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.
3. Progress Report:
This project is related to objective 2 of this in-house project: Elucidate the host-pathogen interactions of Newcastle disease virus infections that impact vaccine efficacy by defining host pathways modulated by Newcastle disease viral infections and by identifying genetic and biological viral determinants that affect the safety and efficacy of Newcastle disease vaccine virus strains. The first specific aim: Co-infection of 900 14 day-old embryonating eggs with a recombinant LaSota vaccine virus containing an avian influenza gene and wild type avian influenza viruses of subtypes H5, H6 or H9 has been completed. Fluid from 800 dead embryonating eggs has been collected and is currently being tested for cytopathic effects and plaque formation in cell culture. The second specific aim: Assessing the potential of recombinant Newcastle disease viruses (attenuated fusion cleavage sites) to revert back to virulence in a host organism has also been completed. 900 embryonating eggs were inoculated with five different Newcastle disease viruses. 106 allantoic fluid samples positive for virus from the inoculated eggs have been tested for cytopathic effects in cell culture. 70 of these 106 samples produced cytopathic effects. 20 of these 70 have been sequenced and so far there has been no reversion to virulence or change in fusion cleavage site. The remaining samples will be sequenced. Specific aim three: Recombinant Newcastle disease virus vaccines are able to infect pigeons and spread to contact birds has been partially completed. The pigeon experiments have been completed but only half of the sparrow experiments and none of the starling experiments. None of the pigeons showed clinical signs after being infected with the Newcastle disease strains, however, the birds shed virus and uninfected pigeons placed in contact with infected wild bird species associated with poultry houses was infected and shed virus for two or more days.