Detection of Hepatitis A Virus by Using a Combined Cell Culture-Molecular Beacon Assay

ABSTRACT Rapid and efficient methods for the detection and quantification of infectious viruses are required for public health risk assessment. Current methods to detect infectious viruses are based on mammalian cell culture and rely on the production of visible cytopathic effects (CPE). For hepatitis A virus (HAV), viral replication in cell culture has been reported to be nonlytic and relatively slow. It may take more than 1 week to reach the maximum production and subsequent visualization of CPE. A molecular beacon (MB), H1, specifically targeting a 20-bp 5′ noncoding region of HAV, was designed and synthesized. MB H1 was introduced into fixed and permeabilized fetal rhesus monkey kidney (FRhK-4) cells infected with HAV strain HM-175. Upon hybridizing with the viral mRNA, fluorescent cells were visualized easily under a fluorescence microscope. Discernible fluorescence was detected only in infected cells by using the specific MB H1. A nonspecific MB, which was not complementary to the viral RNA sequence, produced no visible fluorescence signal. This MB-based fluorescence assay enabled the direct counting of fluorescent cells and could achieve a detection limit of 1 PFU at 6 h postinfection, demonstrating a significant improvement in viral quantification over current infectivity assays.

Inactivation of Hepatitis a Virus by Heat and Formaldehyde

The GBM strain of hepatitis A virus adapted to a fetal rhesus monkey kidney cell line (Frhk-4/R) was treated and inactivated at several different temperatures and with several different formaldehyde concentrations. Heat inactivation showed that no inactivation occurred after treatment for 60 minutes at 37°C. Treatment for 60 minutes at 50°C reduced infectivity by 1 log 10 step ; treatment for 60 minutes at 60°C, by 4 log 10 steps. Treatment for 30 minutes at 70°C resulted in complete inactivation of HAV. Complete inactivation by formaldehyde treatment was achieved only with a 0.35% concentration. Treatment with lower concentrations resulted in incomplete HAV inactivation.