COMPARISON OF RNA HYBRIDIZATION, HEMAGGLUTINATION ASSAY, TITRATION OFINFECTIOUS VIRUS AND IMMUNOFLUORESCENCE AS METHODS FOR MONITORING INFLUENZA-VIRUS REPLICATION IN-VITRO

Rapid and sensitive methods for the monitoring of influenza virus repl ication in vitro are needed to address several research questions. Fou r methods based on different principles were compared. the hemagglutin ation (HA) assay, the measurement of virus infectivity titers in cultu re supernatants, the enumeration of infected cells by immunofluorescen ce and RNA hybridization techniques using digoxigenin (DIG) labeled RN A probes. To this end, MDCK cells were infected at different multiplic ities of infection (moi) with a recent influenza A virus (A/Netherland s/18/94 H3N2) and the kinetics of virus replication were monitored wit h these four assays. At high moi, virus released into the culture supe rnatant of infected cells was detected by the HA assay 12 h post infec tion, whereas at lower moi (less than or equal to 0.01) the first HA a ctivity was not detected before 24 h post infection. The measurement o f infectious viruses in the culture supernatant proved to be more sens itive, since 4-12 h post infection newly produced virus was detected d epending on the moi used. This finding was in agreement with results o btained by the immunofluorescence assay using an antibody preparation specific for the nucleoprotein: single infected cells could be detecte d as early as 4 h post infection. At this time point, positive signals were also obtained when mRNA/cRNA specific hybridization was carried out for the NP gene segment, but not for viral NP RNA or RNA specific for the hemagglutinin, which were only detected at later time points a fter infection. Thus, besides direct measurement of infectious virus a nd immunofluorescence, RNA hybridization proved to be a sensitive assa y for monitoring influenza virus replication in vitro. (C) 1998 Elsevi er Science B.V. All rights reserved.