Performing Department
(N/A)
Non Technical Summary
Over 103 million metric tons of chicken meat was produced in the world in 2023, with 20% produced in the USA, ranking poultry as the second most-produced meat in the world. Americans also consume the most chicken meat in the world, despite having a smaller population than other countries like China or India. The cost of meat and egg production has increased over the years partly due to incidences of diseases such as Marek's disease. Marek's disease is caused by a herpes virus called Marek's disease virus (MDV) and causes cancer and other ailments within the first few weeks after hatching. This ultimately leads to decreased growth of the chickens, susceptibility to other diseases, condemnation of the meat at the processing plant, and death, contributing to the overall cost of production. Most licensed vaccines against MD are administered as cell-associated preparations, also increasing the costs of production since these vaccines require storage and transportation in liquid nitrogen and require a significant amount of primary cell culture for their production. Therefore, a cell-free Marek's disease vaccine that can be prepared as a stable, lyophilized preparation would greatly benefit the poultry industry, especially in countries or rural settings that cannot afford liquid nitrogen storage for current MD vaccines. A major obstacle in designing new cell-free vaccines is the lack of information on viral genes important for this essential step in the virus life cycle. We have identified specific virus genes that are essential for the transmission of MDV and the production of a cell-free virus termed the conserved herpesvirus protein kinase (CHPK). We have identified modifications of this protein kinase during infection in chickens that are not present during replication in cell culture, the technique used to produce Marek's disease vaccines. Our goal here is to better understand these specific modifications of the protein kinase that we can use to produce cell-free Marek's disease vaccines. The long-term impact of this work is the development of novel vaccines that can be used to provide better protection for chickens without the added cost of maintaining a cold chain for administering the vaccine to poultry producers.
Animal Health Component
100%
Research Effort Categories
Basic
70%
Applied
20%
Developmental
10%
Goals / Objectives
We hypothesize the conserved herpesvirus protein kinase (CHPK) of Marek's disease virus (MDV) is a master regulator of cell-free virus (CFV) production. Accumulating data has suggested specific MDV proteins are not expressed or are "inactive" during cell culture propagation; thereby, MDV is unable to complete infectious CFV assembly. These same viral genes are abundantly expressed or "active" in skin cells. This facilitates CFV production and transmission in chickens. Our goal here is to identify these specific viral proteins targeted by CHPK and modify those to be expressed or active in cell culture to produce CFV. Our hypothesis will be examined using three Specific Aims.Specific Aim 1: Determine post-translational modifications (PTMs) on CHPK during semi- and fully productive replication.We do not know the present state of "activation" of CHPK in cell culture and skin cells representing semi- and fully productive replication, respectively. Based on the differential phosphorylation of viral proteins in skin cells during vCHPKwt (fully productive) and vΔCHPK (semi-productive) replication, we hypothesize CHPK is "modified" in skin cells to be "active". We have four objectives to achieve this specific aim.Analyze PTMs on CHPK during fully productive replication in vivo.Analyze PTMs on CHPK during semi-productive replication in cell culture.Test specific PTMs of CHPK during replication in cell culture.Test specific PTMs on pCHPK for in vivo replication and transmission.Specific Aim 2: Determine PTMs on viral proteins during semi- and fully productive replication.Using LC/MS-MS-based proteomics, we detected 20 viral proteins phosphorylated in MDV-infected skin cells, while these same protein peptides were detected in vΔCHPK-infected skin cells but were not phosphorylated. Here, we will examine the modifications of viral proteins during semi-productive replication in cell culture and compare these results to fully productive replication in skin cells. We hypothesize that specific viral proteins, such as pUL47 and pICP27, will be differentially phosphorylated when examined during semi- (cell culture) and fully (skin cells) productive replication. We have four objectives to achieve this specific aim.Analyze PTMs on MDV proteins during fully productive replication.Analyze PTMs on MDV proteins during semi-productive replication in cell culture.Test specific viral protein PTMs in cell culture.Test specific PTMs for in vivo replication and transmission.Specific Aim 3: Determine PTMs on cellular proteins during semi- and fully productive replication.We do not know the present state of activation of CHPK in cell culture and skin cells representing semi- and fully productive replication, respectively; however, Specific Aim 1 is focused on identifying PTMs on pCHPK, while Specific Aim 2 is directed at validating our current information on viral protein PTMs. Based on the differential phosphorylation of cellular proteins in skin cells during vCHPKwt (fully productive) and vΔCHPK (semi-productive) replication, we hypothesize specific cellular proteins or pathways are not present or "active" in cell culture. In contrast, these cellular proteins or pathways are expressed and "active" during fully productive MDV replication in skin cells. Alternatively, it is possible CHPK "activity" is inhibited by cellular proteins or pathway in cell culture cells that is not present in skin cells. We would predict the former to be most likely and this Specific Aim will address these contrasting hypotheses with two objectives.Analyze cellular PTMs during semi- and fully productive replication.Test specific cellular PTMs during replication in cell culture.
Project Methods
Methods to be used include MS-based proteomics studies, generation of mutant MDV, testing for CFV infectivity, and experimental/natural infection studies.We utilized LC/MS-MS-based proteomics with the UIUC Proteomics Core Facility and will continue with the methodology recently published. For all groups, a total of three independent samples will be used. Triplicate analyses are the standard for label-free quantitative proteomics work with good coverage of the proteome without extensive pre-fractionation.To determine the importance of viral proteins for CFV production, we will use our experimental/natural infection model with recombinant viruses. This is a straightforward system for our laboratory. We use two-step Red recombination of bacterial artificial chromosome (BAC) clones containing the complete MDV genome to efficiently modify specific genes. These BAC clones are then reconstituted into infectious viruses, titrated, and then inoculated into chickens to experimentally infect the chickens. These experimentally infected birds are housed with naïve contact chickens that can be infected, through the natural route, to test whether a respective recombinant virus can horizontally transmit. Fluorescently tagged MDV are used to track the virus from chicken to chicken.Using MDV generated in this project, we will test whether specific modifications in the MDV proteins affect their functions in cell culture. Using a refined protocol for CFV extraction in our laboratory, infectivity assays will be used to determine whether modifications led to infectious CFV.