CLINICAL STUDIES Phylogenetic analysis of hepatitis A virus in sera from patients with hepatitis A of various severities Keiichi Fujiwara 1 , Hiroshige Kojima 1 , Yutaka Yonemitsu 1 , Shin Yasui 1 , Fumio Imazeki 1 , Makoto Miki 2 , Kazuyuki Suzuki 3 , Isao Sakaida 4 , Kiwamu Okita 4 , Eiji Tanaka 5 , Masao Omata 6 and Osamu Yokosuka 1 1 Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan 2 Department of Internal Medicine, Yokohama Higashi National Hospital, Kanagawa, Japan 3 First Department of Internal Medicine, Iwate Medical University, Iwate, Japan 4 Department of Gastroenterology and Hepatology, Yamaguchi University School of Medicine, Yamaguchi, Japan 5 Department of Medicine, Shinshu University School of Medicine, Nagano, Japan 6 Department of Gastroenterology, Faculty of Medicine, University of Tokyo, Tokyo, Japan Keywords 2B – 2C – 5 0 NTR – fulminant hepatitis – hepatitis A – hepatitis A virus Abbreviations AH, acute hepatitis; AHs, acute hepatitis severe type; FH, fulminant hepatitis; HAV, hepatitis A virus; PT, prothrombin time. Correspondence Keiichi Fujiwara, MD, PhD, Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Tel: 181 43 226 2083 Fax: 181 43 226 2088 e-mail: fujiwara-cib@umin.ac.jp Received 13 August 2008 Accepted 4 October 2008 DOI:10.1111/j.1478-3231.2008.01919.x Abstract Background: We analysed the association of the 5 0 nontranslated region (5 0 NTR), nonstructural proteins 2B and 2C of the hepatitis A virus (HAV) genome, whose mutations have previously been shown to be important for enhanced replication in cell culture systems, in order to align all our data and examine whether genomic differences in HAV are responsible for the range of clinical severities. Methods: Our accumulated HAV strains of 5 0 NTR [nucleo- tide(nt) 200 and 500], entire 2B and 2C from 25 Japanese patients with sporadic hepatitis A, consisting of seven patients with fulminant hepatitis (FH), five with severe acute hepatitis (AHs) and 13 with self-limited acute hepatitis (AH), in whom the sequences of all three regions were available, were subjected to phylogenetic analysis. Results: Fulminant hepatitis patients had fewer nucleotide substitutions in 5 0 NTR, had a tendency to have more amino acid (aa) substitutions in 2B and had fewer aa substitutions in 2C than AH patients. Four FH and two AHs with a higher viral replication were located in the near parts of the phylogenetic trees, indicating the association between the severity of hepatitis A and genomic variations in 5 0 NTR, 2B and 2C of HAV. Conclusions: Our study suggests that genetic variations in HAV not in one specific region but in 5 0 NTR, 2B and 2C might cooperatively influence replication of the virus, and thereby affect virulence. Viral factors should be considered and examined when discussing the mechanisms responsible for the severity of hepatitis A. Hepatitis A is still a major problem worldwide, not only in underdeveloped countries but also in industrialized nations. Because of improvements in sanitation, there have been no hepatitis A epidemics in Japan in recent years. However, sporadic cases of hepatitis A have not been rare of late. Although the majority of hepatitis A cases are self-limited acute hepatitis (AH), some develop into severe forms of hepatitis (1). In fact, in the past several years, there has been an increase in the numbers of patients with sporadic hepatitis A, especially the more severe kind, visiting our hospital. Our analysis of the possible factors responsible for the disease severity in our patients revealed no significant differences in terms of background including age, suggesting that viral factors might be involved in determining the severity of the disease (2, 3). Hepatitis A virus (HAV) is the sole member of the hepatovirus genus and a member of the Picornavirus family. Virological studies have revealed that HAV is a positive-strand RNA virus comprising approximately 7500 nucleotides and containing a 5 0 nontranslated region (NTR), a single long open reading frame encoding a large polyprotein and a 3 0 NTR. A large polyprotein is cleaved by the viral protease to produce the P1, P2 and P3 regions. The P1 region encodes four structural proteins – VP4, VP2, VP3 and VP1. The P2 and P3 regions encode nonstructural proteins 2A, 2B and 2C, and 3A, 3B, 3C and 3D respectively (4). As far as is known, nonstructural protein 2A participates in virion morphogenesis (5). 2B and 2C play important roles in the replication of viral RNA. 2C is a multifunctional protein and is considered to have helicase and NTPase activities. 2C or 2BC have membrane- and RNA-binding properties (6). 3B is considered to be a genome-linked viral protein (Vpg), 3A a pre-Vpg, 3C a viral protease and 3D an RNA- dependent RNA polymerase. Liver International (2009) 838 c  2009 The Authors. Journal compilation c  2009 Blackwell Publishing Ltd Liver International ISSN 1478-3223