Hepatitis E Virus Fluorescence Reduction Neutralization Test for Measuring Neutralizing Antibody Titers

Virus neutralization assay measures the neutralization potential of an antibody or a mixture of antibodies against the corresponding virus. It is a valuable technique to evaluate the efficacy of vaccine candidates and neutralizing antibodies developed against the virus. The plaque reduction neutralization test (PRNT) is a popular method to evaluate the titer of virus-specific neutralizing antibodies. However, PRNT is not feasible in the case of non-cytopathic viruses. Hepatitis E virus (HEV) is a positive-sense single-stranded RNA virus, belonging to the family Hepeviridae. It is a major cause of acute viral hepatitis worldwide. It does not show any cytopathic effect in mammalian cell culture. A vaccine against HEV is available in China. However, vaccines or therapeutic antibodies against HEV are not available in most of the world. Several laboratories have been working on vaccine candidates against the HEV. A neutralization assay to measure the efficacy of HEV vaccines will be a very useful tool for researchers. Here, we describe a fluorescence reduction neutralization test (FRNT) to estimate the 50% neutralization titre (NT50) of anti-HEV antibodies. Anti-HEV ORF2-neutralized, or rabbit IgG-neutralized HEV, was used to infect Huh7 cells, followed by immunofluorescent staining of the viral ORF2 protein. Fluorescent positive cells were quantified in the ImageJ application, and NT50 was estimated to be 2.1 x 10³. This method may be applied to estimate the NT50 of anti-HEV antibodies present in immunized-mice sera, HEV-recovered individual sera, and laboratory-produced anti-HEV antibodies.