2013 Annual Report
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.