A Sensitive neutralization assay for influenza C viruses based on the acetylesterase activity HEF glycoprotein

Influenza C virus possesses specific neuraminate-O-acetylesterase as a receptor-destroying function. This enzymatic activity of the viral glycoprotein HEF (Hemagglutinin, esterase activity and fusion factor) can be visualized in situ by the use of distinct colour substrates. Hereby the localization, as well as the quantity of synthesized HEF protein is detectable. We further developed the esterase staining technique for a rapid detection and typing of influenza C progeny virus and for sensitive quantification of low viral loads. Neutralizing antibodies, interfering with virus attachment, were utilized to determine the infectivity of the inoculum in an indirect manner. The amount of unneutralized, infectious virus could easily be quantitated by in situ staining of the HEF esterase activity in infected cells. An evaluation of infectivity is presented and is put into relation to hemagglutinating virus units. Both, virus and serum antibody titers can be reliably determined by the esterase-neutralization assay. In this study a serial dilution of human and different animals (swine, dog and rabbit) sera were tested by in situ estrase neutralization assay (ENA) and hemagglutination inhibition (HI) test. The results show that in situ ENA is a sensitive method for titration of infectious rate of virus and quantification of neutralizing antibody against influenza C virus in different sera.