title: The molecular pathogenesis of feline calicivirus infection
Creators: name: Newsham, Emma
Publisher: name: University of Liverpool Repository
Year: 2011

description: Feline calicivirus (FCV) is an important veterinary pathogen of domestic cats. It is highly variable and rapidly evolving such that many different strains of varying pathogenicity exist from avirulent, to mildly virulent and hypervirulent. FCV is one of only a small number of viruses within the Caliciviridae to propagate readily in vitro. This property has made FCV a model for the study of the viruses of the calicivirus family. However, relatively little remains known about the way in which these viruses affect host cellular mechanisms. Despite the increasing use of post-genomic technologies in other areas of science, what is known about the effects of calicivirus replication upon host cells has generally relied on relatively conventional technologies to target known defined pathways. In order to investigate FCV modulation of the host cell proteome a 2D-DIGE experiment was performed upon a single viral strain (FCV-F9) with four time points analysed. The results of this analysis showed that 30 host proteins were differentially expressed in the cells infected with FCV-F9 and of these 30 proteins, 14 were successfully identified using mass spectrometry and 11 of these had cytoskeletal proteins within their identifications. To further evaluate these host proteome changes and to explore whether variations in strain pathogenicity were associated with varying affects in the host proteome, a second 2D-DIGE experiment was performed upon four strains of FCV isolated from cats exhibiting a range of clinical signs. As in the first experiment, host cytoskeletal proteins appeared to be targeted for modulation during infection. Overall, this experiment uncovered 131 modulated host proteins with 61 proteins successfully identified by mass spectrometry and 16 of these had cytoskeletal proteins within their identifications. Also found within this experiment was evidence of the putative virulent FCV strain UKOS-A having a different affect upon 14 of the differentially expressed proteins to the other three FCV strains tested. UKOS-A was found to down-regulate these proteins whereas the other strains had an up-regulatory effect, these proteins could be a potential marker of hypervirulence but would need further study to confirm their validity. To follow the modulation of the identified host cytoskeletal proteins, immunofluorescent staining coupled with confocal microscopy was used to track FCV capsid protein and two cytoskeletal proteins vimentin and tubulin. Results showed that during infection with the four FCV strains tested both vimentin and tubulin were both significantly altered during infection with the intensity of fluorescent staining increasing in all cases. To complement the proteomic analyses, an experiment designed to discover differentially expressed gene transcripts was conducted using RNA-seq technology. Overall, there were 354 genes found to be differentially transcribed within the infected cells at both four and seven hrs post infection (p.i.) with the majority of modulated genes experiencing down-regulation at four hrs p.i. and up-regulation at seven hrs p.i.. Once again, genes within the cytoskeletal regulation pathway were up-regulated alongside genes from the apoptosis regulatory pathway and immune system response pathways. To further characterise the putative virulent strain used in earlier studies, attempts were made to sequence the rest of this virus using polymerase chain reaction and conventional Sanger sequencing technologies. Although almost 50% of the UKOS-A strain was sequenced, the sequence obtained was no more similar or different to other virulent systemic disease (VSD) causing strains. Overall, the most marked effects of FCV infection upon the host were found amongst the cytoskeletal genes and proteins which have been demonstrated to undergo extensive modification. This is a completely novel finding in this family of viruses. Further studies will be needed to identify the consequences of the identified cytoskeletal changes to both the host and the virus.