Meanwhile, the RTCA assay could directly distinguish different strains of the same virus on the basis of our results. For quantitation of virulence, it is necessary to compare different viral stains, determine the number of infectious units required to produce the specific endpoint. Taken together, it is indicated that the RTCA system may provide a feasible in vitro format for assessment of virulence by monitoring CPE kinetics in future investigations of the influenza virus. It may further help to elucidate the relationship between genetic alterations and virulent variations for virological surveillance of influenza. Since virus-induced CPE could be quantitatively monitored using the RTCA system, we also performed a real-time neutralization assay for measuring H1N1-specific antibodies in human sera using the RTCA system. Since the HI test is still used as a standard for epidemiological and immunological studies, as well as for measuring the efficacy of influenza vaccines and potency of neutralizing antibodies, this new assay was evaluated against the standard HI test on a panel of sera collected from adult donors before and after immunization with the H1N1 vaccine. Both assays showed an obvious increase in neutralizing antibodies in the test sera at 3 weeks post-vaccination, and there was a good agreement between the HI and NT antibody titers. However, higher antibody titers were detected using the RTCA-based NT method, indicating the higher sensitivity of this assay. This may have been due to the difference between the CPE-based and HAbased assays, which had been observed in previous reports. Such functional quantitation may provide a valuable platform for serological diagnosis, immunological study, or evaluation of vaccines for influenza. An HI titer of 1:40 is commonly recognized as representing protective immunity. Our results revealed 30% of the HI titers in the pre-vaccination sera were equal to 1:40, and two were even higher. The first H1N1 case was identified in May 2009 in Shanghai, and population-wide vaccination followed in October 2009. Hence, widespread exposure to H1N1 in the society may have caused the higher antibody titers in the donors detected here. In addition, other reports have suggested that serum cross-reactive antibody responses to H1N1 occurred after vaccination with seasonal influenza vaccine and that this also existed in the population during the pre-pandemic period. In many settings influenza is recognized as a major cause of disease and death worldwide, which is the first infectious disease with global surveillance. Not the 2009 Influenza A Virus but other concomitant seasonal or highly pathogenic avian influenza viruses have posed considerable threads to public health. Global surveillance and annual vaccination are both of the key strategies and measures for the prevention and control of influenza.