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.     

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 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.     

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.     

Genotyping of acute hepatitis a virus isolates from China, 2003-2008

Hepatitis A virus (HAV) is usually transmitted by an oral–fecal route and is prevalent not only in developing countries but also in developed countries. In the present study, the phylogenetic characterization of the VP1/2A junction region (321 nucleotides) of China HAV isolates was examined. Anti‐HAV IgM‐positive serum samples were collected from 8 provinces, including 20 cities or counties in China from 2003 to 2008; 337 isolates from 406 HAV patients' serum samples were amplified by RT‐PCR, sequenced at the VP1/2A junction region and aligned with the published sequences from GenBank to establish phylogenetic analysis. All China HAV isolates in this study belonged to genotype I, with 98.8% (333/337) of samples clustering in sub‐genotype IA and 1.2% (4/337) in sub‐genotype IB. In addition, sub‐genotype IA isolates clustered into four groups (92.7–100% nucleotide identity), and the samples collected from all China HAV isolates in this investigation showed 87.5–100% nucleotide identity, but the amino acids in this region were more conserved (95.2–100% identity). Few unique amino acid changes could be deduced (VP1‐253: Glu → Gly; 2A‐34: Pro → Ala; 2A‐33: Leu → Phe). Genetically identical or similar HAV strains existed in some investigated areas in China during different years, suggesting that an indigenous strain has been circulating in those regions. This report provides new data on the genetic relatedness and molecular epidemiology of HAV isolates from China as well as the distribution of sub‐genotype IA and IB in this part of the world. J. Med. Virol. 83:1134–1141, 2011. © 2011 Wiley‐Liss, Inc.     

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.     

河北石家庄2005-2007年甲肝病毒流行株基因分型研究

目的 了解石家庄地区甲型肝炎病毒(HAV)流行株基因型特征,为HAV溯源研究打下基础.方法 收集了2005-2007年石家庄地区部分甲肝患者急性期血清标本,用HAV结构-非结构区VP1-2A基因引物,经核酸提取,RT-PeR,序列测定,对HAV进行基因分型分析.结果 石家庄地区2005-2007年HAV流行株VP1-2A区核苷酸序列同源性为95%～100%,都属于Ⅰ A亚型;该区氨基酸序列几乎相同.结论 石家庄地区存在有多株甲肝病毒流行株,同一株甲肝病毒可以存在不同地区,同一地区可以检测到相同或不相同的HAV毒株.为今后进一步开展甲肝病毒分子流行病学研究及有效控制HAV流行提供了理论和技术支撑.     

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.     

Genetic characterization of hepatitis A virus isolates from Buenos Aires,Argentina

The Hepatitis A virus (HAV) has been classified in seven different genotypes, which include human (I, II, III, and VII) and simian (IV, V, and VI) groups. The sequence analysis of HAV strains contributes to the molecular epidemiology of the virus. Although the infection with HAV is endemic in Argentina and vaccination is being implemented in this country, using both IA and IB strains, there are very few data on the genotypes of the circulating viruses. On the basis of the sequences of 20 isolates collected in Buenos Aires during a 2-year period (extended to 3 years by two additional specimens), we observed the presence of a single sub-genotype, IA, but with a high genetic diversity. We analyzed the VP1-2A junction and also the VP3-VP1 region. Most of the Argentine isolates grouped in at least two clusters. One of these was related to South American strains, thus suggesting a co-circulation of related isolates in neighbor countries. The other cluster was composed only of Argentine specimens. Other sequences were more scattered along the phylogenetic tree. However, we demonstrated that a consistent genetic relatedness of sequences could only be inferred on the basis of a more extensive sequencing of each isolate.     

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 analysis of HAV strains isolated from patients with acute hepatitis in Korea, 2005-2006

Hepatitis A virus (HAV) is a causative agent of acute viral hepatitis, which represents a significant public health problem. HAV is usually transmitted by oral-fecal route and prevalent not only in developing countries but also in developed countries worldwide. To characterize the HAV wild type strains circulating in Korea, the VP3/VP1 and VP1/P2A junction regions were detected by RT-PCR from HAV IgM positives during 2005 and 2006. Among 160 HAV IgM positive sera, 30% (n = 48) were positive for HAV RNA. Additionally, the VP3/VP1 junction regions were detected all six stools, which collected from outbreak in Gyeonggi province. Phylogenetic analysis of the sequences obtained from 54 distinct HAV isolates revealed that most of the strains (n = 45) belonged to genotype IA and the others including nine strains belonged to genotype IIIA. Interestingly, a Q --> S amino acid change was dominantly observed at position 810 of the VP1/P2A junction region in 14 isolates. The molecular epidemiology of HAV infection in Korea has changed with the co-circulation of at least two genotypes and 810Q --> S amino acid substitutions were found to be prevalent. These results strongly suggest that various HAV strains, including genotype IIIA, might be imported from high-endemic countries into Korea.     

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.     

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.     

新疆和田2006年甲肝病毒流行株基因分型研究

目的 了解2006年新疆和田甲型肝炎病毒(HAV)流行株基因型特征,为HAV溯源研究打下基础.方法 收集了新疆和田部分甲肝病人血清标本,用HAV结构.非结构区基因VP1-2A引物,经核酸提取,RT-PCR,序列测定,对HAV进行基因分型研究.结果 新疆和田VP1-2A区HAV核苷酸序列变异为0%～3.9%,分为不同基因簇,但都属1A亚型;VP1-2A区氨基酸序列只有0～2个差异.与已发表的2005年新疆伊犁部分甲肝病毒流行株基因有相同序列或同源性较高.结论 和田有多株甲肝病毒存在,本研究流行可能有多个传染源,多个传播链,在人群免疫水平较低时,引起甲肝流行.结果 表明HAV分子流行病学方法在HAV流行株遗传变异及溯源研究中,以及控制HAV流行中具有重要作用.     

Analysis of the 5' noncoding region versus the NS5b region in genotyping hepatitis C virus isolates from blood donors in France

The 5' noncoding region (5' NCR) of the hepatitis C virus (HCV) has become the standard for genotyping even though several reports show that its use can result in classification errors. The purpose of this study was to perform genotyping based on sequence analysis of the NS5b region in a set of 357 HCV strains isolated from blood donors in France in 2002 and 2003. Results were compared with those previously obtained using 5' NCR analysis, and HCV subtype distribution was reevaluated. Twenty-six of 120 strains (approximately 22%) initially identified as genotype 1b by 5' NCR region sequence analysis were reclassified as genotype 1a by NS5b region sequence analysis. Similarly, 14 of 23 strains (approximately 61%) initially identified as 2a/2c were reclassified as non-2a and non-2c subtypes, and 12 of 22 strains (approximately 45%) initially identified as 4c/4d subtypes were reclassified as non-4c and non-4d subtypes. Sequence analysis of the NS5b region also revealed 5 putative new subtype 2 variants and 2 putative new subtype 4 variants. Although these findings demonstrated full agreement between 5' NCR and NS5b sequence analysis with regard to type classification, genotyping based on phylogenetic analysis of the NS5b region is more accurate for subtype determination than genotyping based on analysis of the 5' NCR. Sequence analysis of the NS5b region is mandatory for epidemiologic studies.     

Nucleotide correlation between HAV isolates from human patients and environmental samples

Hepatitis A disease is endemic in Albania even though records from the Medical Authority show outbreaks involving only limited numbers of people. In the city of Lac, 200 people became ill following an outbreak of hepatitis A started on November 2002. The age distribution of the cases shows a peak in the age group 5-9 (43.5%) followed by the age group 10-14 (28%) and the age group 1-4 (18%). No cases were recorded in the age group 0-1. Drinking water and sewage samples were collected, using electropositive cartridges, during the outbreak. Rotavirus was identified by RT-PCR in two out of five drinking water samples; however, all the samples tested negative for HAV and astrovirus. Rotavirus was also identified in three out of five sewage samples from which four were HAV positive. HAV-RNA was identified in 28 (62.2%) out of 45 IgM anti-HAV positive sera collected during the outbreak. Genotype IA was expressed from all the amplified samples. Sequence analysis of the overlapping VP1-2A region shows 97-99% homology with three Italian strains IT-SCH-00, IT-ZAM-01, and IT-CAP-00 and one Spanish strain (Sa 30/06/95), whereas the phylogenetic tree built from the 168 bp sequence of the VP1-2A shows four clusters, one including 24 out of 28 RT-PCR positive sera. Considering the deduced amino acid sequence, only one substitution was identified and reported previously for genotype IB.