Simrika Thapa

Prion diseases are fatal infectious neurodegenerative disorders affecting both humans and animals. They are caused by the misfolded isoform of the cellular prion protein (PrP^C), PrP^Sc, and currently no options exist to prevent or cure prion diseases. Chronic wasting disease (CWD) in deer, elk and other cervids is considered the most contagious prion disease, with extensive shedding of infectivity into the environment. Cell culture models provide a versatile platform for convenient quantification of prions, for studying the molecular and cellular biology of prions, and for performing high-throughput screening of potential therapeutic compounds. Unfortunately, only a very limited number of cell lines are available that facilitate robust and persistent propagation of CWD prions. Gene-editing using programmable nucleases (e.g., CRISPR-Cas9 (CC9)) has proven to be a valuable tool for high precision site-specific gene modification, including gene deletion, insertion, and replacement. CC9-based gene editing was used recently for replacing the PrP gene in mouse and cell culture models, as efficient prion propagation usually requires matching sequence homology between infecting prions and prion protein in the recipient host. As expected, such gene-editing proved to be useful for developing CWD models. Several transgenic mouse models were available that propagate CWD prions effectively, however, mostly fail to reproduce CWD pathogenesis as found in the cervid host, including CWD prion shedding. This is different for the few currently available knock-in mouse models that seem to do so. In this review, we discuss the available in vitro and in vivo models of CWD, and the impact of gene-editing strategies.

Chronic wasting disease (CWD) is a prion disease found in both free-ranging and farmed cervids. Susceptibility of these animals to CWD is governed by various exogenous and endogenous factors. Past studies have demonstrated that polymorphisms within the prion protein (PrP) sequence itself affect an animal’s susceptibility to CWD. PrP polymorphisms can modulate CWD pathogenesis in two ways: the ability of the endogenous prion protein (PrP^C) to convert into infectious prions (PrP^Sc) or it can give rise to novel prion strains. In vivo studies in susceptible cervids, complemented by studies in transgenic mice expressing the corresponding cervid PrP sequence, show that each polymorphism has distinct effects on both PrP^C and PrP^Sc. It is not entirely clear how these polymorphisms are responsible for these effects, but in vitro studies suggest they play a role in modifying PrP epitopes crucial for PrP^C to PrP^Sc conversion and determining PrP^C stability. PrP polymorphisms are unique to one or two cervid species and most confer a certain degree of reduced susceptibility to CWD. However, to date, there are no reports of polymorphic cervid PrP alleles providing absolute resistance to CWD. Studies on polymorphisms have focused on those found in CWD-endemic areas, with the hope that understanding the role of an animal’s genetics in CWD can help to predict, contain, or prevent transmission of CWD.

Endosomal toll-like receptor-21 and -9 sense CpG DNA activating production of pro-inflammatory mediators with antimicrobial effects. Here, we investigated the induction of antiviral response of in ovo delivered CpG DNA against infectious laryngotracheitis virus (ILTV) infection. We found that in ovo delivered CpG DNA significantly reduces ILTV infection pre-hatch correlating with the expression of IL-1β and increase of macrophages in lungs. As assessed in vitro, CpG DNA stimulated avian macrophages could be a potential source of IL-1β and other pro-inflammatory mediators. Since we also found that in ovo CpG DNA delivery maintains increased macrophages in the lungs post-hatch, we infected the chickens on the day of hatch with ILTV. We found that in ovo delivered CpG DNA significantly reduces mortality and morbidity resulting from ILTV infection encountered post-hatch. Thus, CpG DNA can be a candidate innate immune stimulant worthy of further investigation for the control of ILTV infection in chickens.