The slow growth of hepatitis A virus (HAV) in cell culture is one of the primary pitfalls in the development of sensitive and rapid methods for the detection and quantification of HAV and associated neutralizing antibodies. Currently, in vitro assays frequently require 8 days or more to detect and quantify the presence of HAV neutralizing antibodies. This study describes a rapid immunoassay that allowed the detection of anti-HAV neutralizing antibodies in only 3 days. This microplate-based enzymic assay may be applicable in virological diagnostics, in evaluating the immunogenicity of HAV vaccines and in quantifying neutralizing antibodies during the course of HAV infection.
INTRODUCTIONHepatitis A virus (HAV), a member of the family Picornaviridae, is a non-enveloped, positive-sense RNA virus with a pervasive worldwide transmission (Brown, 1989). HAV causes acute liver infection with a sudden onset of symptoms such as fever and nausea (Jelic et al., 1990;Nainan et al., 2006). The virus is transmitted via the faecal-oral route and infects approximately 1.4 million people every year (Chen & Cantrell, 2006). The HAV genome encodes a single polyprotein of approximately 200 kDa, which is subsequently processed into the structural proteins VP1-VP4 encoded by the viral region P1, and non-structural polypeptides encoded by viral regions P2 and P3 (Endo et al., 2007). HAV grows extremely slowly in cell culture and often replicates without any visible cytopathic effect, unlike other members of the family Picornaviridae such as poliovirus and human rhinovirus (Gauss-Muller et al., 1986;Stapleton et al., 1993;Zahn et al., 1984). Various in vitro assays have been developed to assess the activity of anti-HAV neutralizing antibodies; however, these assays are often lengthy (requiring 8 days-3 weeks) (Beales et al., 1996;Cao et al., 2008;Kim et al., 2004;Konduru et al., 2008) and may be more labour-intensive than microplate-based assays. Thus, a simpler and more rapid method for detecting and quantifying HAV neutralizing antibodies, perhaps amenable to a higher-throughput format, would be beneficial.Over the past few years, cytopathic variants of HAV have been generated (Brack et al., 1998;Emerson et al., 1993). These HAV strains cause acute rather than persistent infection and produce a much higher viral yield than noncytopathic variants. Using a cytopathic variant, we have developed a sensitive, specific and reproducible microplate-based assay for evaluating HAV neutralizing antibody responses. In this immunoassay, only one replication cycle of HAV appeared to be required in order to detect infectivity. To our knowledge, this is the first report that describes a colorimetric assay capable of detecting HAV infectivity and HAV neutralizing antibodies in only 3 days.
METHODSCells and virus. Fetal rhesus monkey kidney (FRhK-4) cells and HM175/18f, a cell-culture-adapted, cytopathic variant of the HM175 strain of HAV (Lemon et al., 1991), were obtained from Dr Syed Sattar (University of Ottawa, Canada). FRhK-4 cells were grown in ...