Recipient Organization
AGRICULTURAL RESEARCH SERVICE
1815 N University
Peoria,IL 61604
Performing Department
(N/A)
Non Technical Summary
Recent research has demonstrated the importance of host proteins for avian influenza virus (AIV) transmission into mammals and the unique capacity of swine to promote AIV infection. Despite extensive characterization of differences within corresponding host proteins between mammalian species, no studies have been reported that focus specifically on natural genetic variation present within domestic swine and the impact on AIV infectivity. Given the immense role that domestic swine play in the adaptation of AIV for mammalian hosts, identification of gene variants that affect swine susceptibility to AIV would provide a plausible avenue for limiting the emergence of novel influenza strains. Therefore, the overall goal of this research is to identify natural host variants that decrease the susceptibility of swine to AIV infection. Specifically, this study will focus on two key host proteins (ANP32A and ANP32B) known to promote AIV replication following infection. First, we will obtain DNA sequences encoding the ANP32A/B proteins from more than 500 pigs representing genetically distinct and commercially relevant breeds to identify natural gene variants. Second, we will use gene editing to systematically modify ANP32A/B in pig cells and fluorescence-based functional assays to quantify the effect of host gene variants on viral replication for different avian and swine influenza strains. The outcomes of this research will directly benefit swine producers by increasing animal welfare through potential genetics-based prevention strategies and alternative solutions to suboptimal vaccination regimes. Additionally, this research has the potential to protect human health as well by limiting the exposure of workers in frequent contact with domestic swine to mammalian-adapted AIV strains capable of human infection.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall goal of this proposal is to provide feasible means of limiting the emergence of mammalian adapted avian influenza virus (AIV) strains by decreasing the suitability of domestic swine populations to serve as endemic AIV and reassortant reservoirs. Specifically, this proposal aims to identify acidic leucine-rich nuclear phosphoprotein 32 (ANP32) variants and isoforms present in domestic swine that modulate AIV polymerase activity through the following objectives: 1) Discovery of ANP32A/B polymorphisms and isoforms across domestic swine using large-scale comparative genomics and 2) Assess the effect of selected ANP32A/B variantsand isoforms on influenza A polymerase binding and activity through in vitro CRISPR-cas9 gene editing and functional assays.
Project Methods
This project will include activities relating to 1) the discovery of ANP32A/B polymorphisms across domestic swine and 2) the effect of these variants on influenza A polymerase binding and activity. The first activity will be to identify and characterize ANP32A/B variants and isoforms via high density comparative analysis of sequences from commercially relevant swine populations. Specifically, we will sequence ANP32A/B genomic and transcript sequences from more than 500 animals representing genetically distinctpurebred and crossbred pig populations using Oxford Nanopore Technology (ONT). This sequencing platform enables rapid multiplexed sequencing and generates individual reads making it ideal for variant and isoform discovery. The putative effect of polymorphisms and isoforms identified by this large-scale comparative analysis will then be subsequently assessed using protein sequence prediction, isoform- or allele-specific RT-qPCR, and motif screening. The second activity will be to generate ANP32A/B double knock-out pig cell lines using CRISPR-Cas9 gene editing. We will then generate plasmids encoding selected ANP32A/B variants for exogenous expression in the knock-out cell line. Lack of endogenous and presence of exogenous protein will be confirmed via western-blot and immunofluorescence assays. Cells exogenously expressing the corresponding ANP32A/B variant proteins will then be transfected with plasmids including each component of the influenza viral ribonucleoprotein (vRNP) or replication complex along with a reporter plasmid including a negative-sense viral RNA (vRNA)-like molecule encoding the firefly luciferase protein. The level of luminescence serves as an indirect measure of viral polymerase activity in the presence of the variant ANP32A/B proteins. Furthermore, co-immunoprecipitation will be conducted to evaluate binding affinity between the variant ANP32A/B proteins and the viral PB2 polymerase subunit. This approach provides feasible means of testing multiple host variants and influenza strains, making it ideal for the scope of this study.A variety of platforms will be utilized to communicate the research results to industry, scientific community, and the general public. Specifically, the PD will present research at meetings to other scientists in the program area (NAPRRS/NC229) and field (local seminars). Following project completion, the results will be published in peer-reviewed journals, such as the Journal of Animal Science, and reported in popular press platforms specific to the swine industry, such as National Hog Farmer. Furthermore, the PD will work with industry partners to ensure project findings are disseminated appropriately to commercial industries, such as swine breeding and genotyping organizations.