Full-length sequences of subgenotype IIIA and IIIB hepatitis A virus isolates: characterization of genotype III HAV genomes

To elucidate the extent of genomic heterogeneity of human hepatitis A virus (HAV) strains and to characterize genotype III HAV strains over the entire genome, the full-length sequence of three subgenotype IIIA isolates (HA-JNG04-90F, HA-JNG08-92F, and HAJ95-8F) and one IIIB isolate (HAJ85-1F) was determined. The HA-JNG04-90F, HA-JNG08-92F, and HAJ95-8F genomes which comprised 7463 or 7464 nt excluding the poly(A) tail, were closest to a reported nearly entire sequence of a IIIA isolate (NOR-21) with identities of 94.4-97.8% over the entire ORF sequence, and the HAJ85-1 genome (7462 nt) to HA-JNG06-90F of IIIB with an identity of 98.6%. The phylogenetic trees constructed based on the complete ORF sequence or the 168-nt VP1/2A junction sequence and comparative analysis with reported HAV isolates suggested the presence of three distinct clusters within IIIA represented by HA-JNG04-90F, HA-JNG08-92F, and HAJ95-8F. The extreme 5' end sequences of IIIA and IIIB were well-conserved, beginning with the sequence UUCAAGAGGG. A single base deletion of G at nt 20, which is involved in the formation of a small loop in domain I, was characteristic of both IIIA and IIIB. Conserved and divergent amino acid sequences as well as amino acids unique to genotype III, IIIA or IIIB were recognized.     

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.     

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.     

Mixed infection of a child care provider with hepatitis A virus isolates from subgenotypes IA and IB revealed by heteroduplex mobility assay

Phylogenetic analysis based on a 168 base segment encompassing the putative VP1/2A junction of the hepatitis A virus (HAV) genome has enabled the classification of HAV isolates into seven genotypes (I-VII). Genotype I, which includes the vast majority of the human HAV isolates, has been divided further into subgenotypes IA and IB. An heteroduplex mobility assay was designed with amplification products from the VP1/2A junction region, and used as a genotyping method able to discriminate HAV isolates belonging to IA, IB and non-I genotypes. The method was used to successfully genotype 48 samples (16 IA and 32 IB). However, one HAV RNA positive serum sample (AUX-23), collected from a 15 year old female employed at a child care center located in Rio de Janeiro, Brazil, showed an unusual pattern. PCR products from sample AUX-23 gave rise to heteroduplex bands when mixed with IA products as well as with IB products, suggesting the presence of HAV isolates from both subgenotypes in the serum. PCR products from sample AUX-23 were then cloned and 20 clones were analyzed by heteroduplex mobility assay. Eleven were subgenotype IA and 9 were IB. Three clones of each subgenotype were then sequenced to confirm the results. These data constitute the first report of mixed infection of a single individual with different HAV isolates.     

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.     

Genetic analysis of hepatitis A virus isolates from Brazil

A limited number of hepatitis A virus (HAV) isolates from South America have been characterised at the genomic level. IgM anti-HAV positive serum samples collected from patients with hepatitis A living in the five geographical regions of Brazil (North, Northeast, Central, South, and Southeast) were used to obtain HAV isolates and determine their genetic relatedness. Of the 232 case isolates, sequence data were obtained from the VP1/2A junction region of the HAV genome. All isolates were classified in genotype I; 231 belonged to subgenotype IA, and one to subgenotype IB. HAV isolates from four States formed distinct clusters of highly related sequences. However, isolates from other states did not cluster and the sequences from those states were intermingled with sequences found in the other states. The amino acid sequences of all but two isolates showed a Leu --> Ile substitution at position 42 in the 2A protein. This substitution appeared to be a characteristic geographic fingerprint of HAV sequences within Brazil.     

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.     

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.     

Identification of recombination between subgenotypes IA and IB of hepatitis A virus

Co-circulation of subgenotypes IA and IB of hepatitis A virus (HAV) has been reported in South Africa, South America, Europe, and the United States. In this study, phylogenetic and recombination analyses were performed for the first time on 31 complete HAV genomes from infected humans and simians. Three potentially significant intra-genotypic recombination events (I–III) were identified by recombination detection analysis. Recombination events I and II occurred between the lineages represented, respectively, by the Japanese isolate AH2 (AB020565, subgenotype IA) and the North African isolate MBB (M20273, subgenotype IB), giving rise to the recombinant Uruguayan isolate HAV5 (EU131373). Recombination event III occurred between the lineages represented, respectively, by the North African isolate MBB (M20273, subgenotype IB) and the German isolate GBM (X75215, subgenotype IA), resulting in the Italian isolate FG (X83302). The findings demonstrate that humans can be co-infected with different HAV subgenotypes and provide valuable hints for future research on HAV diversity.     

Genetic analysis of hepatitis A virus outbreak in France confirms the co-circulation of subgenotypes Ia, Ib and reveals a new genetic lineage

Genetic analysis of selected genome regions of hepatitis A Virus (HAV) suggested that distinct genotype could be defined in different geographic locations. In order to study the degree of genetic variability among HAV isolated during a single epidemic outbreak, sequences from a 148 base pair segment within the VP1 amino terminal region were obtained for eight distinct HAV isolates from an outbreak that occurred in North Bretagne (France). These sequences were compared among themselves and with published sequences from 30 different strains that represented different HAV sub-genotypes that were isolated all over the world. Phylogenetic analysis revealed an extensive genetic heterogeneity among strains belonging to the same outbreak and revealed co-circulation of sub-genotype IA, IB, and the presence of IIIA sub-genotype for the first time in a Mediterranean country.     

Molecular epidemiology of hepatitis A virus in Amsterdam, the Netherlands

The transmission of sporadic community-acquired hepatitis A virus (HAV) among different risk groups in Amsterdam was verified by applying molecular techniques on fecal samples. These were collected in 1997/1998 from 33 persons with HAV infection that was confirmed serologically. From 8 of these persons serial stool samples were collected. Nested RT-PCR targeting the VP3-VP1 and VP1-P2a regions followed by sequence analysis established the duration of fecal HAV RNA excretion in stool and the epidemiological molecular relationships between patients. The samples of 31 patients were RT-PCR positive, of which 24 were positive for both regions. Fecal HAV shedding was found to occur for at least 33 days after onset of disease, which was the longest time span tested. Sequencing showed that the hepatitis A virus subgenotype circulating among persons from Moroccan descent (type IB) was different from the subgenotype circulating among Dutch homosexual men (type IA). If the latter is endemic in the Netherlands, its presence is of importance to the national vaccination strategy.     

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.     

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.     

Epidemiological and genetic analyses of a diffuse outbreak of hepatitis A in Japan, 2010

Background

Hepatitis A virus (HAV) is still one of the most common causative agents of acute hepatitis in Japan. Although a relatively small number of annual acute hepatitis A cases (approximately 100-150, 0.78-1.17 per million) were recently reported, a larger number of cases (346, 2.71 per million) were reported in 2010.

Objectives

To investigate the causes of the 2010 HAV resurgence in Japan by using molecular epidemiological and genetic analyses.

Study design

HAV specimens were obtained from 61 cases from 22 different prefectures. These viral specimens were genotyped by PCR amplification and sequencing of the VP1/2A region of HAV genome.

Results

Phylogenetic analysis revealed that 61 HAV strains could be divided into three genotypes: IA (44 cases), IB (1 case) and IIIA (16 cases). The IA genotype consisted of two genomic sub-lineages. The sequences of one of the two IA sub-lineages (corresponding to 31 cases) were very similar, 26 of these 31 isolates had 100% identity. The other IA sub-lineage corresponded to strains endemic to Japan. The sequences of Japanese IIIA strains were similar to those of strains that caused a large epidemic in the Republic of Korea from 2007 to 2009.

Conclusions

The resurgence of HAV in 2010 can be attributed to importation of two newly emerged HAV genotypes.     

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.     

用核酸序列分析鉴别甲肝病毒疫苗株与野毒株

目的鉴别在接种甲肝减毒活疫苗（Ｈ２株）后２３天发生的一例甲型肝炎,其病原体是疫苗株还是野毒株。方法用细胞培养、ＩＥＭ等确认病原体,接种普通狨猴检定病毒毒力,测定核苷酸序列分析病毒基因型。结果甲型肝炎的确诊依据有：抗ＩｇＭ（＋）,双份血清ＨＡＶ总抗体效价１６倍增长以及粪便排出ＨＡＶ（郑株）。该粪便悬液攻击普通狨猴后,肝脏呈现持续性组织病理改变,证明郑株是强毒ＨＡＶ。郑株的３个片段共１３７７个核苷酸序列分析表明,与１９８８年上海甲肝流行株合－５２的同源性高达（９８．３０～９９．４０）％,判定为ＩＡ基因亚型,与Ｈ２株疫苗病毒的基因亚型不同。结论该例甲型肝炎是接种甲肝减毒活疫苗中交叉感染野毒株引发的偶合病例,与疫苗接种无关