In the Korean group, we found222C to G or T substitution in 8/12 cases of severe disease and222C to G or T and392G to A substitutions in 5/7 and 4/7 cases of mild disease, respectively

In the Korean group, we found222C to G or T substitution in 8/12 cases of severe disease and222C to G or T and392G to A substitutions in 5/7 and 4/7 cases of mild disease, respectively. The number of nucleotide substitutions is shown inFigure 2A & 2B, with the average number between AZD1981 nucleotides 200 and 500 being 9.7 (8.2) [mean (SD)] per case in severe disease and 5.4 (5.2) in mild disease. self-limited cases (25.9%)], all of which were subgenotype IA. Sequence analysis revealed that sequences of severe disease had 93.6%99.0% homology and of self-limited disease 94.3%98.6% homology, compared to subgenotype IA HAV GBM wild-type IA sequence. In this study, confirmation of the 5NTR sequence differences between severe disease and mild disease was not carried out. Comparison with Japanese HAV A10 revealed222C to G or T substitution in 8/12 cases of severe disease and222C to G or T and392G to A substitutions in 5/7 and LPL antibody 4/7 cases of mild disease, respectively, although the nucleotide sequences in this study showed high homology (93.6%100%). In conclusion, HAV 5NTR subgenotype IA from AZD1981 Korea had relatively high homology to Japanese sequences previously reported from Japan, and this region would be considered one of the antiviral targets. Further studies will be needed. == Introduction == Although hepatitis A vaccination is highly effective, providing herd protection and decreasing mortality and morbidity related to the hepatitis A virus (HAV)[1][3], HAV is still a common cause of hepatitis reportedly leading to occasional lethal acute liver failure in many countries of the world[4][7]. Recently, a rise in the frequency of hepatitis A outbreaks was observed in South Korea, which lies adjacent to Japan, while the number of adult hepatitis A cases in Japan has been progressively decreasing during the last several years. There is a concern regarding a possible HAV epidemic in Japan in the near future, as universal vaccination against hepatitis A is not performed in this country. HAV is a member of the genusHepatovirusin thePicornaviridaefamily, and has a positive-sense single-stranded RNA genome approximately 7.5 kb in length[8]. The genome codes a large open reading frame (ORF), which is flanked by 5 nontranslated region (5NTR) and 3NTR. The downstream part of 5NTR represents the internal ribosomal entry site (IRES), which mediates cap-independent translation initiation and is important for HAV replication[9],[10]. 5NTR of HAV is also AZD1981 known as one of the most highly conserved in the HAV genome sequences, making this region one of the likely candidates for antiviral targets[9],[11]. It was reported that nucleotide variations in the central portion of 5NTR of HAV may influence the severity of hepatitis A[12]. Human HAV strains can be grouped into four genotypes (I, II, III and IV) and unique simian strains belong to three additional genotypes (IV, V and VI). Between each of these genotypes, the nucleotide sequence varies by 1520% of the base positions in the P1 region[13]. Genotype I is the most abundant type worldwide, and genotype IA in particular has been reported from North America, AZD1981 Korea, China, Japan and Thailand[14]. The aim of this study is to characterize the recent HAV genotype I 5NTR sequences in Korea, to compare them with those reported from Japan and to clarify this region as a target candidate for anti-HAV drugs. == Materials and Methods == == Patients == Fifty-four patients infected with HAV subgenotypes IA and IB were included in this study. Serum samples were collected at four hospitals located in the Seongnam city area, near Seoul, South Korea. Our study was approved by the Seoul National University Bundang Hospital Institutional Review Board (IRB), and we obtained written informed consent from every patient enrolled during Sep 2008 to Aug 2008. We collected serum or plasma samples immediately after hospital admission, and they were stored at 70C. The 54 patients comprised 27 with severe disease, defined as prolonged prothrombin time [international normalized ratio (INR) > or = 1.5] and 27 with mild disease: self-limited acute hepatitis in this study (Table S1A & S1B). == Primers for PCR and Direct Sequencing == For amplification of HAV sequences and bidirectional direct sequencing of the amplified segments, we prepared several primers for PCR and sequencing as previously described[12]. These primers were prepared with the sequence reported by Cohen et al[8]. == Detection of Hepatitis A Virus RNA in Serum == RNA was extracted from sera using the acid guanidinium-phenol-chloroform method. Reverse transcription was performed with HAV genome specific antisense primer (5-AGTACCTCAGAGGCAAACAC-3) as previously described[12]. In the first round PCR, 1 l of 20 l of the cDNA solution was used. The first round PCR was performed with 50 l of reaction mixture containing 25 pmol of outer antisense primer (5-AGTACCTCAGAGGCAAACAC-3) and sense primer (5-TCTTGGAAGTCCATGGTGAG-3), 200 M of each dNTP, 50 mM KCl, 10 mM Tris HCL (pH 8.3), 1.5 mM MgCl2, 0.001% gelatin, and 2.5 units of Ex Taq polymerase (Takara Bio Inc., Ohtsu, Shiga, Japan). Amplification conditions consisted of 35 cycles of 95C for one minute, 50C for one minute, and 72C for one minute, and 1 l of the first round product was used for the second round of PCR with the same PCR mixture, except 1.0 M of inner.