Andrew Routh, PhD
Assistant Professor, Department of Biochemistry & Molecular Biology
Tel: (409) 772-3663
Campus Location: 6.136B T.G. Blocker Med. Res. Bldg
Mail Route: 1061
The Routh Lab is 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
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.
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 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.