Detection Of Infectious HAV Using ICC/Strand-specific RT-PCR And Construction Of Two HAV Chimeras

Hepatitis A virus, the only member of the Hepatovirus genus in the Picornaviridae family, is unique among picornaviruses with regard to its specific features of growth and molecular biology. HAV normally is not cytopathic in infected cells, and its growth is a lengthy, inefficient process with low virus yield, which usually takes 3 to 4 weeks. These factors made the detection and titration of infectious HAV difficult, lengthy and laborious tasks. Presently no rapid assay is available. HAV IRES is also distinguishing from other picornavirus IRESs and assigned to a separate minor group. In this paper, the establishment of a new, rapid method for the specific detection of infectious HAV and the feasibility to exchange HAV IRES and 5' NTR with its counterparts from poliovirus were investigated.A new and rapid method, namely integrated cell culture/ strand-specific RT-PCR (ICC/ strand-specific RT-PCR), was described for the specific detection of infectious HAV, based on the demonstration of viral replication in infected cells by detecting the formation of negative-strand RNA replicative intermediate using strand-specific RT-PCR. This method, a combined cell cultural and molecular biological technique, involved initial propagation of infectious virus particles in cell culture and then detection of negative-strand RNA replicative intermediate. It could distinguish between infectious and non-infectious HAV, and no positive occurred in the presence of high concentrations of formalin-inactivated HAV. ICC/ strand-specific RT-PCR could detect infectious HAV at inoculation level of 10°TCID₅₀/ml in spiked water samples after 84 hr of incubation. Using ICC/ strand-specific RT-PCR, the titer of live vaccine sample Ref was determined as 10^7.83 TCID₅₀/ml after 5 days and 10^8.0 TCID₅₀/ml after 8 days of cell culture incubation. The results were very close to that from ELISA-based assay (10^7.67 TCID₅₀/ml). Coupled with a suitable virus concentration and purification system, ICC/ strand-specific RT-PCR should be a practical approach to monitor virological safety of food and environmental samples and to study the molecular epidemiology of hepatitis A. It also could be adopted to test the effective inactivation of inactivated virus vaccines as well as to evaluate the efficacy of disinfection of HAV and enteric viruses...