Association between severity of type A hepatitis and nucleotide variations in the 5' non-translated region of hepatitis A virus RNA: strains from fulminant hepatitis have fewer nucleotide substitutions

Abstract

Background: Type A hepatitis is still a considerable problem in both underdeveloped and developed countries. Why some patients progress to fulminant type A hepatitis and others do not is unclear.

Aims: To determine if nucleotide differences in the genome of hepatitis A virus (HAV) are responsible for the range of clinical severities, we analysed the 5' non-translated region (5'NTR) of the HAV genome, which has an internal ribosomal entry site and is important for cap independent translation of the viral message.

Methods: Serum samples from 84 Japanese patients with sporadic type A hepatitis from five distant regions of Japan, comprising 12 patients with fulminant hepatitis (FH), 13 with severe acute hepatitis (AHs), and 59 with acute hepatitis (AH), were examined for HAV RNA. The fragment between nucleotides 75 and 638 of the 5'NTR was amplified by reverse transcription-polymerase chain reaction, and the nucleotide sequence was determined by direct sequencing.

Results: Comparison of sequences of the 5'NTR revealed relatively fewer nucleotide substitutions in FH and AHs patients compared with the considerable sequence variations found in strains of AH. This tendency was most prominent between nucleotides 200 and 500. Strains from FH and AHs cases had fewer nucleotide substitutions (p<0.001) in this region.

Conclusions: Nucleotide variations in the central portion of the 5'NTR of HAV may influence the severity of type A hepatitis.

Figure 1

Serial changes in the clinical forms of the 84 patients with type A hepatitis analysed for hepatitis A virus 5`non-translated region between 1984 and 1999.

Figure 2

Phylogenetic tree of the sequence between nucleotides 200 and 500 of the 5`non-translated region of the hepatitis A virus genome, as determined by the neighbour joining method. To confirm the statistical reliability of the tree, bootstrap analysis was performed 1000 times. Numbers beside the phylogenetic roots are the results of bootstrap analyses. Horizontal lines indicate the nucleotide sequence distance between the sequences.

Figure 3

Nucleotide sequence of the central portion of the 5`non-translated region of the hepatitis A virus between nucleotides 200 and 500. Horizontal lines indicate identical nucleotides between the individual strains and hepatitis A virus prototype (HAVPT). Vertical lines indicate positions where nucleotide changes occurred in the individual strains compared with HAVPT. FH cases were A10, A1, A413, A425, A502, A601, A204, A5, A206, A414, A165, and A205, respectively. AHs cases were A16, A159, A197, A811, A156, A196, A6, A200, A201, A702, A161, A302, and A160, respectively. AH cases were A141, A180, A404, A406, A713, A809, A819, A19, A39, A62, A168, A301, A304, A402, A711, A802, A814, A8, A32, A36, A38, A51, A55, A68, A303, A306, A407, A422, A424, A807, A816, A97, A157, A195, A207, A503, A9, A162, A707, A305, A307, A409, A75, A812, A7, A40, A65, A71, A80, A712, A93, A28, A20, A77, A85, A158, A423, A408, and A58, respectively.

Figure 4

Number of nucleotide substitutions between nucleotides 200 and 500. Bars represent mean (SD). FH, fulminant hepatitis; AH, acute hepatitis; AHs, severe acute hepatitis.

Figure 5

Distribution of nucleotide substitutions between nucleotides 200 and 500 of the 5`non-translated region. Bars indicate the percentage of cases that have substitutions at each nucleotide position. FH, fulminant hepatitis; AH, acute hepatitis; AHs, severe acute hepatitis.

Figure 6

Portions of the proposed secondary structure of the 5`non-translated region of hepatitis A virus. Arrows indicate positions of nucleotide substitutions. (A) Nucleotide 324 and (B) nucleotide 372 where substitutions are found more frequently in acute hepatitis than in fulminant hepatitis and severe acute hepatitis.