2011 Annual Report
1a.Objectives (from AD-416)
Identify swine influenza virus strain specific antigenic epitopes to support the development of serological assays for surveillance in swine. Compare pathogenesis and transmissibility of selected isolates of the wild-type novel A/HINI virus from animals (e.g., Canadian, Chilean and Argentinean viruses) and correlate with genetic and antigenic changes. Evaluate a reverse genetics-derived modified live vaccine in pigs and other susceptible animal hosts against pandemic A/H1N1.
Additional objective: Generate reverse genetic derived mutants as amino acid residues demonstrated to be important for transmission and/or virulence to test in mice, ferrets, and/or swine.
1b.Approach (from AD-416)
Conduct immunological investigations of influenza A virus components that lead to immune responses against specific epitopes that may enable serological surveillance for the 2009 A/H1N1 in swine and determine whether heterologous immunity against endemic swine influenza viruses interferes with serological surveillance methods. Conduct an animal study to determine the pathogenesis and transmissibility of selected isolates of the wild-type novel A/HINI virus from animals and correlate with genetic and antigenic changes. Conduct an animal study utilizing a reverse genetics-derived modified live vaccine in pigs and other susceptible animal hosts against pandemic A/H1N1.
Additional Approach: Reverse genetics mutants will be generated to define amino acid residues in the hemagglutinin (HA) gene (or other if relevant) that are proposed to be important for aerosol transmission and/or virulence in multiple susceptible species. Naturally occurring amino acid changes were identified in the HA of pandemic H1N1 viruses from 2009. One change, S183P, has been increasing in prevalence in the human and swine populations and was present as a low frequency quasispecies in the original human isolate A/CA/04/2009. HA containing P183 appear to have an advantage in aerosol transmission based on preliminary data in swine studies at NADC and in ferret studies at University of Maryland. This study proposes making single amino acid changes at the 183 position and/or other positions demonstrated to play a role in the observed phenotype. Virus clones will be tested in vitro, in tissue explants, and in vivo in mice, ferrets, and/or swine for comparison.
This past year we investigated the contributions of a possible virulence trait, PB1-F2, to the virulence of influenza viruses in the swine host. PB1-F2 is a proapoptotic protein expressed by certain influenza A viruses that enhances viral virulence in the mouse model but was not found naturally occurring in the 2009 H1N1 pandemic virus. Some endemic swine influenza viruses are known to carry this gene and it was important to know if a reassortment event between an endemic swine influenza virus and the 2009 H1N1 pandemic virus were to occur whether this might alter the pathogenicity of the pandemic virus in pigs. Given that pandemic H1N1 (pH1N1) isolates do not express PB1-F2, we restored the PB1-F2 ORF in the PB1 gene of A/California/04/2009 (H1N1)(Ca/04). All mutations introduced were silent in the PB1 ORF. We then developed an ex vivo organ culture model of the pig respiratory tract that maintains an air–liquid interface to study the replication of PB1-F2 recombinant viruses. These tissue explants retained their cytoarchitecture and supported productive replication of the recombinant influenza viruses. Knocking out PB1-F2 in the context of an H3N2 strain decreased its replication in swine explants, whereas knocking in PB1-F2 in the background of the H1N1 pandemic virus improved its replication in nasal turbinate and tracheal explants. However, in the H3N2 strain, the PB1-F2 mutation had no effect on replication in ex vivo tissues. We next evaluated the virulence of these viruses in swine. The expression PB1-F2 did not affect viral shedding in any of the strains tested. Similarly, virus titers in BALF were not affected by PB1-F2 in the context of the H3N2 viruses, but knocking-in PB1-F2 did increase BALF titers in the pandemic H1N1 backbone. Upon necropsy, PB1-F2 had no effect on the gross pathology caused by H3N2 or H1N1 pandemic. Microscopic pneumonia and the pulmonary levels of IFN-alpha and IL-1beta were increased in the pH1N1 encoding a functional PB1-F2. Our results indicate that PB1-F2 has pleiotropic effects in the swine host, which are expressed in a virus strain-dependent manner. Progress is monitored via email and conference calls.