| Summary: | <p>Abstract</p> <p>Background</p> <p>Hepatitis A virus (HAV), an atypical Picornaviridae that causes acute hepatitis in humans, grows poorly in cell culture and in general does not cause cytopathic effect. Foreign sequences have been inserted into different parts of the HAV genome. However, the packaging size limit of HAV has not been determined. The purpose of the present study is to investigate the maximum size of additional sequences that the HAV genome can tolerate without loosing infectivity.</p> <p>Results</p> <p><it>In vitro </it>T7 polymerase transcripts of HAV constructs containing a 456-nt fragment coding for a blasticidin (Bsd) resistance gene, a 1,098-nt fragment coding for the same gene fused to GFP (GFP-Bsd), or a 1,032-nt fragment containing a hygromycin (Hyg) resistance gene cloned into the 2A-2B junction of the HAV genome were transfected into fetal Rhesus monkey kidney (FRhK4) cells. After antibiotic selection, cells transfected with the HAV construct containing the resistance gene for Bsd but not the GFP-Bsd or Hyg survived and formed colonies. To determine whether this size limitation was due to the position of the insertion, a 606 bp fragment coding for the Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence was cloned into the 5' nontranslated (NTR) region of HAV. The resulting HAV-IRES retained the EMCV IRES insertion for 1-2 passages. HAV constructs containing both the EMCV IRES at the 5' NTR and the Bsd-resistance gene at the 2A-2B junction could not be rescued in the presence of Bsd but, in the absence of antibiotic, the rescued viruses contained deletions in both inserted sequences.</p> <p>Conclusion</p> <p>HAV constructs containing insertions of approximately 500-600 nt but not 1,000 nt produced viable viruses, which indicated that the HAV particles can successfully package approximately 600 nt of additional sequences and maintain infectivity.</p>
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