Molecular characterization of hepatitis A virus isolated from acute gastroenteritis patients in the Seoul region of Korea

Hepatitis A virus (HAV) is a major public health problem throughout the world. As a result of declining HAV endemic in Korea, an increasing number of children and adolescents have become susceptible to HAV infection. HAV is related with sanitation conditions of the environment and is transmitted via the fecal–oral route, either through person-to-person contact or by contaminated water and food. The present study has been carried out to determine the phylogenetic analysis and circulating patterns of HAV strains detected from hospitalized patients with acute gastroenteritis (AGE) in the Seoul region of Korea. In total, 2,782 stool specimens from hospitalized patients with AGE collected in October 2006 to September 2007 in Seoul were tested for HAV. A pair comparison of the nucleic acid sequence of a 159-bp base region at the putative VP1/2A junction of 85 Seoul isolates revealed that the most common HAV strain circulating in the region during 2006–2007 was subgenotype IA. HAV phylogenetic studies can provide important information on the genetic characteristics of HAV from AGE patients who may subsequently become the source of infection in 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 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.     

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.     

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.     

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.     

Hepatitis A virus genetic diversity in Venezuela: Exclusive circulation of subgenotype IA and evidence of quasispecies distribution in the isolates

Hepatitis A virus (HAV) infection is highly prevalent in Latin America, including Venezuela. Subgenotype IA seems to circulate in an almost exclusive fashion, except in Brazil. The aim of this study was the molecular characterization of the HAV infection in Venezuela, in order to characterize the circulating strains and to analyze the presence of quasispecies in sporadic cases and an epidemic outbreak. A total of 125 (113 sera and 12 feces) samples positive for anti‐HAV IgM from sporadic cases and epidemic outbreak, were submitted to hemi‐nested RT‐PCR for amplification of the VP1 N terminus or complete region of the HAV genome. Sequences obtained from 96 Venezuelan isolates were used for phylogenetic analysis. The quasispecies distribution was evaluated by cloning of HAV amplicons. Phylogenetic analysis of HAV sequences from Venezuela showed the exclusive circulation of subgenotype IA, but with co‐circulation of two lineages, not found in other countries. The genetic variability found among Venezuelan strains was also analyzed by single‐strand conformation polymorphism (SSCP). This technique allowed the detection of intra‐strain variability, which was indeed related to the presence of quasispecies populations in the isolates. The quasispecies heterogeneity was higher in some isolates derived from sporadic cases compared to the one observed in the outbreak. The molecular characterization of HAV isolates from Venezuela showed the circulation of a unique subgenotype IA, but with the presence of diverse strains and quasispecies inside the viral populations. J. Med. Virol. 82:1829–1834, 2010. © 2010 Wiley‐Liss, Inc.     

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 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.     

Acute hepatitis A is the chief etiology of acute hepatitis in Egyptian children: a single-center study

Acute hepatic illness is an important health issue in children. Our work aimed to determine the prevalence of viral hepatitis in symptomatic children. It is a prospective cohort study of 268 children presented with acute hepatitis. Complete blood count, liver panel, and anti-hepatitis A virus (HAV) IgM were done initially. Cases negative for HAV were tested for anti-hepatitis E (HEV) IgM, anti-Epstein-Barr virus viral capsid antigen (EBV VCA) IgM, anti-cytomegalovirus virus IgM, hepatitis B surface antigen, anti-hepatitis B core IgM antibody, and anti-HCV antibody. Anti-HCV was repeated after 12 weeks to exclude seroconversion. In cases with negative viral serology, ceruloplasmin, total immunoglobulin G, antinuclear antibody, and abdominal ultrasound were done. Follow-up visits were bimonthly till recovery, then after 6 months. The mean age?±?SD was 7.1?±?3.7 years (1.5–18), and 56% were males. Acute HAV infection was diagnosed in 260 (97%) of cases and acute EBV infection in one case (0.4%). HAV/HEV coinfection was excluded in 70 HAV-positive cases. Six (2.2%) children remain undiagnosed and one child lost follow-up. About 80% of HAV-cases had normal laboratory results within 45 days. Unusual presentation of HAV infection was noticed in six children: four (1.5%) were relapsing, one had a cholestatic course, and one case had severe hemolytic anemia. Acute HAV infection was the chief etiology of acute hepatitis in our Egyptian children. The majority of the presentations were mild and children recover within a few weeks. An unusual pattern of HAV in children can be observed in endemic areas.     

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.     

Epidemiological and genetic analysis of a sustained community-wide outbreak of hepatitis A in the Republic of Korea, 2008: A hospital-based case–control study

BackgroundThe epidemiological shift of hepatitis A has contributed to a sustained community-wide outbreak in Korea during 2008.ObjectivesTo assess the risk factors associated with hepatitis A virus (HAV) propagation, and to analyze the circulating genotype in the sustained community-wide outbreak.Study designThe hospital-based case–control study was conducted in an 850-bed university hospital in Seoul from April to August, 2008. For molecular analysis of HAV isolates, a 488-bp gene fragment of the VP1 region was amplified and sequenced.ResultsIn the multivariated logistic regression model, the risk factors of HAV infection adjusted by age were contacts with hepatitis A case (OR 3.98, 95% CI: 1.36–11.66), residence with child aged ≤5 years (OR 3.43, 95% CI: 1.32–8.87), consuming uncooked lettuce (OR 3.98, 95% CI: 1.83–8.68) or carrot (OR 2.38, 95% CI: 2.38–5.09), drinking tap water (OR 3.68, 95% CI: 1.62–8.37) or portable spring water (OR 2.71, 95% CI: 1.11–6.62) supplied by water purifiers, and eating out (OR 3.87, 95% CI: 1.53–9.78). All isolates analyzed belonged to genotype IIIA. There were 42 nucleotide differences in the sequenced VP1 region among the isolates. Amino acid sequences were identical with each other.ConclusionsOur study suggests that sporadically contaminated food- or water-borne sources as well as person-to-person transmission might lead a sustained community-wide HAV outbreak and pre-existing dominant genotype IA might be replaced with genotype IIIA as a major epidemic strain in Korea. Our findings urge the health authority to make public guidelines for HAV vaccination and outbreak control.     

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.     

Hepatitis A viral load in relation to severity of the infection

A correlation between hepatitis A virus (HAV) genomes and the clinical severity of hepatitis A has not been established. The viral load in sera of hepatitis A patients was examined to determine the possible association between hepatitis A severity and HAV replication. One hundred sixty‐four serum samples from 91 Japanese patients with sporadic hepatitis A, comprising 11 patients with fulminant hepatitis, 10 with severe acute hepatitis, and 70 with self‐limited acute hepatitis, were tested for HAV RNA. The sera included 83 serial samples from 20 patients. Viral load was measured by real‐time RT‐PCR. The detection rates of HAV RNA from fulminant, severe acute, and acute hepatitis were 10/11 (91%), 10/10 (100%), and 55/70 (79%), respectively. Mean values of HAV RNA at admission were 3.48 ± 1.30 logcopies/ml in fulminant, 4.19 ± 1.03 in severe acute, and 2.65 ± 1.64 in acute hepatitis. Patients with severe infection such as fulminant hepatitis and severe acute hepatitis had higher initial viral load than patients with less severe infection (P < 0.001). Viremia persisted for 14.2 ± 5.8 days in patients with severe infection and 21.4 ± 10.6 days in those with acute hepatitis after clinical onset (P = 0.19). HAV RNA was detectable quantitatively in the majority of the sera of hepatitis A cases during the early convalescent phase by real‐time PCR. Higher initial viral replication was found in severely infected patients. An excessive host immune response might follow, reducing the viral load rapidly as a result of the destruction of large numbers of HAV‐infected hepatocytes, and in turn severe disease might be induced. J. Med. Virol. 83:201–207, 2011. © 2010 Wiley‐Liss, Inc.     

Infection with hepatitis A, B, C, and delta viruses among patients with acute hepatitis in Mongolia

One hundred ten consecutive patients (60 males and 50 females; age, mean +/- standard deviation [SD], 22.6 +/- 6.4 years; range 16-48 years) who were clinically diagnosed with sporadic acute hepatitis between December 2004 and January 2005 in Ulaanbaatar, Mongolia, were studied. IgM antibodies to hepatitis A virus were detected in 18 patients (16.4%), IgM antibodies to hepatitis B core (anti-HBc IgM) in 38 patients (34.5%) including two patients with concurrent hepatitis delta virus (HDV) infection, and hepatitis C virus RNA in nine patients (8.2%). There were 30 hepatitis B virus (HBV) carriers who had detectable hepatitis B surface antigen and antibodies to HDV but were negative for anti-HBc IgM, suggesting that they acquired type D acute hepatitis due to superinfection of HDV on a background of chronic HBV infection. None had IgM antibodies to hepatitis E virus (HEV). Consequently, 16.4, 32.7, 6.4, 1.8, and 27.3% of the patients were diagnosed as having acute hepatitis of type A, B, C, type B + D (HBV/HDV coinfection), and type D (superinfection of HDV), respectively. The cause of hepatitis was not known in the remaining 17 patients (15.5%). All 18 HAV isolates were genotyped as IA, all 9 HCV isolates were genotyped as 1b, and all 32 HDV isolates were classified into genotype I. The distribution of HBV genotypes among the 67 HBV isolates was A (1.5%, n = 1) and D (98.5%, n = 66). The present study indicates that de novo infections of HAV, HBV, HCV, and HDV are prevalent among young adults in Mongolia.     

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.