Use of Highly Pathogenic Avian Influenza A(H5N1) Gain-Of-Function Studies for Molecular-Based Surveillance and Pandemic Preparedness

Davis, C. Todd; Chen, Li-Mei; Pappas, Claudia; Stevens, James; Tumpey, Terrence M.; Gubareva, Larisa V.; Katz, Jacqueline M.; Villanueva, Julie M.; Donis, Ruben O.; Cox, Nancy J.

Laboratories worldwide have employed reverse genetics to study the mechanisms by which HPAI H5N1 and other zoonotic influenza viruses evolve and how these mechanisms influence host receptor specificity, antigenic variation, replication, pathogenesis, drug susceptibility, and transmission (8) (9) (10) (11) . Besides being used to create vaccine viruses for the development of live, attenuated (12) and inactivated prepandemic H5N1 influenza vaccines (13), reverse-genetics methodologies also have been used for many years to study the phenotypic consequences of particular mutations, including genetic changes that confer a gain of function (GOF). Influenza virus GOF studies have focused on several research areas: in vitro and/or in vivo replication in mammalian cell culture or animal hosts, adaptive mutations conferring changes in host susceptibility, alteration of receptor binding profiles and/or tropism for mammalian airway tissues, enhanced polymerase activity, changes in host antiviral response (e.g., cell signaling pathways), susceptibility to antiviral drugs, and pathogenesis and/or transmissibility in mammalian animal models. Such GOF experiments have elucidated key biological principles and provided the scientific basis for genomic sequence-based risk assessment of zoonotic viruses with pandemic potential. For example, the molecular basis for avian versus mammalian influenza virus receptor binding (␣2,3 versus ␣2,6 sialylated glycans) has been elucidated largely through GOF experiments, and some recent studies that identified specific HA mutations conferring a switch from avian to mammalian host receptor specificity also demonstrated the impact of these mutations on the ability of H5N1 virus to more efficiently infect the human upper respiratory tract (14) (15) (16) (17) (18) (19) . Mutations conferring enhanced virulence in mammalian models or inhibition of the host antiviral response with the potential to cause more serious human illness have been described in other studies (20) (21) (22) . Still other GOF work characterized mutations that confer resistance to neuraminidase (NA) inhibitors (23) (24) (25) (26) . These data are critical to make effective drug treatment decisions and to inform stockpiling of antiviral medications. Finally, many publications have described mutations that confer adaptation of H5N1 viruses to mammalian hosts and transmissibility in guinea pigs or ferrets (19, 24, 27) . It should be noted that in many cases, this research can demonstrate loss of function, which is also valuable for risk assessment. These types of studies provide vital data with which to monitor circulating viruses for features that may suggest increased capability for human-to-human transmission or more long-term adaptation of H5N1 viruses in humans or other mammalian hosts, such as pigs.