Hepatitis A virus (HAV) exclusively infects the gastrointestinal tract of primates and does not naturally infect the central nervous system. To test HAV's ability to replicate in neural tissue, cultured human fetal brain tissue (HFBT) was infected with HAV/7. In vitro cytopathic effects were not seen after infection. Semiquantitative slot blot analyses, using RNA dilutions of 1:10 and 1:100, documented HAV/7 replication in HFBT up to 19 days after the initial infection. Immunofluorescent staining using a monoclonal antibody against the viral capsid demonstrated the presence of HAV/7 and counterstaining with a monoclonal neurofilament (68kd) antibody identified HAV/7 in the cytoplasm of neurons. To test the safety, efficacy and in vivo ability of HAV/7 to replicate in the central nervous system, 5 x 106TCID50/ml was directly inoculated into the thalami and lumbar spinal cords of four Mucaca mulatta monkeys. The animals were euthanized at 1 hour, 1 week, 2 weeks and 4 weeks after inoculation. RT- PCR was used to detect the presence of small quantities of the negative strand RNA replication intermediate (RI). To overcome the high degree of secondary structure present in the highly conserved 5' NCR of the HAV RNA, a thermostable reverse transcriptase (rTth DNA polymerase) permitted transcription to proceed at a higher temperature. The PCR products were hybridized with either a 32P-labeled full length HAV cDNA (10kb) or an overlapping PstI HAV cDNA (687 bp) fragment. Both probes identified a specific 251-bp fragment. RT-in situ PCR identified the RI in the cytoplasm of neurons in widespread areas of the brain and spinal cord in tissue samples from these monkeys. Histological evaluation of the adjacent spinal cord and brain areas did not show gliosis, vacuolization or an inflammatory response. The 5' NCR region of HAV/7 was cloned into the plasmid pALTER-1 preparation for oligonucleotide-directed mutagenesis downstream from the IRES. Two unique restriction enzyme sites (9 base pairs) were cloned at nt 776 by annealing a 50 base pair mutagenic oligonucleotide to a double-stranded DNA template. The CAT gene was inserted in-frame at this site and transcription was driven by the plasmid T7 promoter. Fetal rhesus monkey kidney cells were lipofected with the DOTAP reagent. CAT activity was measured by ELISA at days 7, 12, 16, 19 and 26 post-transfection. Low levels of CAT (20-30 pg/ug protein) expression were detected at day 16 post-transfection. These studies suggest that HAV/7 can act as a vector for viral-mediated gene transfer.
