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United States Department of Agriculture

Agricultural Research Service

Research Project: H1n1 Influenza a Virus in Swine Supplemental Research Program
2013 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.

3.Progress Report:

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 coding sequence in the PB1 gene of A/California/04/2009 (H1N1)(Ca/04). All mutations introduced were silent in the PB1 coding sequence. 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 lung fluids were not affected by PB1-F2 in the context of the H3N2 viruses, but knocking-in PB1-F2 did increase lung fluid 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.

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.

Reverse genetic clones of the swine H2N3 isolate were generated at the 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. 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.

Although avian influenza viruses typically are adapted for avian tissues, many recent H9N2 Asian influenza isolates from poultry were shown to bind human receptors and occasionally infect swine and humans, providing the opportunity for reassortment between H9N2 and H1N1pdm09. To test if these viruses would be adapted to mammalian respiratory tracts, ferret adapted H9N1 avian influenza virus surface genes were combined with the H1N1pdm09 internal genes by reverse genetics in 2+6 (rH9N2) or 1+7 (rH9N1) combinations and used to infect 4 week old pigs. Compared to the H1N1pdm09, the reassortants showed reduced pathogenicity. Swine infected with H1N1pdm09 showed higher percentages of lung lesions at both 3 and 5dpi compared to the 2 reassortant viruses, consistent with what was seen in ferrets with similar viruses. Both reassortant H9 viruses replicated and transmitted to contact pigs whereas the wild type avian H9N2 did not. This study highlights the role pigs may play as mixing vessel species and underscores the potential threat that H9N2 viruses could pose to swine and ultimately humans as a future pandemic subtype. These findings emphasize the need for better surveillance of influenza in swine and the need for future research on how the swine host can effect H9N2 reassortment or adaptation.

Since 1999, plasmid-based reverse genetics (RG) systems have revolutionized the way influenza viruses are studied. However, it is not unusual to encounter cloning difficulties for one or more influenza genes while attempting to recover virus. To overcome this, we sought to develop partial or full plasmid-free RG systems. The influenza gene of choice was assembled into a RG competent unit by virtue of overlapping PCR reactions - herein referred to as Flu PCR amplicons. Transfection of tissue culture cells with either HA or NA Flu PCR amplicons and 7 plasmids encoding the remaining influenza RG units, resulted in efficient virus rescue. Likewise, transfections including both HA and NA Flu PCR amplicons and 6 RG plasmids also resulted in efficient virus rescue. In addition, influenza viruses were recovered from a full set of Flu PCR amplicons without the use of plasmids.

Although the ferret model has been extensively used to study pathogenesis and transmission of influenza viruses, little has been done to determine whether ferrets are a good surrogate animal model to study influenza virus reassortment. To address this issue, we performed coinfection studies with recombinant influenza viruses carrying the surface genes of a seasonal H3N2 strain in the background of an H1N1pdm09 strain and vice versa. After serial passages in ferrets, a dominant H1N2 virus population was obtained with a constellation of gene segments, most of which, except for the neuraminidase (NA) and PB1 segments, were from the H1N1pdm09 strain. Our studies suggest that ferrets recapitulate influenza virus reassortment events. The H1N2 virus generated through this process resembles similar viruses that are emerging in nature, particularly in pigs, and a pig study is the natural progression to evaluate these viruses.

Last Modified: 4/19/2014
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