Influenza Virus Inactivation for Studies of Antigenicity and Phenotypic Neuraminidase Inhibitor Resistance Profiling

Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and β-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and β-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.Host switching of viruses from animals to humans may result in an epidemic among humans and can be particularly dangerous for the new, immunologically naïve host. Examples are the introduction of human immunodeficiency virus, severe acute respiratory syndrome coronavirus, and pandemic influenza A viruses in humans. In particular, avian influenza A virus subtypes H5N1, H9N2, and H7N7 have been transmitted directly to humans in the past decade, exhibiting the zoonotic potential of influenza viruses (4, 11, 19, 25). Moreover, the recent introduction of swine origin influenza A(H1N1)v virus in humans initiated the first influenza pandemic of the 21st century (16, 35). Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to assist in the determination of the impact on public health and the development of protective measures. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions hampers timely characterization of such viruses.Several virological and immunological assays are used for the characterization of a virus and the immune response induced. For antigenic characterization of influenza viruses, hemagglutination assays and hemagglutination inhibition (HI) assays are the “gold standard” tests. In addition, since the global emergence of antiviral-resistant influenza viruses is becoming an increasing problem, the characterization of influenza virus susceptibilities to the neuraminidase (NA) inhibitors (NAIs) oseltamivir and zanamivir is a clinical necessity (2, 9, 13, 17, 23). For investigating the immune response against influenza viruses, the HI assay determines protective humoral responses (8). Finally, in addition to HI assay results, assessment of the human T-cell responses against influenza virus infection has been reported previously to provide an important marker of protection (3, 10, 22). Until now, these assays have been performed mostly by applying live virus, hence necessitating the use of BSL-3 conditions for studying (potential) pandemic influenza virus. Although numerous studies of virus inactivation, e.g., by means of virucidal compounds, UV light, or gamma irradiation treatment, have been performed, these studies have not comprehensively documented the preservation of influenza virus protein function and antigenic characteristics following inactivation (5-7, 14, 18). Specifically, these studies have not addressed whether inactivated virus can be used for phenotypic determination of susceptibilities to NAIs and for characterization of T-cell responses.In this study, we evaluated the inactivation of influenza viruses of human, avian, and swine origins by heat, formalin, Triton X-100, or β-propiolactone (β-PL) and the retention of hemagglutinin (HA) and NA glycoprotein functions and antigenic integrity. The optimal procedures have been used to demonstrate the proof of principle in antiviral susceptibility assays, antigenic characterization, and T-cell response assays with both seasonal and pandemic influenza A(H1N1) viruses.