Location: Endemic Poultry Viral Diseases Research
Project Number: 6040-32000-083-029-I
Project Type: Interagency Reimbursable Agreement
Start Date: Mar 1, 2026
End Date: Feb 28, 2030
Objective:
We propose to investigate the role of a newly discovered viral gene in the pathobiology of infectious laryngotracheitis virus. This research is based on mining RNA sequencing data generated from Leghorn male hepatoma (LMH) cells infected with virulent and vaccine strains of ILTV. The polypeptide translated from the 6-exon spliced mRNA shows a high degree (>70%) of homology to interleukin 4 (IL-4) of gallinaceous birds. The hypothesis of this proposal is that ILTV acquired the IL-4 gene between 60 and 70 million years ago, which enhanced its fitness by suppressing host immunity. Avian immune responses and protective outcomes induced by the delivery of a recombinant lacking the viral interleukin gene (vIL4) will be evaluated before and during challenges with virulent ILT virus. The specific objectives are (1) create a recombinant containing a deletion in the gene encoding vIL4 (2) evaluate the safety and immunogenicity of the vaccine candidate in specific pathogen-free (SPF) chickens (3) determine the protective indices of vaccinates against ILTV challenge (4) define the transcriptome of lymphocytes from disease susceptible and resistant lines of vaccinated/challenged SPF chickens.
Approach:
Our approach will include the use of CRISPR/cas9 technology to delete the gene encoding a virokine (homolog to interleukin 4, called vIL4) from the genome of a virulent strain (1874C5) of ILTV. We have evidence that this interleukin mimic is structurally and functionally similar to IL4 encoded by the host. Two guide RNAs specific for exons 3 and 6 will be design, synthesized and cloned into a plasmid containing a codon optimized cas9 gene. Leghorn male hepatoma cells will be transfected with this plasmid and a plasmid containing a marker gene (encoding red fluorescent protein) flanked by homologous sequences needed for marker rescue purposes. Following transfection, cells will be infected with virulent ILTV and overlaid with methylcellulose. Seventy-two hours post infection, plaques will be harvested and subjected to rounds of plaque purification using a limited dilution scheme. Recombinants containing deletions in the vIL4 gene will be identified using the polymerase chain reaction with primers specific for regions flanking the deletion. Large virus stocks will be generated, titered and used to inoculate specific pathogen-free (SPF) chickens. Clinical signs will be scored, and the level of neutralizing antibodies determined. Vaccinates will then be challenged with a virulent strain of ILTV and likewise clinical signs will be assessed, and the level of protection determined. In a separate study, lymphocytes will be collected from disease resistant and disease susceptible birds that were vaccinated and challenged with virulent ILTV. Lymphocytes will also be collected from vaccinated/nonchallenged birds, nonvaccinated/challenged birds and nonvaccinated/non challenged negative control birds. We will use cell sorting to separate T and B cells using fluorescent cellular markers. RNA will be isolated from the separated cells and subjected to short and long read RNA sequencing. A bioinformatic approach will be used to determine the level of transcription of both host and viral genes.