CHAPTER 2: Materials and Methods 42
3.1 Introduction 58
Many recombinant enteroviruses have been isolated and characterised from field samples following outbreaks of infection (Kew et al., 2002, Zhang et al., 2010, Oberste et al., 2004a). Early studies have highlighted potential triggers found in the RNA sequence and RNA secondary structure that provides preferential sites for recombination (section 1.3). However, the
mechanisms of recombination still remain poorly understood. The field isolates studied in all examples have been exposed to a highly selective and dynamic environment, and may not necessarily represent the initial recombinant molecules produced. Characterising the
mechanisms that drive recombination will help us understand a basic evolutionary mechanism. Additionally, potential strategies to limit the occurrences of such events can be developed. This would be helpful in the fight to eradicate poliovirus for example; production of a live attenuated vaccine strain that doesn’t recombine would be very beneficial to this process.
Recombination is a rare event, with intertypic recombinants estimated to have frequencies of 1x 10-‐6 (Kirkegaard and Baltimore, 1986b, Jiang et al., 2007). Identification of recombinant
genomes in a population that is predominately made up of parental viruses has therefore been an issue in all cell based studies. Early in vitro approaches have largely relied upon crossing parental genomes carrying different selectable markers. Those that ‘escape’ the selection process will have inherited the mutations from the two parental strains and would be recombinant viruses (Kirkegaard and Baltimore, 1986a). There is no doubt that such studies have provided some interesting observations, most notably the ‘copy-‐choice’ model of
recombination (section 1.3). Although, identification of the recombinant viruses isolated under such selective pressures was limited to only a small region of the genome as defined by the selectable markers and may again not have represented the initial recombination event. An additional risk of reversion is also a possibility in such approaches, with a point mutation reverting a genome back to wild type around 10-‐100 fold more likely that an intertypic recombination event (Crotty et al., 2001). To overcome such issues, a novel in vitro assay has
been developed by a previous student (Kym Lowry) that uses two defective parental genomes that are each unable to generate infectious virus. Only a recombination event can produce a genome that can be potentially infectious.
Methodology
The first parental RNA is a sub-‐genomic replicon that has a large deletion of the virus genome. The capsid region (P1) was replaced with a reporter gene; in this study it is a firefly luciferase gene. This type of genome can translate and replicate successfully as it contains the key cis-‐
acting elements and is a well established approach used to study polioviruses (Barclay et al., 1998, Percy et al., 1992). The lack of a capsid region ensures no virus particles are produced when transfected into permissive cells alone. In any recombination assay this genome would provide the non-‐structural P2 and P3 regions, and is therefore considered the ‘donor’. The second genome (the acceptor in a recombination event) would be required to produce the capsid-‐coding region, but carry a defect in the non-‐structural region preventing production of a viable virus upon transfection. A suitable candidate is the well characterised genome that carries eight synonymous mutations in the highly conserved 2C CRE that disrupts positive strand RNA synthesis (Goodfellow et al., 2003a). This genome can produce negative strand RNA so is considered suitable for a replicative recombination assay, as it is believed recombination occurs during negative strand synthesis (Kirkegaard and Baltimore, 1986b). This approach relies upon delivering both RNA templates into cells, via transfection, where subsequent recombination and genomic re-‐arrangements can occur. Donation of a capsid from one parental genome and a functional CRE from the other parental genome would in principle produce a viable virus, and is termed the CRE-‐REP assay (figure 3.1). In principle, this provides a region of 1.058 Kb, between the end of VP1 and the 2C CRE, where recombination can occur. Importantly, the use of rodent cell lines like the murine L929 or baby hamster BsrT7 cells allows minimal selection of recombinant virus as is possible. Both cell types lack the poliovirus receptor CD155, but can support RNA replication. They are therefore deemed permissive but not susceptible to infection. Any recombinant virus produced and released from the cell into the media
isolated and characterised as near to the recombination event as possible. Additionally, by minimising selection, this approach allows isolation of a range of recombinant regardless of growth advantages. This is in contrast to previous in vitro cell based approaches that have largely used HeLa cells that are susceptible to re-‐infection due to the availability of receptor. This may have biased the identification of recombinant viruses to the ones that had a growth advantage over others. Quantification of any recombinant virus is by plaque assay, media supernatant is isolated from transfected rodent cells following an incubation period and subsequently used with permissive and susceptible cell lines like HeLa (section 2.1 for full description).
The CRE-‐REP has allowed various combinations of enterovirus partners to be used previously (Lowry et al., 2014). The primary enterovirus serotypes studied in this investigation were poliovirus type 1 (Mahoney) and type 3 (Leon). This was due to the availability of cDNA, the subsequent re-‐producibility of the CRE-‐REP assay when using these partners (outlined in text later), and the vast amount of past research into the life cycle of this virus.
Aims
The aim was to build upon the findings of a previous student, Kym Lowry. These included: comparing intratypic and intertypic poliovirus recombination frequencies, characterisation of early intratypic and intertypic recombinant isolates, cell specificity and characterisation of early intertypic recombinants to those following serial passage. In addition, it was felt important to develop a non-‐replicative recombination assay that would contrast the CRE-‐REP assay, which was presumed to be replicative i.e. recombinant virus genome produced during replication of the parental RNA. This would determine if the recombinant progeny was produced via a replicative mechanism like ‘copy-‐choice’, whilst also highlighting the relative contribution of non-‐replicative recombination to the overall yield of virus. The results in this chapter have contributed to the manuscript Lowry et al., 2014.
Figure 3.1: Overview of the CRE-‐REP assay
Two genomes, upper [dark shading] bears a defective CRE indicated as a broken line with a superimposed X in the 2C-‐coding region. Lower genome is a luciferase-‐encoding sub-‐genomic replicon (light shading). Following co-‐transfection into permissive cells (indicated by an arrow), a recombinant replication competent genome may be recovered of the generic structure shown, consisting of the 5’ part of the genome derived from the CRE-‐defective parent (the recipient) and the 3’ part of the genome from the luciferase-‐encoding replicon (the donor). The graduated shading between the 3’ end of the VP1-‐coding region and the 2C CRE indicates the area within which recombination must occur to produce a functional genome.