With the RNA-seq experiment performed in this chapter mirroring the FCV strain and time points used in the proteomic experiments, a comparison of differentially expressed genes and proteins should be possible.
Looking at all of the identifications of the differentially expressed proteins of the two 2D-DIGE experiments (chapters 2 and 3, Appendix I and II) and the differentially transcribed genes in the RNA-seq study (Appendix VII) it would appear that there is very little correlation between the genes identified and the proteins expressed. There are also no significant matches in the GO analyses with the DIGE experiments proving to have a much larger proportion of cytoskeletal proteins than any of the RNA-seq comparisons. The pathway analysis of the RNA-seq genes, however, shows that there are some significant genes up-regulated within the actin cytoskeletal regulation pathway. This does correlate with the findings of the previous 2D-DIGE
and yeast often show good correlation whereas in mammalian samples the correlation becomes much weaker. The authors hypothesise that the transcriptional and translational networks have evolved independently within the cell and so they are not directly linked which may explain the often seen discrepancies between transcriptomes and proteomes. Another analysis performed by Wang et al, (2010) gave a hypothesis that transcription and translation contained a time-delayed component and once this is taken into account then the correlation is much greater. The overall results from this study show that there are a large number of genes whose transcription is significantly altered as a result of FCV-F9 infection and although this investigation was only possible upon genes previously annotated upon the feline genome there was still a vast amount of data generated. Future interrogation of the data will rely heavily upon bioinformatics methods but now the data has been generated it may be useful in driving forward the annotation of the cat genome.
6
Chapter six
Sequence analysis of a UK outbreak of virulent systemic FCV
6.1
Introduction
Infection with FCV has been shown to be associated with a wide range of clinical signs although until recently these clinical signs have generally been found to be self limiting with a low mortality rate. In recent times outbreaks of new, highly virulent strains of FCV have been reported in the USA and Europe which show much greater mortality rates and clinical signs.
The first three outbreaks were reported in the USA (Hurley et al., 2004; Pedersen et al., 2000; Schorr-Evans et al., 2003) and followed similar patterns. Cats infected during the outbreaks showed clinical signs such as fever, anorexia, oedema of face and limbs, oral ulceration, ocular and nasal discharge, jaundice and a very high mortality rate of up to 50%. There have also been two similar outbreaks in Europe; one in the UK (Coyne et al., 2006b) and one in France (Reynolds et al., 2009). In France the outbreak occurred at a veterinary teaching hospital in 2005 and the clinical signs observed were similar to those from the US outbreaks. The overall mortality rate was even higher than the previous outbreaks reported with 63% of cats dying or being euthanised.
The viruses used in this chapter have been isolated from the UK outbreak of VS- FCV, reported by Coyne et al in 2006. The UK outbreak was traced back to three kittens being initially fostered from a local rescue shelter into the home of one of the workers at the shelter. During the fortnight following the fostering two of the kittens died. Subsequently, three of the four cats already living in this foster household went on to exhibit signs of infection with VS-FCV. All three of these cats suffered pyrexia
euthanised giving an overall mortality rate of 71% which again is in keeping with the previous reports. VS-FCV was isolated from the three dead adult cats and given the nomenclature UKOS-A, UKOS-B and UKOS-W. Capsid sequencing was performed upon these three isolates and the results were compared to the other outbreak strains, other published genomes and some unpublished lab strains using phylogenetic analysis (Fig. 6.1, reproduced from Coyne et al (2006b)).
Also as part of the Coyne et al study into this outbreak, FCV isolates were collected 58 days after the outbreak began from 28 of 42 cats housed in the local rescue shelter where the 3 kittens were fostered from. These types of shelters have previously been shown to have higher prevalences of FCV than private cat homes and that there are often a number of strains found circulating within them (Radford et al., 2001) (Bannasch & Foley, 2005). As such there are also more chances of recombination
Figure 6.1. Phylogenetic tree based upon capsid sequence. Included in this analysis are the three UKOS strains; FCV-Ari (Pedersen et al., 2000), FCV-Kaos (Hurley et al., 2004), FCV-Diva (Schorr-Evans et al., 2003). Also included are FCV-F9 (Genbank M86379), LS012 (Genbank AF109467), LS015 (Genbank AF109464), F65 (Genbank AF109465), CF168 (Genbank U13992), 255(Genbank U07131) and unpublished field viruses (S090 and V818) (Coyne et al., 2006b).
events occurring within these shelters and colonies which can aid rapid evolution of FCV strains (Coyne et al., 2006c). It has been hypothesised that such environments provide the ideal environment for the evolution of the hypervirulent phenotype (Coyne et al., 2006b). Selection pressures within the colony favour the selection of rapid high titre replication as such viruses are likely to have a high transmission advantage. Within the confines of the colony, cats in the shelters acquire and evolve immunity in a progressive, step-wise fashion, in parallel with this viral evolution, allowing the replication of these viruses to be controlled within relatively low limits, preventing manifestation of VS-FCV symptoms. However, when these virus strains are introduced to naïve populations, these newly infected cats have no strain- matched acquired immunity and so are unable to fight off the infection in the same manner, such that the shelter viruses are able to fulfil their high titre/fast replication rate phenotype leading to the development of the severe clinical signs associated with VS-FCV. Such a mechanism would allow each outbreak of VS-FCV disease to be associated with a new strain of FCV.
The collection of isolates from the UK outbreak has for the first time, allowed some analysis to be made on the relatedness of the UKOS strains to those found within the rescue shelter from where the putative index case kittens originated. This has already been done using sequence analysis of a small region of the polymerase gene (Coyne, 2005), showing that the rescue shelter contained at least five strains of FCV. The most frequently recognised strain in the rescue shelter was FCV-UKOS, confirming the shelter as the likely source of this outbreak, despite the fact that the cats in the shelter were generally healthy. Within the shelter, viruses belonging to this strain were highly diverse, with some viruses being extremely similar to this found in the dead cats, whilst others were less similar.
The aim of this chapter was two-fold. Firstly, further sequence analysis of the genome of one of the strains isolated from a cat which died during the outbreak
Secondly, we wish to capitalise on the unparalleled availability of viral strain variants of FCV-UKOS in the rescue shelter, to further characterise the diversity and evolution of FCV in this environment. In particular, by analysing sequence of the capsid gene (complimenting the already available polymerase sequence), it was hoped to discover how much strain variation was present in the shelter and whether there were any recombination events occurring that may have aided evolution of a hypervirulent strain.