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.     

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.     

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.     

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.     

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.     

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 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.     

Analysis of the full-length genome of a subgenotype IIIB hepatitis A virus isolate: primers for broadly reactive PCR and genotypic analysis

Among six known subgenotypes (IA, IB, IIA, IIB, IIIA, and IIIB) of human hepatitis A virus (HAV), the complete genomic sequence has not been determined for IIIB. In this study, the full-length genomic sequence of a IIIB HAV isolate (HA-JNG06-90F) recovered from a Japanese patient who contracted sporadic hepatitis A in 1990, was determined. The HA-JNG06-90F genome, which comprised 7462 nt excluding the poly(A) tail, was related most closely to NOR-21 of subgenotype IIIA with an identity of 89.1%, and was only 82.6-83.4% similar to human HAV isolates of genotypes I and II over the entire genome. Comparison of full-length genomic sequences of 20 reported isolates and HA-JNG06-90F generated optimal results for separation of different levels: the nucleotide identities were 80.7-86.6% at the genotype level, 89.1-91.9% at the subgenotype level, and 94.6-99.7% at the isolate level. Similar ranges of nucleotide identity were observed when comparing partial nucleotide sequences of the VP1-2B (481 nt; primer sequences at both ends excluded) and 3C/3D (590 nt) regions, which were amplifiable by PCR with primers designed from well-conserved areas of the HAV genome. All 66 samples with IgM-class HAV antibodies tested positive for HAV RNA by both VP1-2B (481 nt)-PCR and 3C/3D (590 nt)-PCR: subgenotype assignment was concordant in all samples tested (IA [n = 61], IB [n = 1], IIIA [n = 2] and IIIB [n = 2]). These results suggest that two broadly reactive PCRs using primers derived from the VP1-2B and 3C/3D regions, respectively, may be applicable to universal detection and phylogenetic analysis of various HAV strains.     

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.     

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 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.     

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.