Publications

Avian leukosis virus (ALV) induces B-cell lymphomas and other malignancies in chickens through insertional activation of oncogenes, and c-myc activation has been commonly identified in ALV-induced tumors. Using ALV-transformed B-lymphoma-derived HP45 cell line, we applied in situ CRISPR-Cas9 editing of integrated proviral long terminal repeat (LTR) to examine the effects on gene expression and cell proliferation. Targeted deletion of LTR resulted in significant reduction in expression of a number of LTR-regulated genes including c-myc. LTR deletion also induced apoptosis of HP45 cells, affecting their proliferation, demonstrating the significance of LTR-mediated regulation of critical genes. Compared to the global effects on expression and functions of multiple genes in LTR-deleted cells, deletion of c-myc had a major effect on the HP45 cells proliferation with the phenotype similar to the LTR deletion, demonstrating the significance of c-myc expression in ALV-induced lymphomagenesis. Overall, our studies have not only shown the potential of targeted editing of the LTR for the global inhibition of retrovirus-induced transformation, but also have provided insights into the roles of LTR-regulated genes in ALV-induced neoplastic transformation.

The introgression of genetic traits through gene drive may serve as a powerful and widely applicable method of biological control. However, for many applications, a self-perpetuating gene drive that can spread beyond the specific target population may be undesirable and preclude use. Daisy-chain gene drives have been proposed as a means of tuning the invasiveness of a gene drive, allowing it to spread efficiently into the target population, but be self-limiting beyond that. Daisy-chain gene drives are made up of multiple independent drive elements, where each element, except one, biases the inheritance of another, forming a chain. Under ideal inheritance biasing conditions, the released drive elements remain linked in the same configuration, generating copies of most of their elements except for the last remaining link in the chain. Through mathematical modelling of populations connected by migration, we have evaluated the effect of resistance alleles, different fitness costs, reduction in the cut-rate, and maternal deposition on two alternative daisy-chain gene drive designs. We find that the self-limiting nature of daisy-chain gene drives makes their spread highly dependent on the efficiency and fidelity of the inheritance biasing mechanism. In particular, reductions in the cut-rate and the formation of non-lethal resistance alleles can cause drive elements to lose their linked configuration. This severely reduces the invasiveness of the drives and allows for phantom cutting, where an upstream drive element cuts a downstream target locus despite the corresponding drive element being absent, creating and biasing the inheritance of additional resistance alleles. This phantom cutting can be mitigated by an alternative indirect daisy-chain design. We further find that while dominant fitness costs and maternal deposition reduce daisy-chain invasiveness, if overcome with an increased release frequency, they can reduce the spread of the drive into a neighbouring population.

Guidance surrounding equine slaughter varies globally and lacks published evidence, limiting practical application, causing industry confusion and potentially compromised welfare at all stages of the slaughter process, both ante- and post-mortem. Existing research in this field was systematically reviewed, with gaps in the literature assessed. Four databases were searched: PubMed, CAB Abstracts, Science Direct and Google Scholar, using a combination of different search terms. Predetermined inclusion and exclusion criteria were applied with studies required to be novel research, on the welfare of equids or comparable species, relevant to the research question. Full articles were assessed for reliability, repeatability, potential bias and study design.

In total, 2194 articles were screened, and an additional 35 articles were identified via peer-networks and after a snowball search of reference lists. After screening, 105 studies were identified for inclusion in the review. Of these, 101 (96%) were peer-reviewed journal articles and 4 (4%) were grey literature. Thirty-two (30%) looked at equid slaughter specifically with conflicting findings regarding slaughter efficacy. Similar to other species, there was overall agreement on horses showing stress-related behaviour prior to slaughter. Most studies (n = 76, 72%) were conducted in High-Income Countries and not countries where equid slaughter is estimated to be most prolific. There was no published research on the efficiency of stunning or slaughter of donkeys or mules. In conclusion, this systematic review found a shortage of published research assessing equid welfare at slaughter, particularly donkeys and mules in low-income countries, highlighting the need to urgently develop an evidence base for improving guidance in this area.

Kobuviruses are an unusual and poorly characterized genus within the picornavirus family and can cause gastrointestinal enteric disease in humans, livestock, and pets. The human kobuvirus Aichi virus (AiV) can cause severe gastroenteritis and deaths in children below the age of 5 years; however, this is a very rare occurrence. During the assembly of most picornaviruses (e.g., poliovirus, rhinovirus, and foot-and-mouth disease virus), the capsid precursor protein VP0 is cleaved into VP4 and VP2. However, kobuviruses retain an uncleaved VP0. From studies with other picornaviruses, it is known that VP4 performs the essential function of pore formation in membranes, which facilitates transfer of the viral genome across the endosomal membrane and into the cytoplasm for replication. Here, we employ genome exposure and membrane interaction assays to demonstrate that pH plays a critical role in AiV uncoating and membrane interactions. We demonstrate that incubation at low pH alters the exposure of hydrophobic residues within the capsid, enhances genome exposure, and enhances permeabilization of model membranes. Furthermore, using peptides we demonstrate that the N terminus of VP0 mediates membrane pore formation in model membranes, indicating that this plays an analogous function to VP4. IMPORTANCE To initiate infection, viruses must enter a host cell and deliver their genome into the appropriate location. The picornavirus family of small nonenveloped RNA viruses includes significant human and animal pathogens and is also a model to understand the process of cell entry. Most picornavirus capsids contain the internal protein VP4, generated from cleavage of a VP0 precursor. During entry, VP4 is released from the capsid. In enteroviruses this forms a membrane pore, which facilitates genome release into the cytoplasm. Due to high levels of sequence similarity, it is expected to play the same role for other picornaviruses. Some picornaviruses, such as Aichi virus, retain an intact VP0, and it is unknown how these viruses rearrange their capsids and induce membrane permeability in the absence of VP4. Here, we have used Aichi virus as a model VP0 virus to test for conservation of function between VP0 and VP4. This could enhance understanding of pore function and lead to development of novel therapeutic agents that block entry.