1a.Objectives (from AD-416)
Evaluate three risks associated with recombinant Newcastle disease viruses being used as live vaccines in poultry and to provide data to regulatory agencies (Center for Veterinary Biologics) and researchers to allow them to consider whether this class of vaccine is safe and effective for use in the U.S. market.
1b.Approach (from AD-416)
We will use commercially available live vaccines from China or Mexico formulated with Newcastle disease viruses (NDV) containing the H5 hemagglutinin (HA) protein for avian influenza and/or NDV recombinants with H5 inserts made in our laboratory. We will utilize a NDV that has been modified by reverse genetics to include an attenuated hemagglutinin-neuraminidase (HN) or fusion (F) and HN genes from a virulent NDV. An established cell culture protocol that uses products from egg based studies will be performed to determine if. 1)the avian influenza HA gene inserted in the NDV genome can recombine, by homologous or non-homologous recombination, with low pathogenic H5 and non H5 influenza viruses and. 2)if the recombinant NDV (rNDV) containing an attenuated HN and/or F and HN genes from a virulent strains can revert back to a virulent virus. A wild type NDV, documented to have increased in virulence in nature in 1998, will be tested alongside the rNDV. The protocol uses 14-day-old specific pathogen free (SPF) embryonated chicken eggs (ECE) and favors the growth of virulent viruses in cell culture, avoiding having to make multiple passages of egg fluids. Any viruses that form plaques in cell culture without the addition of an extraneous protease potentially have an increase in virulence and will have the HN and F genes sequenced to compare with parent virus. Selected viruses will be evaluated in embryos and birds to define the change in virulence. To assess non-target species infection for specific aim three, the three most common wild avian species associated with poultry houses; pigeons, starlings, and house sparrows, will be tested experimentally with rNDV and rNDV-H5 used in the first two specific aims to determine susceptibility to infection and for the potential of the virus to transmit and change within these species. Selected viruses recovered after infection will be viewed in the same egg based study to evaluate virulence.
This research is related to inhouse objective 2: Development of improved Newcastle disease control strategies addressing issues important to virus transmission, vaccines and vaccination, diagnostics, or international trade. Develop models to show vaccination is a viable method of controlling avian paramyxovirus outbreaks.
A post-doctoral research associate has been hired to complete the objectives of this grant and we are waiting for select agent clearance. Preliminary data showing that two different strains of Newcastle disease virus (NDV) can co-infect the same cell have been obtained. Additional avian influenza virus (AIV) strains have been located from a different institution and will be obtained through a material transfer agreement (MTA) to show co-infection of one cell with NDV and AIV is possible. Next studies: Initial experiments are being currently being planned and will be initiated in the upcoming months.