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2. Replication in CellCulture Since no CPE was observedi n HAV-infected cultures, viruswas detected by immunotluorescent localization of HAV antigen in the cytoplasm ( 1 19,124). Upon initial isolation, HAV antigen accumulates slowly, but with increasing passage the time required for maximum antigen production can be shortened (133,134). However, in all instances maximum yields of virus were not obtained before 3 weeks post infection. The lack of CPE resulted i n the establishment of persistently infected cell cultures which have been used to produce large quantities of virus ( 135- 139).

Nested PCR using a second round of primers internal to the first set has been used to increase the sensitivity of detection by an order of magnitude and provide the same degree of speciticity observed with hybridization of amplification products. 49) (B. H. , in preparation); however, the infectivity of virus excreted at the later time points has not been determined. The level of detection of HAV by IC-RT-PCR has been as low as 0. 01 radioinmunofocus units or plaque-forming units (PFUs) using laboratory prepara- 38 al.

Through con- Cromeans et al. 28 taminated food and/or water, or from person-to-person contact) large outbreaks of disease have occurred. An example of the impact of the changing epidemiology of HAV infection occurred in1988 in Shanghai, China. Seroprevalence studies had shown low rates of infection in 1988, a very large epidemic among young children and young adults (74). However, of hepatitis A occurred over several months (more than 300,000 cases) because adults ate partiallycookedclamsthatwereharvestedfromareascontaminatedwithrawsewage (77,78).

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A retrospective and prospective survey of Monte Carlo Method by Halton

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