Location: Endemic Poultry Viral Diseases Research
Project Number: 6040-32000-083-005-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jul 1, 2020
End Date: Jun 30, 2025
Objective:
Objective 1. Identify and test in vitro candidate genes encoded by Marek's disease virus (MDV) whose gene products inhibit the production of the type I interferons (IFNA/B). Genes likely to encode inhibitors of the production of type I IFNs in infected cells were identified by homology with their orthologues present in the HHV-1 genome (see Table 1) and are referred to as “candidate genes’ (CGs). As the prototype alphaherpesvirus, HHV-1 has been extensively studied and selected inhibitors of type I IFN production have been identified and ablated, resulting in strains suited as live attenuated virus vaccines produced (e.g., Halford et al., 2011). We have identified a total of 10 genes that show significant homology with their MDV orthologues. These CGs will be expressed from expression constructs in our in vitro model of type I IFN production. When double-stranded DNA (dsDNA) is transfected into HD11 cells, they respond with a dramatic upregulation of production for IFNA and IFNB. The utility of this model is that it engages the same nucleic acid sensor of dsDNA as does an actual herpesvirus infection, but in the absence of other viral virulence factors. This system allows us to assay individual viral gene products for their ability to inhibit the production of the type I IFNs. Using this system we will examine each of the wild type genes selected by orthology with HHV-1 genes which inhibit the production of the type I IFNs. Positive genes will be those which result in reduced IFNA/B mRNA upon transfection of stimulatory dsDNA.
Objective 2. Genes identified in Objective 1 as inhibitors of type I IFN production will be subjected to random mutation. Of the 10 genes identified, eight of them are essential for viral replication. Their orthologues in HHV-1 are examples of “multitasking virulence factors” (Ayllon and García-Sastre, 2014) in that they each have known essential functions that are in addition to their roles as inhibitors of the production of the type I IFNs. It will therefore be necessary to dissect their interferon-inhibitory activity away from their other essential functions. These virulence factor genes will be subjected to extensive random mutation (Boeke laboratory) and returned to us for in vitro testing to verify that they do indeed allow increased type I INF production in cells induced to produce the type I IFNs by transfection of dsDNA. Finally, these mutagenized genes will be assayed for complementation in permissive cells electroporated with a rMd5 B40 mutant that lacks the candidate gene being tested (also produced in the Boeke laboratory). The idea that we will be able to separate type I IFN inhibition and other functions by domain on the candidate gene product has been demonstrated recently in MDV by Gao and colleagues (Gao et al., 2019). Using the example of the VP23 protein already identified as an inhibitor of the production of type I IFNs in MDV, Gao and colleagues successfully separated the type I IFN-inhibitory activity from the capsid organizing function of this protein by analysis of truncation mutants.
Approach:
1) Identification of Marek's disease virus (MDV) virulence factors which suppress the production of the type I IFNs.
We have identified candidate MDV genes by homology with HSV-1 genes (see Table 1) that have been shown to suppress the production of IFNA/B. As an example, MDV UL18/VP23 is an integral viral capsid protein, which has recently been shown to interfere with the association of IRF7 with TBK-1, thereby inhibiting the phosphorylation of IRF7 and ultimately suppressing the production of IFNA/B (Gao et al., 2019). We will detect genes that inhibit the production of IFNA/B by transiently expression of the candidate gene from an expression plasmid in chicken cells followed by stimulation with dsDNA to elicit the production of IFNA/B. We have developed a cell-based model of type I IFN production upon stimulation with dsDNA in HD11 cells, a macrophage-like chicken cell line. See the section entitled “preliminary data” for a detailed explanation of this model and supporting data.
Candidate genes thus identified (phase 1a) and verified as suitable targets for ablation (this step, phase 1b) will advance to phase 2.
2a) Production of candidate gene mutants through random mutagenesis (Boeke laboratory)
As previously stated, eight of the 10 genes identified by substantial homology with their orthologous genes in HHV-1 are essential for viral replication. For this reason it will not be possible to construct MDV recombinants that are simply deleted for every candidate genes because such mutants would not be replication competent and isolation after reconstitution by electroporation into competent cells would be problematic without “helper” viruses or cell lines engineered to express the essential gene in trans. It is therefore necessary to specifically target a particular candidate gene product’s ability to suppress the production of type I IFN, while leaving untouched other, critical functional areas.
3a) In vivo pathogenesis assessment. In this the final step, these recombinant viruses will be tested for their pathogenicity and vaccinal utility. A preliminary study will be conducted to assess the pathogenicity (if any) of the MDV mutant strains. The number of animals used in this initial test will be 15 birds per MDV recombinant strain tested. The animals used in these experiments will be maternal antibody negative and of a susceptible strain (e.g., 15I(5) x 7(1) to maximize our ability to detect differences in viral pathogenicity.
3b) In vivo protection assessment. Viruses that do not cause significant morbidity (determined by the assessment of pathogen described above) will be assayed for their ability to protect against a challenge, again from the very virulent (vv) MDV Md5 strain.
