An official website of the United States government.
We hypothesize that influenza A viruses of genetically different HA type bind to swine respiratory epithelial cells expressing either the NA alpha 2,3 Gal or the NA alpha 2,6 Gal swine influenza viruses and that receptor distribution is critical for these different influenza A viruses to adapt, replicate and undergo reassortment within swine respiratory epithelial cells. To test this hypothesis we propose two specific objectives: 1) Identify and assess the distribution of sialic acid residues on swine epithelial cells isolated from various locations within the respiratory tract. This objective will have a two pronged approach. The sialic acid moieties associated with influenza receptors will be defined and quantified on airway epithelial cells in the nasal cavity, trachea, bronchi and bronchioles. In addition we will compare the in vivo receptor distribution to the receptor populations in the using an in vitro differentiated epithelial culture system developed from the equivalent airway regions. 2) Evaluate the ability of swine influenza viruses (SIV) with genetically distinct HA proteins to attach to the two sialic acid moieties and infect swine respiratory epithelial cells from various locations within the respiratory tract. Using the same in vitro cultures as in objective one, we will be able to assess the ability of 4 genetically diverse SIV viruses to bind receptors on the surface of the same cell, which is the first step in the reassortant process.
NON-TECHNICAL SUMMARY: Assess the receptors for swine influenza viruses plays in the ability of different influenza viruses to infect, adapt, replicate, and resort within the swine cells.
<p>
APPROACH: Objective one: Sections will be collected from the nasal turbinates, trachea, bronchi and bronchioles of uninfected neonatal pigs euthanized at birth. Digoxigenin labeled Maackia amurensis lectin specific for NA alpha 2,3Gal or Sambucus nigra lectin specific for NA alpha 2,6Gal will be used to identify and assess the distribution of sialic acid residues on swine epithelial cells within the respiratory tract. Following incubation with the digoxigenin labeled lectins the sections will be incubated with fluorescein and rhodamine conjugated anti-digoxigenin antibodies for evaluation by fluorescent microscopy and confocal microscopy. The distribution in vivo will be compared to epithelial cells isolated from the same regions that will be propagated in an in vitro differentiated respiratory epithelial cell culture system. The epithelial cells will be grown on membrane supports utilizing an air-liquid interface as previously described. Briefly, swine nasal, tracheal and bronchial epithelial cells will be isolated by enzymatic digestion and grown on microporous membrane inserts using an air-liquid interface feeding system. The cultured cells form a confluent monolayer and retain most of the morphologic characteristics of intact epithelial cells. A similar human airway epithelium cell culture system has been shown to support infection of human and avian influenza viruses. <p>Objective two: Evaluate the ability of SIV with genetically distinct HA proteins to infect swine respiratory epithelial cells. Mature differentiated epithelial cultures will be infected with a classical H1N1, triple reassortant H1N1, H1N2 and H3N2 SIV isolates consisting of genetically distinct HA proteins. <p>These 4 viruses differ in the genetics of the various segments containing different swine, avian and human components. We have characterized all 4 of these isolates and determined their ability to produce antibodies that cross react in serological assays. The cells will be infected at a multiplicity of infection of 1. At 24 hours post infection the cells will be fixed and stained and the infected cells will be identified using anti-subtype specific SIV antibodies developed in our lab and fluorescently labeled secondary antibodies. The specific sialic acid moieties will be stained as described in Objective one and the epithelial cells will be evaluated for both receptor and subtype infection by detection of fluorescence using confocal microscopy.