Gene editing sheep for pestivirus resistance

Pestiviruses are a global economic burden for livestock production, although exact figures are often difficult to ascertain. Whilst Classical Swine Fever virus (CSFV) has been eradicated in the UK, both Bovine Viral Diarrhoea virus (BVDV) and Border Disease virus (BDV) continue to cause significant economic impact. It is estimated that BVDV costs the UK cattle sector around £36.6M annually (APHA). While the financial impact of BDV on sheep is difficult to quantify, around 30% of UK sheep flocks have incidence of BD with an estimated 2-8% reduction of lamb production in affected flocks (FAS). CSFV remains a disease of global impact and is notifiable to the world organisation of animal health. Reintroduction of the virus to the UK could be devastating; the eradication of a widespread outbreak in the Netherlands in 1997/98 reportedly cost of $2.3 billion1. Most pestiviruses are named based on the host species from which they were first isolated, but these closely related viruses can display interspecies transmission. In the UK it has been reported that as many as 20% of cases identified as BD are actually caused by BVDV infections2, suggesting that cross-species transmission could impede control strategies. While there are effective vaccines available for CSFV and BVDV, there is currently no vaccine against BDV. Vaccines are not cross-protective. Pestiviruses are a genus within the Flaviviridae family of positive-sense, enveloped RNA viruses that can be directly transmitted or vector-borne. Host tropism within the family is broad and many of the pathogens can cause severe illness. It was previously shown that the vertebrate host chaperone protein DNAJC14 is required for replication of the flavivirus Yellow Fever virus (YFV) in vitro3,4. It was also shown that knockout of the DNAJC14 gene in pig and cattle cell lines renders them non-permissive to replication of both CSFV and BVDV5. However, data generated in cellular systems does not always translate to the context of a whole organism6. Our previous work has shown that genome editing can be a viable tool to introduce genetic resistance to disease7,8. Within this project, we aim to generate ruminants with edited DNAJC14, which we hypothesise will be non-permissive to pestivirus (and potentially flavivirus) replication. In parallel, we aim to interrogate virus evolution in vitro prior to validating resistance in vivo. Taking into account the generation interval and financial cost of working with either cattle or sheep, the number of available viral isolates for testing in either species, together with the UKRI funding cycle, we propose to generate a cohort of sheep with edited DNAJC14 to test our hypothesis. The objectives are: To generate genome edited founder (F0) and F1 sheep with ablated DNAJC14 function To assess resistance to and evolution of BDV, BVDV, CSFV and selected flaviviruses in DNAJC14 edited primary cells and cell lines To determine in vivo efficacy of the DNAJC14 edit in the context of BDV challenge of F1 sheep Findings from this project have the potential to provide a genetic control option for pestiviruses and flaviviruses either through genome editing or genetic selection. It may also provide the basis to develop other antiviral strategies, such as small molecule compounds. This project contributes to the BBSRC priority of "Bioscience for sustainable agriculture and food".