Download Detection and diagnosis of new pathogens

Under the Microscope
Detection and diagnosis of new pathogens
Theo P Sloots
Queensland Paediatric Infectious
Diseases Laboratory
Department of Infectious Diseases
Sir Albert Sakzewski Virus
Research Centre
Royal Children’s Hospital
Brisbane, QLD 4029
Tel (07) 3636 8833
Fax (07) 3636 1401
Email [email protected]
Molecular laboratory techniques are widely used to
detect new or previously unrecognised agents which
are implicated in infectious disease. In many cases,
the traditional microbiology techniques are inadequate
and commonly fail to uncover the aetiologic agent,
particularly new viruses that continue to challenge the
human population. Metagenomics-based tools, such as
microarrays and high-throughput deep sequencing are
increasingly applied and are ideal for the identification of
new human pathogens, particularly viruses.
Pan-viral microarrays, containing representative sequences from
all known virus families, have been used to detect novel
and distantly related variants of known viruses. Alternatively,
sequencing-based methods have been employed to detect
genomic sequences of new microbial agents and have the
potential to detect the full spectrum of viruses, including viral
quasi-species.
Indeed, the discovery of new viruses has been a major
consequence of the developments in molecular technology
over the last few years. Particularly, new viruses associated
with the human respiratory and gastrointestinal tracts. In both,
approximately 40% of cases of acute infectious episodes remain
undiagnosed, leading to speculation that as yet undiscovered
pathogens may be responsible for disease. This is supported by
the fact that, since the discovery of human metapneumovirus in
2001 1, six previously undescribed viruses have been identified
by molecular analysis of clinical respiratory specimens, and
an additional number of novel viruses have been associated
with acute diarrhoea in humans. These findings have been
widely reported and include the discovery of three new human
coronaviruses (HCoV); the severe acute respiratory syndrome
(SARS)-associated coronavirus in 2003 2, coronavirus NL63
(HCoV-NL63) in 2004 3, coronavirus HKU1 in 2005 4, human
bocaviruses in 2005 5 and 2009 6,7, the recently described human
polyomaviruses KI and WU (WUV) in 2007 8,9 and Merkel cell
polyomavirus in 2008 10, and novel astroviruses MLBI and VAI 11,12
as well as a novel picornavirus related to cosaviruses 13.
1 2 4
These novel viruses were discovered using a diverse range
of molecular methods, including VIDISCA 3,14, pan-viral
DNA microarrays 15 and high-throughput sequencing 8,9. A
comprehensive review of these and other methods has previously
been published by Ambrose and Clewley in 2006 16.
Virus-Discovery-cDNA Amplified Fragment
Length Polymorphism
In 2004, van der Hoek and colleagues used a modification
of a sequence-independent primer amplification technique,
called Virus-Discovery-cDNA-AFLP (VIDISCA; Figure 1), to
detect a new human coronavirus, HCoV-NL63, from the human
respiratory tract. This technique employs two primers in the PCR
amplification step and includes an amplified fragment length
polymorphism (AFLP) method previously described 3.
DNA is digested with two restriction enzymes, for example, MseI
and HinP1I, both of which have four base pair recognition sites.
This produces DNA molecules with MseI and HinP1I overhangs
at either end, as well as some with MseI–MseI and HinP1I–HinP1I
overhangs. Only the MseI and HinP1I fragments are amplified
in the subsequent PCR as each adapter binds to one specific
end of the DNA fragment, according to its complementary
overhang. Two primers specific to each adapter are then used in
an exponential amplification reaction by PCR. A second selective
nested PCR amplification can be used to simplify the resultant
PCR products from a DNA smear to specific bands. By extending
Figure 1. VIDISCA method. Schematic overview of the stages
involved (adapted from 3).
MICROBIOLOGY AUSTRALIA • SEPTEM B E R 2 0 1 0
Under the Microscope
the 3’ end of the primers by one to three nucleotides, a subset
of the PCR products is generated which is subject to further
characterisation by nucleotide sequencing 17.
Pan-viral DNA microarrays
Wang et al. 18 designed comprehensive DNA microarrays for virus
discovery and applied these in the identification of the novel
coronavirus associated with SARS 15 and the discovery of WUV 9.
These arrays consisted of oligonucleotides representing highly
conserved sequences, derived from reference sequences of
existing viral families obtainable from public sequence databases.
Ten 70-mers were used for each virus, totalling approximately
10,000 oligonucleotides. Viral sequences that hybridised to the
individual array elements were recovered and sequenced, to
identify novel viruses.
Other viral-specific microarrays have been developed to detect
PCR amplicons from sequence-independent amplification
reactions. Boriskin et al. 19 described a diagnostic DNA microarray
specific for central nervous system viral infections and applied
it to the examination of CSF and non-CSF specimens. The
array contains 38 gene targets for 13 viral causes of meningitis
and encephalitis. Other arrays have been described for the
rapid detection and serotyping of acute respiratory diseaseassociated adenoviruses 20, and for the simultaneous detection of
herpesviruses, enteroviruses and flaviviruses 21. Comprehensive
microarrays representing the most up-to-date sequence
information for all viral families offer much promise for the
detection of previously unidentified viruses, provided these have
sufficient homology to known viral sequences 22.
of the invading viral RNA genome using small sequence assembly
software. The ability to assemble these fragments is a spinoff of the new genome-sequencing platforms, which by their
nature produce very short sequence reads that have to be
computationally stitched back together. Results produced using
this technology positively identified RNA from ssDNA and
dsDNA reverse transcribing viruses, generally covering 80%–95%
of the genome and, in some cases, the entire genome. This
method presents a novel approach which can identify known
viral pathogens occurring at extremely low titers and also novel
viruses for which previously identified genome sequence is not
available 25,26.
Conclusion
With the development of new molecular technology, our ability
to detect and characterise new viral agents has greatly improved.
As a result, genome sequences have been described for new
viruses that are associated with the human respiratory tract,
gastrointestinal tract as well as new blood-borne viruses. Some
of these are recognised as significant human pathogens causing
disease in certain population groups. Others can be found in
clinical specimens without definitive evidence for their role as the
causative agent of disease, and yet others, like TT-(torqueteno)
Random PCR amplification and highthroughput sequencing
In some instances, it is advantageous to amplify viral nucleic acids
by random PCR amplification before these can be identified using
microarrays 18. Generally, random PCR uses one primer with a
unique nucleotide universal sequence at the 5’ end (Figure 2).
This sequence contains restriction enzyme sites for subsequent
cloning. On the 3’ end this primer contains a degenerate
hexa- or heptamer sequence 17,23. A second primer, which is
complementary to the 5’ universal region of the first random
primer, is used in subsequent PCR amplification. PCR products
are then cloned and sequenced (Figure 3). Random PCR can be
used to detect both DNA and RNA viral genomes 24.
Virus discovery by deep sequencing and
assembly of virus-derived small interfering
RNAs
Most recently, a novel molecular method was described utilising
small interfering RNA (siRNA) produced by cells, from replicating
viral genomes, as an important part of their antiviral immune
response 25. These siRNA were found to be overlapping in
sequence and were assembled into long, contiguous fragments
M I C R O B I O L O G Y A U S T RALIA • SEPTEMBER 2010 Figure 2. Random PCR. In the first round PCR, Primer 1 with a unique
5’ end and degenerate 3’ end sequence is used. The degenerate
segment binds to template sequences that occur stochastically
throughout the viral genome, and primer extension occurs with T4
polymerase. Double-stranded DNA is formed, containing unique
sequences on each strand. Primer 2 hybridises to the unique
sequence and is used for amplification of fragments of the viral
template DNA (adapted from 24).
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Under the Microscope
Figure 3. High-throughput sequencing. Randomly amplified PCR products are cloned into a suitable plasmid vector and subsequently
sequenced.
virus 27,28 and mimivirus 29,30 have been loosely associated with
clinical disorders in humans.
Many putative viral pathogens cannot be isolated from patients
with overt disease, and researchers have as yet been unable to
satisfy the modified Koch’s postulates that will prove a causal
relationship. It is important, therefore, that further extensive
clinical studies be continued in an attempt to define the role of
these agents in human disease.
Still, for a significant proportion of clinical infectious disease of
suspected viral origin, a pathogen cannot be identified. Although
new molecular methods are increasingly used to investigate these
unknown causes of disease, they remain technically challenging
and prone to the amplification of non-viral related sequence
artefacts. However, with continuing advances in molecular
technology and the development of more reliable, robust and
reproducible molecular techniques, it seems certain that more,
new viral pathogens of humans will be discovered.
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Biography
Theo Sloots (PhD, Grad Cert Management, MASM) has more
than 25 years’ experience in medical microbiology and is
currently the Director (Research) at the Queensland Paediatric
Infectious Diseases (QPID) Laboratory of the Royal Children’s
Hospital, Brisbane, as well as Consultant Virologist to Pathology
Queensland Central. Research at the QPID Laboratory has focused
on examining the significance of human metapneumovirus as a
newly recognised respiratory pathogen and the discovery of new
viral agents associated with respiratory disease in children.
Theo is a chief investigator on three separate Research Project
Grants funded by the NHMRC, and also holds academic
appointments with the Biological and Chemical Sciences Faculty,
University of Queensland and the School of Biomolecular and
Physical Sciences, Griffith University, Brisbane.
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