutes the first report of a full-length HAV sequence in Argentina and the third in South America, after the sequence of the IA isolate HAV5 from Uruguay and the IB isolate HAF-203 from Brazil. The availability of new sequence data covering the complete HAV genome will help establish a more consistent genetic relatedness among HAV isolates and the role of recombination in its evolution.     

Molecular epidemiology of an outbreak of hepatitis A in Italy

The relationship of hepatitis A virus (HAV) isolates associated with an outbreak in Genoa, Italy, in 1993 was examined using direct sequencing of amplicons derived by antigen capture PCR (AC/ PCR) from faecal samples of the infected persons. Forty samples recovered from 38 primary and two secondary cases were examined. The latter were household contacts of the primary cases. In addition, faecal material of 2 unrelated persons infected simultaneously with hepatitis A in Genoa were tested. The PCR products derived rom the P1/P2 junction of the HAV genome were analysed. A 100% nucleotide identity was detected between the viral isolates originating from the primary as well as the secondary cases. The viral isolates recovered from the faecal samples of the two unrelated cases differed from the virus causing the outbreak as well as from each other. These results indicate that a single HAV strain caused the outbreak. The virus might have been transmitted by ingestion of contaminated food or water since all hepatitis A infected employees of the factory had eaten in the same canteen. Definitions of HAV genotypes are based on numerous genetic comparisons of different strains. The sequence comparison of the investigated isolates with published HAV sequences of the P1/P2 genome region revealed that the virus associated with the outbreak belongs to HAV subgenotype IA, whereas the strains recovered from the viral isolates of the unrelated cases belong to subgenotype IB. © Wiley-Liss, Inc.     

Molecular epidemiological studies show that hepatitis A virus is endemic among active homosexual men in Europe

Large outbreaks of hepatitis A have occurred in Denmark, Germany, the Netherlands, Norway, Spain, Sweden, and the United Kingdom during the period 1997-2005 affecting homosexual men. A collaborative study was undertaken between these countries to determine if the strains involved in these hepatitis A outbreaks were related genetically. The N-terminal region of VP1 and the VP1/P2A region of the strains were sequenced and compared. The majority of the strains found among homosexual men from the different European countries formed a closely related cluster, named MSM1, belonging to genotype IA. Different HAV strains circulated among other risk groups in these countries during the same period, indicating that specific strains were circulating among homosexual men exclusively. Similar strains found among homosexual men from 1997 to 2005 indicate that these HAV strains have been circulating among homosexual men for a long time. The homosexual communities are probably too small within the individual countries to maintain HAV in their population over time, whereas the homosexual communities across Europe are probably sufficiently large to sustain continued circulation of homologous HAV strains for years resulting in an endemic situation among homosexual men.     

Increased circulation of hepatitis A virus genotype IIIA over the last decade in St Petersburg, Russia

The current study, covering the period 2004–2009, is a part of long‐term monitoring for hepatitis A virus (HAV) strains circulating in St Petersburg, Russia. The HAV RNA was isolated directly from the sera of hepatitis A patients and RT‐PCR was carried out using primer pairs for VP1/2A and VP1 genomic regions. PCR products were sequenced and 324 nucleotides from VP1/2A and 332 from the VP1 region were used for phylogenetic analysis. The results show that the IA subtype was the most common circulating subtype during the follow‐up period, as found in the previous study: almost 90% of the isolated HAV strains belonged to the IA subtype. The large hepatitis A food‐borne outbreak in St Petersburg in 2005 was caused by HAV IA. However, the proportion of HAV isolates belonging to subtype IIIA significantly increased in the period 2001–2009 (7.9%) compared to the period 1997–2000 (none found). The subtype IIIA was first found in St Petersburg in 2001 among a group of intravenous drug users. The increase in its circulation during the decade suggests that this previously unusual genotype has been permanently introduced into the general population of St Petersburg. These results indicate the usefulness of molecular epidemiological methods for studying changes in the circulation of HAV strains. J. Med. Virol. 84:1528–1534, 2012. © 2012 Wiley Periodicals, Inc.     

Hepatitis A virus genotype and its correlation with the clinical outcome of acute hepatitis A in Korea: 2006-2008

Korea has recently experienced a nationwide outbreak of hepatitis A. This study aimed to investigate hepatitis A virus (HAV) genotypes and to compare clinical features between patients infected with HAV genotype IA and those with genotype IIIA. From September 2006 to August 2008, 595 patients with symptomatic hepatitis A were enrolled prospectively in four hospitals in Korea. Among them, 556 patients participated in this study by providing serum or stool samples for genotypic analysis. HAV RNA was detected in 499 patients (89.7%). Major genotypes included IA (n = 244, 48.9%) and IIIA (n = 244, 48.9%), and the remaining genotype was IB (n = 11, 2.2%). From September 2006 to August 2007, the distribution of genotypes IA and IIIA were 64.6% and 35.6%, respectively, which changed to 42.3% and 54.6%, respectively, from September 2007 to August 2008, indicating change of circulating HAV genotypes in the study period from IA to IIIA. Major patterns of amino acid substitution in the VP3/VP1 junction region were observed at position 512 (P → L) in genotype IA and at 520 (R → K) in genotype IIIA. Patients with genotype IIIA infection showed significantly higher aminotransferase levels, prothrombin time, and leukocyte count, with more severe symptoms than those with genotype IA at the time of admission. These results suggest the occurrence of a change of circulating HAV genotypes in recent community‐wide outbreaks of hepatitis A in Korea, and genotype IIIA infection, compared with genotype IA infection, might show more severe clinical manifestations. J. Med. Virol. 83:2073–2081, 2011. © 2011 Wiley Periodicals, Inc.     

Outbreaks of hepatitis A in England and Wales associated with two co-circulating hepatitis A virus strains

During 2002, an upsurge in frequency of hepatitis A outbreaks among injecting drug users was observed in England and Wales. As lack of risk factor information and the high mobility of the cases made linkage of outbreaks difficult, the relationship of nucleotide sequences in the VP1/2PA junction of the hepatitis A virus (HAV) genome amplified from serum of case-patients was investigated. A total of 204 HAV RNA positive sera obtained from a network of 23 laboratories were studied. Comparison of the sequences identified two principal strains: ES1 (n=95) belonging to type IB, and ES2 (n=72) to type IIIA. Of the remaining samples, 15 were type IA, 11 were type IB and 11 were type IIIA. ES1 predominated in Doncaster and other towns in Trent and northern England, and ES2 in the Midlands and southern England; the difference in geographical distribution between these two strains was significant (P<0.0001). In comparison to the sporadic cases, cases infected by either ES1 or ES2 tended to be younger, injecting drug users, people in contact with injecting drug users, or those with a history of incarceration in prisons or homelessness (P<0.0001). Cases infected by ES1 tended to be younger than those by ES2 (P<0.0001). The association of the outbreaks to two geographically restricted strains implicates two principal transmission pathways associated with injecting behavior. Identifying these routes may be conducive to preventing further outbreaks.     

Molecular epidemiology of hepatitis A virus in patients in the Ahwaz region of Iran

Hepatitis A virus (HAV) is one of the etiologic agents of acute viral hepatitis, an important public health problem worldwide. The aim of this study was to investigate the genetic diversity of HAV in Southwest Iran (Ahwaz). A total of 59 sera were collected from acutely ill patients with anti-HAV IgM antibodies during 2009 and 2010 were tested also by RT-PCR targeting the 5' NCR for molecular diagnosis and examined in the VP1-2A and VP3-VP1 regions for genotyping. Twelve (20%) patients were detected VP1-2A by RT-PCR and 10 patients had VP3-VP1. The resulting amplicons were sequenced for genotype identification. All HAV strains were identified as subgenotype IB. Phylogenetic analysis revealed an extensive genetic heterogeneity among the strains. Seven hundred sixty-five S→F and 788 K→R amino acid substitutions in IRI49 isolate were found. It is concluded that subgenotype 1b is the sole genotype HAV in this region.     

Presumed common source outbreaks of hepatitis A in an endemic area confirmed by limited sequencing within the VP1 region

Hepatitis A virus isolates from anti-HAV IgM positive sera of 70 hepatitis cases in two outbreaks and 216 other cases in Central America, 136 sporadic cases and 53 cases from an hyper-endemic region in Costa Rica, were compared by phylogenetic analyses within the VP1 region. The outbreaks in all 531 cases, in 1992 and 1999, respectively, were presumed water borne. In the first outbreak, HAV RNA could be detected in 70% of the cases sampled during 6 weeks after onset of jaundice. In the hyper-endemic region of San Ramón in Costa Rica, 1,932 cases were registered between 1972 and 1985. All isolates belonged to subtype 1A. Background isolates from Costa Rica and El Salvador tended to form separate subclusters in the phylogenetic tree construction and were mostly unrelated to subtype 1A strains from other parts of the world. Based on their amino acid sequences, four HAV strains, all related to CR326 sampled in Costa Rica in 1960, were found to have circulated in the area during the last three decades. However, on the basis of nucleotide variability the isolates from the outbreaks could be distinguished from the strains from sporadic cases and sequence analysis could confirm the epidemiological homogeneity of both outbreaks. In the hyper-endemic region, 16 different sequences were encountered forming one single subcluster. Thus, limited sequencing within the VP1 region proved useful to identify outbreaks of hepatitis A in a highly endemic area, where most strains were local and only one subtype was prevalent.     

Exposure to multiple subgenotypes of hepatitis A virus during an outbreak using matched serum and saliva specimens

Matched serum and saliva samples were collected simultaneously from 124 subjects exposed during a hepatitis A virus (HAV) outbreak at a daycare center in Rio de Janeiro, Brazil. All samples were tested for IgM and total anti-HAV antibodies by enzyme immunoassay (EIA). HAV was detected by nested PCR in serum, saliva, and water samples employing primers for the VP1/2A region of the viral RNA; all positive products were then sequenced. The viral load of the matched samples was determined by real-time PCR using the TaqMan system. HAV-RNA was identified by nested PCR in 37.7% of the saliva samples, 29% of the serum samples, and one drinking water sample. The mean HAV viral load was similar in the serum and saliva specimens (10(3) copies/ml). HAV genotypes IA and IB were detected in both specimen types, and the water sample isolate was classified as genotype IB, indicating the existence of more than one source of infection at the daycare center. In six infected patients, a different HAV subgenotype was found in their serum than in their saliva, and this unusual pattern of mixed HAV infection was investigated further by molecular cloning followed by nucleotide sequencing. All clones derived from the saliva samples belonged to subgenotype IB and shared 96.5-100% identity. However, clones derived from their corresponding serum sample belonged to subgenotype IA and shared 90.5-100% identity. This study showed the important role that non-invasive saliva samples can play in the molecular epidemiological analysis of a hepatitis A outbreak.     

Epidemiology of hepatitis A in Finland in 1990–2007

The seroepidemiology of hepatitis A virus (HAV) for the period 1990–2007 and the molecular epidemiology for the period 1994–2007 in Finland were studied. The incidence of hepatitis A has been very low since 1990, at 0.3–3.6/100,000 inhabitants, excluding two outbreaks in 1994–1995 and 2002–2003, both of which were connected to intravenous drug use. Serum samples (3,217) collected in the period 1997–1998 were tested for hepatitis A antibodies to assess the percentage of seropositive Finns. More than 50% of Finns aged over 55 were seropositive for hepatitis A, while less than 5% of those aged under 40 were seropositive. In addition, patient samples (52,012) from the period 1990 to 2007 were assessed for antibodies against HAV. In these samples the proportion of acute HAV infections stayed at around 2% per year (excluding outbreaks), whereas the overall seropositivity for hepatitis A increased from some 30% to 45%, which was most likely due to increased vaccinations. For molecular epidemiology, samples from 1994 to 2007 were analyzed by RT‐PCR and sequencing. The results showed that most of the strains (82%) of HAV were of genotype IA but with an increasing number of genotypes IB and IIIA appearing during the last years of the study. All the cases seemed to be travel related and there was no endemic strain circulating in Finland. The low seroprevalence, especially in younger age groups, makes the population vulnerable to infection, which can be compensated for by increasing the number of vaccinations. J. Med. Virol. 82:934–941, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of a novel variant of human hepatitis E virus in Hungary.

First human case of hepatitis E infection detected in Hungary is reported. This hepatitis E virus (HEV), Hungary1, belongs to genotype 3 and had 95% and 90% nucleotide identity within the capsid region of the European swine and human (Greece2) strains, respectively. Hungary1 represents a potential novel human variant of HEV in genus Hepevirus.