Nucleotide Variability and Translation Efficiency of the 5′ Untranslated Region of Hepatitis A Virus: Update from Clinical Isolates Associated with Mild and Severe Hepatitis

Abstract

Mutations in the internal ribosome entry site (IRES) of hepatitis A virus (HAV) have been associated with enhanced in vitro replication and viral attenuation in animal models. To address the possible role of IRES variability in clinical presentation, IRES sequences were obtained from HAV isolates associated with benign (n ‫؍‬ 8) or severe (n ‫؍‬ 4) hepatitis. IRES activity was assessed using a bicistronic dual-luciferase expression system in adenocarcinoma (HeLa) and hepatoma (HuH7) cell lines. Activity was higher in HuH7 than in HeLa cells, except for an infrequently isolated genotype IIA strain. Though globally low, significant variation in IRES-dependent translation efficiency was observed between field isolates, reflecting the low but significant genetic variability of this region (94.2% ؎ 0.5% nucleotide identity). No mutation was exclusive of benign or severe hepatitis, and variations in IRES activity were not associated with a clinical phenotype, indirectly supporting the preponderance of host factors in determining the clinical presentation.Hepatitis A virus (HAV) is a nonenveloped RNA virus of the Picornaviridae family. The viral genome consists of an approximately 7,500-nucleotide (nt)-long, positive-stranded RNA divided in three parts: a 5Ј untranslated region (5Ј UTR), a single open reading frame that encodes both structural and nonstructural proteins, and a 3Ј UTR with a short poly(A) tail. By sequencing of the VP1-2A junction and the VP1 gene, 3 genotypes (I, II, and III) divided into A and B subtypes have been described in humans (7,27). HAV is the main cause of acute viral hepatitis worldwide. The majority of cases follow a benign course, but some may be present with fulminant forms, characterized by acute liver failure (factor V levels of Ͻ50% and encephalopathy). HAV-induced liver disease appears to result primarily from immunologic mechanisms, chiefly on the basis of in vitro studies. Most HAV strains have no detectable cytopathic effect in cell culture and no apparent effect on cell growth or metabolism (16), and HAV-infected cells are lysed by cytotoxic T cells isolated from the liver of acutely infected patients (30,31). Clinical studies have suggested that host factors such as age and underlying liver disease were involved in the severity of liver diseases (32, 33) and that the host immune response also played a role in the fulminant forms of hepatitis A, as evidenced by markedly low viral loads (26).Nevertheless, the existence of viral determinants of hepatitis A severity is suggested by both experimental and clinical studies. Indeed, mutations within the VP1-2A and 2C genes have been shown to enhance virulence in tamarins (9). It has also been suggested that 5Ј UTR mutations associated with viral adaptation to cell culture were also responsible for viral attenuation in vivo (15). The 5Ј UTR of HAV is about 735 nucleotides long and is considered the most conserved region of the genome. The 5Ј UTR is involved in genome replication and translation initiation. Folding predictions and biochem...

Comparative analysis of disease severity between genotypes IA and IIIA of hepatitis A virus

“…They suggested that differences in HAV genetic variations of 5 0 non-coding regions 2B and 2C between fulminant and self-limited hepatitis A might cooperatively influence viral replication and virulence. Mackiewicz et al [2010] reported the association of mutations in the internal ribosome entry site of HAV with enhanced in vitro replication, despite lack of relevance to a clinical phenotype. Several studies reported that the viral load might be closely correlated with liver damage and disease severity in mild cases of hepatitis, but not in severe cases [Rezende et al, 2003;Sainokami et al, 2005;Fujiwara et al, 2009].…”

Improving virus production through quasispecies genomic selection and molecular breeding

“…First, the mutations born in haplotypes ɸ3 (U359C; most frequent), ɸ4 (A513G; negative control), ɸ5 (U590C; alternative IRES structure), ɸ7 (U726C; mutation in pY2), ɸ10 (U590C and U726C) and ɸ16 (U359C, U590C and U726C) were introduced in the G1RL0 bicistronic vector, a modified version of the G1RC21 plasmid, in which the translation of the renilla luciferase gene ( RLuc ) is cap-dependent and that of the firefly luciferase gene ( FLuc ) is dependent on the HAV IRES (Supplementary Fig. 1).…”

“…In vitro assays of IRES activity21 and translation efficiency36 of the VP1 region under study suggested that these minor variants could have higher fitness than the dominant mutants. Additionally, following population dynamics principles, these variants were predicted to be better competitors and colonizers than the major variants, although they were numerically suppressed by them14.…”

“…While no clear relationship between the severity of hepatitis A infection and mutations in the IRES has never been reported2142, their role in increasing virus replication in cell culture is well documented43. However, the replication rise observed in the HM175-HP population has no paragon among the HAV strains, and is due to the combined effect of an efficient IRES with codon usage modifications in a context of cellular shut-off.…”

Scientific Opinion on an update on the present knowledge on the occurrence and control of foodborne viruses

“…Among these viral factors it may be pointed that some mutations at the 5'NCR of HAV or at the VP1X2A and 2C regions have been associated with fulminant hepatitis (Fujiwara et al, 2002;Fujiwara et al, 2001;Fujiwara et al, 2003) or higher virulence in tamarinds (Emerson et al, 2002), respectively. However, there is no consensus whether the VP1X2A-derived genotypes are clinically different, although some strains belonging to the former genotype VII now included in genotype II were associated with fulminant cases (Ching et al, 2002;Costa-Mattioli et al, 2002;Mackiewicz et al, 2010).…”