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: 2. Development of improved Newcastle disease control strategies addressing issues important to virus transmission, vaccines and vaccination, diagnostics, or international trade. (Aim 1) To determine if a novel virulent virus can be created through homologous or non-homologous recombination of a recombinant Newcastle disease virus (rNDV) expressing the AIV hemagglutinin (HA) gene and a wild type avian influenza virus of low pathogenicity (LPAI). We infected 14-day old embryos with rNDV-AI/HA and LPAI (recombinant H5, or wild type H6 or H9) and assess recombination via sequencing and pathogenicity studies. 14-day old embryos have a stronger immune response and only the more virulent viruses are able to grow. Allantoic fluids containing virus from embryos with mortality were evaluated on MDCK (canine kidney cells) for their ability to cause cytopathic effect without the addition of trypsin, which is also a sign of a stronger AIV. NDV does not grow well on MDCK cells. 111 samples were further incubated with antibodies to NDV to remove the NDV and leave only the AIV in the sample. AIV RNA was isolated from 74 samples and these samples were tested with the real time RT-PCR assay for AIV to confirm that they contain AIV. Subsequently, the samples were evaluated in a PCR assay using primers specific for the virulent HA of H5 to assess if recombination occurred. 12 of 74 samples have been tested and no recombination has been observed. (Aim 2) To determine the relative potential of NDV of low virulence to mutate to a virulent NDV in a host organism we infected 14-day old embryos with rNDV or wild type NDV strains to evaluate if the stronger immune response would facilitate an increase in virulence. The fusion cleavage site was sequenced for the 104 allantoic fluids containing NDV from embryos with mortality and there were no changes in fusion cleavage site that would increase virulence. The mean death time (MDT) assay was completed on 88 of the samples and while some approached the level suggesting an increase in virulence, all 88 samples remained of low virulence. (Aim 3) To determine the impact of rNDV and NDV-vectored vaccines on non-target species with respect to infection and transmission pigeons were infected with rNDV and placed with contact pigeons. The rNDV strains infected the pigeons and were transmitted to the control pigeons. Next studies: For aim 1 PCR assays will be performed on the remaining 62 samples to assess if a virulent HA gene exists due to recombination. Any bands existing at the correct, and expected location will be sequenced to evaluate the HA cleavage site. MDT will be performed on the 16 samples remaining for aim 2. Intracerebral pathogenicity index assays will be performed on 5-6 isolates from aim 2 with lower MDT results. Some or all of the allantoic fluids from aim 2 may be passed in additional embryos and the cleavage site reevaluated to mimic a field situation. European Starlings and House sparrows will be tested with the same protocol as the pigeons for the completion of aim 3. The amounts of virus from the pigeon swabs will be quantified. Monitoring: The ADODR has had regular email, telephone conversations, and meetings with the post-doctoral research associate and co-investigators on research planning.