We are always looking for bright and enthusiastic people to work with us. For details of current open positions, please contact Andrew Routh by email.
RNA viruses are masters of evolution; rapidly adapting to new environments, evading immune responses and side-stepping anti-viral therapies. Next-generation sequencing (NGS) has transformed our ability to probe and characterize the biology and evolution of viruses, and is still a burgeoning field. From lab-adapted viral strains to on-going pandemics, NGS allows us to map with exquisite accuracy the variations and mutations that define a viral population. By characterizing the changes in these variations over time, we may unpick the processes that underpin viral evolution and progression.
In my lab, we combine molecular and cellular virology, next-generation sequencing and computational biology to study well-controlled and highly characterized model systems such as Flock House Virus and Cricket Paralysis Virus, as well as human pathogens including Human Rhinoviruses and HIV. We study systems ranging from controlled cell culture, through animal models, into clinical specimens. This multi-strata approach is aimed at gaining a molecule’s-eye view of the mechanisms of RNA replication and recombination in order to understand virus evolution on a population scale.
“Viral evolution and adaption”
Four main project areas are described here:
1. Virus structure and assembly
We study the structure and assembly of virus particles with a focus on the role of the packaged nucleic acids. The recognition, selection and packaging of viral RNA from among total cellular RNA present is critical for a viral progression. However, by sequencing the RNA encapsidated by purified Flock House Virus (FHV) particles, we have observed that FHV can package host-derived RNA transcripts including retro-transposons (Routh et al., PNAS 2012). As well as providing important insights into viral assembly and the mechanisms of RNA packaging, this revealed a potentially important role for small RNA viruses in the evolution of their hosts through the horizontal transmission of transposons. Retro-transposons were also found in virus-like-particles of FHV, which may have important implications for VLP-based therapies. We are studying the role of specific amino acids in the capsid of FHV that are known to determine faithful viral RNA packaging, as well as investigating the role of functional RNA motifs found within the RNA genome of FHV.
“Amino acids in the capsid protein of Flock House Virus are important for the packaging of correct viral RNAs”
2. Virus evolution and genetics
It has long been speculated that viruses can evolve a reduced virulence to prolong the period during which the host is infectious through the co-transmission of defective-interfering RNAs (DI-RNAs). DI-RNAs attenuate viral infections via a variety of proposed mechanisms and have been proposed to promote the transition of acute to chronic infections. Until recently, DI-RNAs had only been captured individually via classical cloning techniques, limiting our understanding of the diversity of DI-RNAs. Despite the well-established abilities of DI-RNAs to attenuate virus replication in cell culture and their observation in a number of clinical settings (e.g. measles, dengue and chronic HCV infection), little is understood about the action of DI-RNAs in live animals and their effect upon disease progression.
We are characterizing the step-wise evolution of DI-RNAs in cell culture using model systems, including Flock House Virus. In turn, we are comparing them to the DI-RNAs that arise spontaneously during live animal infections and determining their effect upon the outcome of viral infection, for instance by inducing persistence or by providing protection to super-infection. In the long-run, the ability of DI-RNAs to attenuate viral infections raises the tantalizing prospect of developing live-attenuated vaccines.
3. Next-Generation Sequencing techniques.
I recently developed a click-chemistry based next-generation sequencing library generation method called “ClickSeq” (Routh et al. 2015 JMB). Here, we supplement randomly-primed RT-PCR reactions with small amounts of 3’-azido-nucleotides to randomly terminate cDNA synthesis and release a random distribution of 3’-azido blocked cDNA fragments (a process akin to classical Sanger sequencing using dideoxynucleotides). These are then ‘click-ligated’ to 5’ alkyne-modified DNA adaptors via copper-catalysed cycloaddition. This generates ssDNA molecules with unnatural yet bio-compatible triazole-linked DNA backbones that can be used as PCR templates to generate RNAseq libraries. By virtue of removing the fragmentation and enzymatic ligation steps, artifactual recombination is reduced to fewer than 3 events per million reads allowing us to confidently detect rare recombination events and replication intermediates.
“ClickSeq: ‘Click-ligation’ of alkyne modified adaptors to azido-terminated cDNAs”
ClickSeq relies on the random incorporation of 3’azido-nucleotides into ssDNA during RT-PCR. However, this basic process also occurs in live cells that have been treated with the anti-viral drug, azidothymidine, AZT. So, can we adapt ClickSeq to capture and sequence DNA that has been replicated by viral polymerases or reverse transcriptases? This may enrich rare DNA transcripts from novel retroviruses, enabling their discovery. There are also similar potential applications for other AzT-sensitive polymerases such as endogenous retroviruses, telomerase, and bacterial DNA polymerases, which may be profiled in a number of settings such as in cell culture, animal models, or from clinical samples (‘ex vivo’ ClickSeq).
4. Computational Virology:
We are utilizing and developing computational pipelines for the analysis of NGS data of viral samples.
- “ViReMa” (Viral Recombination Mapper) is a versatile and flexible computational pipeline for the discovery of viral recombination events in NGS datasets that employs a novel ‘moving-seed’ approach for sequence alignment (Routh et al 2014 NAR). In addition to improved speed and sensitivity over other algorithms using canonical ‘fixed-seed’ approaches, ViReMa detects substitutions and non-reference insertions, multiple recombination events and virus to host recombination. This flexibility has proven critical for mapping viral recombinations as these events rarely conform to predefined (or known) rules. Using ViReMa, we have found that after resistance to protease inhibitors had developed in HIV positive patients, virus populations haboured short duplications proximal to the proteolysis sites in the GAG protein.
- “CoVaMa” (Co-Variation Mapper) takes NGS alignment data and populates large matrices of contingency tables that correspond to every possible pairwise interaction of nucleotides in the viral genome or amino acids in the chosen open reading frame (Routh et al. 2015 Methods). These tables are then analysed using classical linkage disequilibrium to detect and report evidence of epistasis. CoVaMa found epistatically linked loci in FHV genomic RNA grown under controlled cell culture conditions as well as correlated amino acid substitutions in the protease genes among a large cohort of HIV infected patients undergoing anti-retroviral therapy.
“CoVaMa reveals networks of correlated mutations in drug-resistant HIV protease. Colour-map indicates levels of linkage-disequilibrium.”