Hepatitis a virus: molecular detection and typing.

Abstract

Hepatitis A virus (HAV) infection is the leading cause of viral hepatitis throughout the world. HAV infection is mainly propagated via the fecal-oral route, and waterborne and foodborne outbreaks of the disease have been reported.HAV, the prototype of the genus Hepatovirus, belongs to the family Picornaviridae. Its 7.5-kb single-stranded RNA genome bears different distinct regions: the 5' and 3' noncoding regions (NCR), the P1 region, which encodes the structural proteins VP1, VP2, VP3, and a putative VP4, and the P2 and P3 regions encoding nonstructural proteins associated with replication. A single HAV serotype has been described, although seven genotypes have been defined. Since environmental samples usually contain low numbers of viral particles, sensitive methods such as molecular techniques based on nucleic acid amplification are required for their detection. However, even with the adoption of these techniques, the choice of the most adequate target is of relevant importance. The target region should be highly conserved, to increase the chance of detection, and should have an appropriate structure and length to allow sensitivity high enough for these kind of samples. As a target region, we have chosen a fragment of the 5'NCR flanked by highly conserved sequences that have been used for the primer design (forward primer from position 68 to position 85; reverse primer from position 222 to position 240 in the HM175 strain of HAV; GenBank accession number M14707). The internal part of this region, however, may present a certain degree of variation mainly owing to insertions and/or deletions, causing a variable size of the amplimer obtained, i.e., the wild-type HM175 strain gives a size of 174 bp whereas the cell-adapted pHM175 strain gives a size of 186 bp. For this reason it is extremely important to include a confirmative method such as Southern blot hybridization with an internal probe from a region not affected by the insertions/deletions.