Evaluating Differences in Innate Immune Response to Highly Pathogenic H5N1 Viruses - Abstract H5N1 Highly Pathogenic Avian Influenza virus (HPAIV) spreads globally through migratory birds. Outbreaks of avian influenza cause significant financial losses for particularly for the poultry and dairy industries. The first human infection with HPAIV H5N1 was in 1997, followed by an additional 17 cases and a total of 6 deaths. Since 2003, there have been over 900 human cases with a mortality rate exceeding 50 percent. Currently, no effective vaccine is available, and Tamiflu does not work well against clade 2.3.4.4b H5N1. It is pivotal to understand the risk of the virus acquiring efficient cross-species transmission and further sustained human-to-human transmissibility. Adaptive mutations have emerged during infection of humans and a broad spectrum of mammals, posing a huge threat to public health. As a host restriction factor, myxovirus resistance 1 (Mx1) protein is recognized as a crucial gatekeeper candidate. Studies have shown Mx1 protein exhibits antiviral activity against HPAIV H5N1. The proposed research will test the hypothesis that different H5N1 isolates have different sensitivity to different Mx1s, which is associated with mutations of the viral genome. Preliminary data showed that murine Mx1 (muMx1) provides impressive resistance against H5N1 infection. Mx1 expression increased the lethal dose by at least 1000-fold when comparing that between wild type B6 and Mx1 (+/+) A2G mice. Meanwhile, the Mx1 sensitivity differs significantly between a human isolate and a sea lion isolate, carrying limited variation between two viral genomes. I will test the sensitivity of different H5N1 viruses to muMx1 in vivo and investigate any correlation to specific genotypic polymorphisms (Aim 1). It is anticipated that the data will show different H5N1s carry different muMx1 sensitivity. Based on the genome alignment, the genomic determinant will be identified and related to Mx1 sensitivity. I will investigate the impact of these new biomarkers on virus sensitivity to Mx1. The results will provide insight into the risk of H5N1 breaking the species barrier with novel genetic markers. Additionally, I will assess the sensitivity of H5N1 viruses to Mx1 from different hosts in vitro, serving as a tool to assess potential host range (Aim 2). A549 STAT1 knockout cell line will be used as a platform to express exogeneous Mx1 proteins to examine Mx-mediated antiviral phenotype. It is anticipated that the results will show viruses from different hosts display different Mx1 sensitivities, indicating each virus has its own susceptible hosts. This Mx1 library can be expanded with new Mx sequences from mammalian species of interest, assessing the potential transmissibility of certain H5N1s among different mammalian species. The overall project will provide risk assessment of the current HPAIV H5N1 to public health and in vitro and in vivo models for the future study. Completion of this proposal will provide training in (1) advanced molecular biology approaches, (2) virology concepts and methods, (3) next generation sequencing skills, (4) mass spectrometry techniques, and (4) biosafety procedures for working with select agents – equipping me with essential expertise needed to develop an independent research program on emerging infectious diseases.
