Abstract:
Nelson Bay orthoreovirus (NBV) was first discovered in the 1960s in Nelson Bay, Sydney, Australia. Subsequently more isolates of NBV was found in patients and flying foxes in China, Hong Kong, Japan and Malaysia. Surveillance studies in Malaysia have found that this virus has spilled over to humans, with more than 10% of the samples tested being positive. This is a worrying trend as there is limited knowledge on the long term impact of human health by this virus. With more knowledge on this virus gathered, the name NBV was changed to Pteropine orthoreovirus (PRV) to reflect the host carrying the virus. The cell attachment protein for Mammalian orthoreovirus (MRV) is σ1 while for Avian orthoreovirus (ARV) σC is the cell attachment protein. Since the size and genome encoding both these proteins are closely related to σC of PRV, it is hypothesised that σC is the cell attachment protein. For MRV, the cellular receptor that binds to σ1 is Junctional Adhesion Molecule A (JAMA) while chicken embryo fibroblast receptor was identified for ARV. We aim to identify the cell receptor that binds to σC of PRV. Through our experiment, we have successfully produced σC of Pulau virus (PulV), Kampar virus (KamV) and NBV using bacteria after fusing it with Maltose Binding Protein (MBP). However, when we perform immunoprecipitation we were unable to immunoprecipitate any protein. We hypothesised that bacterial-produced protein might lack biological activity due to lack of post translational modification hence we used mammalian cells to produce σC. After cloning sigma C gene into a mammalian vector, we determined that σC was expressed through western blot. The subsequent immunoprecipitation ended in failure. This caused us to use a technique known as Virus Overlay Protein Binding Assay (VOPBA) and we successfully identified human cytokeratin 1 and pyruvate kinase as the proteins that bind to PulV and KamV. We were not able to identify the receptor for NBV as we do not have live NBV. Based on existing literature, it is found out that HeLa cells are less permissive to PRV and HeLa cells are known not to produce human cytokeratin 1. This supports our finding that human cytokeratin 1is the cellular receptor that PRV binds to for cell entry. Pyruvate kinase is a protein found in the cytoplasm and nucleus of a cell. Due to that, pyruvate kinase is not responsible for viral cell entry. However, its binding with PulV and KamV raises the possibility that it might be important in the subsequent pathogenesis of PulV and KamV. Further studies in this area are required before realizing the importance of pyruvate kinase in the pathogenesis of PulV and KamV.
