Adrianus Boon, Ph.D.
Project Overview:
The influenza virus kills nearly 500,000 individuals each year and this number can increase dramatically during a pandemic with a novel strain. Influenza virus infections can be prevented by antiviral drugs such as oseltamivir, or by vaccinations. However, antiviral drugs are not prescribed prophylactically and vaccination is not monitored for efficacy, providing ample opportunity for influenza to infect and cause disease. If we can identify individuals who are at increased risk of severe influenza virus we can tailor our care to provide drugs prophylactically to improve health and longevity.
The goal of our research is to define genetic biomarkers that predict susceptibility to influenza disease. The interferon pathway is very important for restricting influenza virus replication and therefore disease. Individuals with unique or rare polymorphisms in essential genes in this pathway are more susceptible to influenza virus and more likely to require hospital care or succumb to infection. Because of the importance of this pathway we want to study polymorphisms in two interferon pathway genes that have previously been shown to affect viral diseases and antiviral immunity. These polymorphisms are relatively common in the population, with about 5% to 10% of all people carrying two copies of the minor allele.
To test if these genetic differences between individuals predispose to more severe disease we will use cultured primary human airway epithelial cells as a model to look at the role of these polymorphisms on disease parameters. Airway epithelial cells are the primary target cell of influenza virus and we know that increased virus replication and attenuated antiviral immunity correlate with severe disease. We will determine virus replication and antiviral gene-expression in cells obtained from individuals with different IFN genotypes. Analysis of the data between cells containing the two alleles will reveal if this genetic marker can be used to identify individuals that are genetically susceptible to influenza virus. The identification of prognosticators of influenza disease and survival is of special interest to the Foundation and leads to fewer influenza associated deaths and hospitalizations.
Final Report
With our collaborators (Dr. Steven Brody at Washington University and Dr. Matthias Salathe at the University of Miami) we cultured and expanded human primary airway epithelial cells from 23 different donors. We also developed genotyping protocols for polymorphisms in the IRF7 (rs12805435) and IFNL3 (rs12979860) host genes. Among the 23 samples, we identified 10 donors homozygous for the major allele (T) of IRF7, eight homozygous for the minor allele (C), and five that were heterozygous. Fourteen samples were also genotyped for IFNL3, and we identified six that were homozygous for the major allele (C), four for the minor allele (T) and four that were heterozygous.
Cells from 14 different donors were selected based on the genotype for IRF7 and were homozygous for the major or minor allele. Confluent monolayers were inoculated with a low dose of A/California/04/2009 H1N1 virus and supernatant from the infected cells was collected at 24, 48, and 72 hours post-infection. The viral titer in the supernatant of these cells was quantified by virus titration assays. At 24 hours post infection, we observed a more than 1,000-fold difference in virus titer between cells derived from different donors. This difference was maintained at 48 and 72 hours post infection. These findings suggest a role for host genetic variation on influenza virus replication in primary airway epithelial cells.
Association between polymorphisms in IRF7 and IL28b and influenza virus replication: The average virus titer was compared between the different IRF7 and IFNL3 genotypes. We found no difference in virus titer from cells that were homozygous for the major or minor allele of IRF7. Similarly, we also observed no difference in virus titer from cells that were homozygous for the major or minor allele of IFNL3. Interestingly, the four samples that were heterozygous for IFNL3 produced the least amount of influenza virus at 24 hours post infection. These data suggest that polymorphisms in IRF7 do not impact virus replication in primary airway epithelial cells in vitro. The impact of genetic variation in IFNL3 is unclear and warrants further investigation.
