PCR-SSCP Analysis of 5"-Nontranslated Region of Hepatitis A Viral RNA:

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Abstract
The recent development of the sensitive reverse transcription-polymerase chain reaction (RT-PCR) method allowed us to detect the presence of hepatitis A virus (HAV) RNA in sera from hepatitis A patients. To determine whether differences in HAV are related to the wide range of clinical severity, we used PCR–single strand conformation polymorphism (SSCP) to analyze the amplified product of the 5"-nontranslated region (5"NTR), where relatively homologous sequences are reported and the internal ribosomal entry site is considered to exist, from these various levels of hepatitis A. Twenty-seven patients admitted to Chiba University Hospital (between 1988 and 1997) were examined for HAV RNA in their sera by RT-PCR with primers located at 5"NTR of HAV RNA. The nucleotide (nt) sequence of a central part (nt 277–551) of 5"NTR was amplified and the product was examined by PCR-SSCP. HAV RNA was detected in all 27 hepatitis A patients examined: in 3 with fulminant hepatitis, 2 with severe acute hepatitis, and 22 with self-limited acute hepatitits. The amplified sequence revealed by PCR-SSCP showed that samples from fulminant and severe acute hepatitis patients had similar mobility in the gel, whereas that from acute hepatitis patients demonstrated a considerable variety in mobility patterns. From the analysis of the amplified product of 5"NTR of HAV, the bands are likely to be similar in the more severe forms of hepatitis A compared to those from uncomplicated self-limited acute hepatitis.