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.     

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.     

中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点分析

目的 分析中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点.方法 收集42份甲肝患者急性期血清标本,经核酸提取、逆转录及巢氏PCR,测得结构-非结构蛋白VP3-VP1-2A区序列,进行序列同源性比较并分析其基因特点.结果 42株HAV病毒株在VP1-2A连接处核苷酸和氨基酸序列同源性分别为89.1%～100%和97.3%～100%;在全长结构蛋白VP3-VP1区的核苷酸和氨基酸序列同源性分别为87.6%～100%和98.8%～100%.VP1-2A连接处序列相同的病毒株在全长结构蛋白VP3-VP1区的核苷酸同源性为98.4%～100%,0～2个氨基酸位点不同.本实验所得序列在中和抗原位点处氨基酸序列均未变异.结论 42株病毒株均属于I型,40株是IA亚型,2株IB亚型.本实验所用HAV流行株在结构蛋白VP3-VP1区的核苷酸存在差异但是氨基酸序列高度保守且没有中和抗原位点处的变异.VP1-2A结合处核苷酸序列相同的分离株在全长结构蛋白VP3-VP1区核苷酸序列相同或相近,氨基酸序列保守.     

中国部分甲肝病毒流行株结构蛋白VP3-VPl区基因特点分析

目的分析中国部分甲肝病毒流行株结构蛋白VP3-VPl区基因特点。方法收集42份甲肝患者急性期血清标本,经核酸提取、逆转录及巢氏PCR,测得结构一非结构蛋白VP3-VPl-2A区序列,进行序列同源性比较并分析其基因特点。结果42株HAV病毒株在VPl-2A连接处核苷酸和氨基酸序列同源性分别为89．1％～100％和97．3％～100％;在全长结构蛋白VP3-VPl区的核苷酸和氨基酸序列同源性分别为87．6％～100％和98．8％～100％。VPl-2A连接处序列相同的病毒株在全长结构蛋白VP3．VPl区的核苷酸同源性为98．4％～100％,0～2个氨基酸位点不同。本实验所得序列在中和抗原位点处氨基酸序列均未变异。结论42株病毒株均属于I型,40株是IA亚型,2株IB亚型。本实验所用HAV流行株在结构蛋白VP3．VPl区的核苷酸存在差异但是氨基酸序列高度保守且没有中和抗原位点处的变异。VPl-2A结合处核苷酸序列相同的分离株在全长结构蛋白VP3-VPl区核苷酸序列相同或相近,氨基酸序列保守。     

中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点分析

目的 分析中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点.方法 收集42份甲肝患者急性期血清标本,经核酸提取、逆转录及巢氏PCR,测得结构-非结构蛋白VP3-VP1-2A区序列,进行序列同源性比较并分析其基因特点.结果 42株HAV病毒株在VP1-2A连接处核苷酸和氨基酸序列同源性分别为89.1%～100%和97.3%～100%;在全长结构蛋白VP3-VP1区的核苷酸和氨基酸序列同源性分别为87.6%～100%和98.8%～100%.VP1-2A连接处序列相同的病毒株在全长结构蛋白VP3-VP1区的核苷酸同源性为98.4%～100%,0～2个氨基酸位点不同.本实验所得序列在中和抗原位点处氨基酸序列均未变异.结论 42株病毒株均属于I型,40株是IA亚型,2株IB亚型.本实验所用HAV流行株在结构蛋白VP3-VP1区的核苷酸存在差异但是氨基酸序列高度保守且没有中和抗原位点处的变异.VP1-2A结合处核苷酸序列相同的分离株在全长结构蛋白VP3-VP1区核苷酸序列相同或相近,氨基酸序列保守.     

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.     

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.     

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.     

Hepatitis in Albanian children: molecular analysis of hepatitis A virus isolates

Hepatitis A is a common disease in developing countries and Albania has a high prevalence of this disease associated to young age. In spite of the occurrence of a unique serotype there are different genotypes classified from I to VII. Genotype characterisation of HAV isolates circulating in Albania has been undertaken, as well as the study of the occurrence of antigenic variants in the proteins VP3 and VP1. To evaluate the genetic variability of the Albanian hepatitis A virus (HAV) isolates, samples were collected from 12 different cities, and the VP1/2A junction amplified and sequenced. These sequences were aligned and a phylogenetic analysis performed. Additionally, the amino half sequence of the protein VP3 and the complete sequence of the VP1 was determined. Anti-HAV IgM were present in 66.2% of all the sera. Fifty HAV isolates were amplified and the analysis revealed that all the isolates were sub-genotype IA with only limited mutations. When the deduced amino acid sequences were obtained, the alignment showed only two amino acids substitutions at positions 22 and 34 of the 2A protein. A higher genomic stability of the VP1/2A region, in contrast with what occurs in other parts of the world could be observed, indicating high endemicity of HAV in Albania. In addition, two potential antigenic variants were detected. The first at position 46 of VP3 in seven isolates and the second at position 23 of VP1 in six isolates.     

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.     

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.     

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.     

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.     

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 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.     

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.     

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.