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.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...

Methods to detect infectious human enteric viruses in environmental water samples

“…Faster than conventional cell culture; less susceptible to PCR inhibitors Costly; carryover detection of nucleic acid of inactivated viruses; non-appropriate for non-culturable viruses, specific to certain type of viruses Ko et al (2003Ko et al ( , 2005 Nuclease and proteinase treatment before PCR Rapid; may confirm damaged viral capsid; no need for cell culture Unable to assess the thermal inactivation that may occur at 37 • C; use capsid integrity as the criterion for the infectivity Cliver (2002, 2003) Sano et al (2010) ml obtained by the IFA were approximately 2 log 10 less than the values in genome copies per ml obtained by qPCR (Calgua et al, 2011). A different approach based on the detection of the newly synthesized viral nucleic acid by molecular beacon combined cell culture assay was described (Yeh et al, 2008a). Molecular beacons (MBs) are single stranded hairpin shaped oligonucleotide probes, labelled at the 5 and 3 ends with reporter and quencher dye, respectively.…”

“…In the presence of the target sequence, they unfold, bind and fluoresce. Yeh et al (2008a) used hepatitis A virus as a surrogate for enteric viruses. The detection of infectious HAV with a detection limit of 1 PFU within 6 h post-infection was achieved so the reported fluorescence assay combines a 32-fold reduction in detection time with a quantitative capability similar to that for the 8-day conventional plaque assay for HAV at the same cells.…”

Visualizing the dynamics of viral replication in living cells via Tat peptide delivery of nuclease-resistant molecular beacons

“…The spontaneous hybridization between MBs and their target sequences is highly specific and can even distinguish a single nucleotide mismatch (2)(3)(4). Recently, MBs have been used to detect the presence of viral RNA in infected cells, with positive responses to even a single infectious viral particle (5). However, the long-term, real-time monitoring of viral RNA in living cells has not been possible because of the relatively short half-life (Ϸ30 min) of MBs due to endogenous nuclease degradation, resulting in false-positive signals (6,7).…”

Use of Flow Cytometry for Rapid, Quantitative Detection of Poliovirus-Infected Cells via TAT Peptide-Delivered Molecular Beacons

“…Molecular beacons have been employed for various applications, such as in vitro hybridization assays (9-11), RNA imaging (12), and real-time detection of DNA-RNA hybridization in living cells (13). Although MBs have been commonly used for intracellular detection of gene expression, their application for the in vivo detection of viral RNA have only been reported recently (14). The use of nuclease-resistant MBs enables the real-time detection of viral replication in living cells via Tat peptide delivery (15).…”