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Characterization of an Influenza Infection Model in Porcine Airway Epithelial

Undergraduate #103
Discipline: Biological Sciences
Subcategory: Microbiology/Immunology/Virology

Aliyah Glover - University of Arkansas at Pine Bluff
Co-Author(s): Jennifer Bartlett and Paul McCray, University of Iowa, Iowa City, IA



Background: Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Our goal is to develop an infection model using a medically relevant virus (influenza), which we can use to study the antiviral responses of cultured airway epithelia from CF and non-CF pigs. Influenza causes acute respiratory infections responsible for seasonal epidemics and occasional pandemics. In a previous study, Xu and colleagues (2006) showed that CF airway cells have a delayed/reduced immune response to influenza A infection. We hypothesized that influenza A would have a dose-dependent ability to infect and replicate in well-differentiated primary cultures of airway epithelia from wild-type pigs.
Methods: We are using influenza virus, A/Memphis/4/82 (H1N1), thought to have originated in swine. Well-differentiated porcine airway epithelial cultures grown at an air-liquid interface were infected with this virus at a range of MOIs. At 12 hr, 24 hr, 48 hr and 72 hrs post infection, we collected: 1) washes from the apical surface, and 2) whole-cell lysates. We assessed viral infection/replication by performing western blots on the cell lysates. We also assessed viral replication by performing plaque assays to detect shed virus in the apical washes.
Results: Western blotting showed that viral antigen was present in lysates at 48 hr and 72 hr post infection. Viral progeny were detected in apical washes from the cultures at all time points, with a peak in viral shedding at 48 hr post infection.
Conclusions: We found that infecting cultured porcine airway epithelia with influenza A/Memphis/4/82 at an MOI of 0.001 is sufficient to produce efficient infection and replication in our culture model, with maximal replication and viral production occurring at 48 hr post infection. We will use the results from this pilot study to guide future experiments using airway epithelia from CF and non-CF pigs. This viral infection model will help us gain insight into antiviral host defense mechanisms in CF airways.

Funder Acknowledgement(s): This project is funded by: NIH grants P01 HL-51670 and P01 HL-091842; HHMI; CFF; Roy J. Carver Charitable Trust. We would like to thank the McCray Laboratory, Papajohn Biomedical Institute for their help. My research was supported by Summer Undergraduate MSTP Research program Carver College of Medicine, The University of Iowa.

Faculty Advisor: Anissa Buckner, bucknera@uapb.edu

Role: I had a hand in every step of the way with this research project under the guidance of my two co-authors. My laboratory skills were enhanced through calculating multiplicity of Infection, calculating dilutions, conducting apical washes on cells, fixing cells, tittering cells, making lysates, performing Western Blot Assays, and immunofluorescence staining cells.

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This material is based upon work supported by the National Science Foundation (NSF) under Grant No. DUE-1930047. Any opinions, findings, interpretations, conclusions or recommendations expressed in this material are those of its authors and do not represent the views of the AAAS Board of Directors, the Council of AAAS, AAAS’ membership or the National Science Foundation.

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