2012 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.
Development of a novel live attenuated influenza vaccine against swine influenza. A novel live attenuated influenza vaccine (LAIV) that is safe and efficacious for use in swine was previously generated. Ultimately, we would like to produce LAIVs that do not require the virus to be grown either in vitro or in eggs. Instead, using reverse genetics it can be envisioned that LAIVs could be generated in vivo in the vaccinated animals. For such an approach to work, the use of adequate vRNA transcription units is needed; specifically the use of RNA polymerase I promoters that correspond to the animal species in question. Towards that goal, we produced a plasmid-based reverse genetics system for swine influenza viruses using vRNA transcription units under the control of a swine RNA polymerase I promoter. Future studies are aimed at determining whether in vivo generation of the virus is possible in pigs. For the purpose of creating an influenza reverse genetic vector capable of expressing both viral mRNA and negative sense viral genomic RNA in swine cells, the human RNA polymerase I promoter element in pDP2002 was replaced with the porcine promoter sequence. Results indicate the successful generation of influenza virus using a swine RNA polymerase I promoter element to synthesize negative sense vRNA.
Project 1: Generate reverse genetic derived mutants possessing amino acid residues demonstrated to be important for transmission and/or virulence to test in mice, ferrets, and/or swine. Reverse genetic clones of the swine H2N3 isolate were generated in a University of Maryland lab by a visiting USDA-ARS postdoctoral research associate. Future pathogenesis and cross-species transmission studies involving the gene combinations of this virus are planned. In addition, the constructs of the unique surface genes can be readily available for future pathogenesis and/or vaccine trials utilizing different vaccine platforms.
In a separate experiment, three reverse engineered reassortant viruses with the same genetic backbone and hemagglutinin (HA) gene but differing in the neuraminidase (NA) gene were constructed. The goal was to generate test antigens for the neuraminidase inhibition assay (NI). All reassorted viruses were successfully rescued, propagated and stored at -80oC. These viruses will be used as test antigens in the NI assay to test reference swine sera generated against the different swine influenza virus subtypes and strains to study the anti-neuraminidase cross reactivity and antigenic drift of the NA protein and the potential contribution of NI antibodies to protection and involvement in vaccine-associated enhanced respiratory disease.
Project 2: Role of NP in vaccine-associated enhanced respiratory disease. In order to determine the role of influenza A virus nucleoproteins (NP) in vaccine-associated enhanced respiratory disease (VAERD) a collection of reverse genetic derived reassortant viruses were prepared. The viruses are currently being evaluated to establish the contribution of various gene constellations (with particular emphasis on the NP, hemagglutinin and neuraminidase gene products alone or in combinations) on VAERD.