A small molecule library containing 480 known bioactive compounds was screened for antiviral activity against poliovirus (PV) using a cellular fluorescence resonance energy transfer (FRET) assay for viral protease activity. The infected reporter cells treated with the viral replication-suppressing compounds were examined via fluorescence microscope 7.5 h postinfection. Twelve molecules showed moderate to potent antiviral activity at concentrations less than 32 microM during the primary screening. Three compounds, anisomycin, linoleic acid, and lycorine, were chosen for validation. A dose-dependent cytotoxicity assay and a secondary screening using conventional plaque assay were conducted to confirm the results. The developed method can be used for rapid screening for molecules with antiviral activity.

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Two newly developed protocols for infective virus detection were compared to the plaque assay. An immunomagnetic separation procedure coupled with real-time reverse transcription-PCR of viral nucleic acids was developed to identify intact enteroviral particles, and a reporter cell system responding to viral replication based on fluorescent resonance energy transfer for detection of infectious enteroviruses was tested. Both new procedures detected infective viruses in environmental samples at the same level as the plaque assay.

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.Hepatitis A virus (HAV), a positive single-stranded RNA virus, was previously classified as enterovirus type 72 (14) and has now been classified in the Hepatovirus genus of the Picornaviridae family (15). These single-plus-stranded RNA viruses can directly translate plus-strand RNA genomes into protein using the host ribosomes. The plus strand is transported to the infected cell via specific membrane vesicles, where it is copied into full-length minus strands. These minus strands then serve as templates for the synthesis of plus-strand genomic RNA molecules (1, 2).HAV is known to cause acute liver infection with a discrete onset of symptoms (e.g., fever, malaise, and nausea), followed in several days by jaundice. Person-to-person transmission through the fecal-oral route is the primary means of HAV transmission. Outbreaks and periodic cases also occur from exposure to fecally contaminated food or water (Centers for Disease Control and Prevention). Most picornaviruses are lytic, causing distinctive cytopathic effects and replicating in an 8-h cycle under one-step growth conditions. The growth of HAV in cell culture systems, however, is much slower and nonlytic and does not produce a detectable cytopathic effect in infected cells (13). Cromeans et al. reported the isolation of a cytopathic HAV variant from the rapidly replicating isolate HM-175 that is lytic for fetal rhesus monkey kidney (FRhK-4) cells (4). However, complete lysis still did not occur until 5 to 6 days postinfection (p.i.).Conventional methods for detecting infectious HAV rely on viral propagation in cell culture, radioimmunoassay, immunofluorescence, or plaque assay; these methods are difficult to perform, and it may take weeks before the viruses reach sufficiently high amo...