Host cell determinants of BoHV-1 pathogenesis: a genome wide analysis.

Infectious bovine rhinotracheitis (IBR) is highly contagious respiratory and genital tract disease of cattle caused by a herpesvirus, Bovine herpesvirus -1 (BoHV-1). Clinical symptoms of the respiratory disease include high fever, weight loss, coughing, excessive salivation, nasal discharge, conjunctivitis, inflamed nasal passages, and breathing difficulties. Abortions can also occur in pregnant cattle. IBR results in significant distress to the animal and significant economic losses to the cattle industry.
Like most viruses BoHV-1 relies on the host cell to provide the machinery need to replicate the virus. However, infected cells also make proteins which inhibit virus replication and the outcome of infection depends on the balance between pro and anti-virus cell factors. Cells contain about 20-30,000 genes and little is known about which of these genes encode proteins that inhibit, or are essential for, BoHV-1 replication. An understanding of the host factors involved in viral replication is important in developing not only genetic approaches to disease resistance, but also new vaccines and treatments.
Previously we were only able to study the effects of one or two genes at a time, but new technologies allow us to investigate the function of all genes in a cell in a small number of experiments. Genome editing technology allows us to precisely target single genes within a cell and either block the ability of that gene to make a protein (turn off the gene), or make the gene produce more protein than normal (turn on the gene). We will create two populations (libraries) of cells; in one library we will use genome editing to produce a population of cells in which each individual cell has a different single gene turned off, and in the second library a population in which each individual cell has a different single gene turned on.
We will use these libraries to look at how cells in which individual genes are turned on or off respond to infection with BoHV-1. We will infect the libraries with BoHV-1 and measure how the infection proceeds compared to unmodified cells. We can select populations of cells in which the virus replicates better or worse and identify which genes have been turned on or off in that population. We predict that if a gene makes a protein which is essential for virus replication, then the virus will replicate less well in the turned off library, but it will replicate better in the turned on library. If a gene makes a protein which inhibits virus replication, then the virus will will replicate better in the turned off library, but it will replicate less well in the turned on library. This approach will allow us to better understand how the virus and cell interact and how the cells control virus infection. It has the potential to identify new genes that inhibit BoHV-1 infection, which could be included in breeding programmes to increase resistance to this important animal pathogen or provide the basis for better vaccines or drugs.

BoHV-1, a herpesvirus, is the causative agent of infectious bovine rhinotracheitis (IBR) and causes significant economical losses to the cattle industry. Little is known about the cellular factors which inhibit, or are essential, for BoHV-1 replication. An understanding of the host factors involved in viral replication is key to developing not only genetic approaches to disease resistance but also new vaccines and therapeutics. We will develop two genome wide CRISPR libraries for cattle: 1) Loss of function library (LOF) where the expression of individual genes is knocked down: 2) Gain of function library (GOF) where the expression of individual genes is switched on. We will screen both libraries following infection with BoHV-1 to identify host genes essential for replication or resistance. We will infect cells at both low and MOI. For low MOI infection we will harvest surviving cells after 4 days. Using PCR for the CRISPR sgRNAs and sequencing we will identify the genes targeted in these cells. Genes required for BoHV-1 replication will be overrepresented in LOF cells; genes that restrict BoHV-1 replication will be overrepresented in GOF cells. For high MOI we will harvest cells at 3 times and FACs sort into High, Median and Low BoHV-1 antigen expressing pools Genes targeted in these pools will be identified as above. In the LOF pools genes required for BoHV-1 replication will be overrepresented in the low pool; genes that restrict BoHV-1 replication will be overrepresented in the high pool. In the GOF pools genes required for BoHV-1 replication will be overrepresented in the high pool; genes that restrict BoHV-1 replication will be overrepresented in the low pool. We will validate a number of candidate genes by creating cell lines in which only the candidate gene is affected. This study will provide detailed information on cellular factors essential for replication or resistance to BoHV-1 and will inform future studies on the development of resistant animals.

Impact Summary
(i) The academic community. Scientifically, the project constitutes a step change in the approach to investigating host pathogen interactions at the cellular level. It will bring together virologists whose expertise lies in understanding pathogenesis with geneticists whose expertise lies in genome manipulation to understand cell and organism phenotype. This impact will be delivered via publication in journals, presentations at conferences, seminars, and by making data and the bovine sgRNA libraries available. Little is known about bovine genes in contrast to e.g. human and mouse. The reagents and data produced will therefore also benefit colleagues in the wider bovine research community by providing a better understanding of bovine gene function.
(ii) The entire chain of users of animal products, including meat packers, milk processors, retailers and consumers will benefit because the knowledge generated will increase our understanding of the pathogenesis of this economically important disease leading to new intervention strategies including possible future scientific breeding programmes aimed at developing resistant animals. This will lead to an improvement in animal welfare with decreased production costs to producers, increased production yields and food security.
iii) UK Treasury will benefit from increased tax revenues through increased profitability of the farm animal supply chain.
(iv) UK science infrastructure and capacity. The proposed methods and resultant materials will provide a platform for increased research capabilities in the UK, maintaining its scientific reputation and associated institutions, with increased capability for sustainable agricultural production. The post-docs working on the project will have the opportunity to be trained at a world-class institute in a cutting edge area of research that unifies two fields.
(v) Society. This project will demonstrate the value of genome editing for identifying the genetic basis of disease resistance, which will help the general public to understand and appreciate the great value of genome editing technology. The knowledge will also feed into educational programs.
(vi) International. The impact of BoHV1 is felt worldwide. The international scientific and agriculture sector will benefit from the knowledge generated in this project.
(vii) Commercial. The opportunity for commercial follow-up on targets identified in this project offers the opportunity for entrepreneurial activity.