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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). 125 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. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. van den Hoogen, B.G. et al. (2001) A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat. Med. 7, 719–724. Ksiazek, T.G. et al. (2003) A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348, 1953–1966. van der Hoek, L. et al. (2004). Identification of a new human coronavirus. Nat. Med. 10, 368–373. Woo, P.C. et al. (2005) Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J. Virol. 79, 884–895. Allander, T. et al. (2005) Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc. Natl. Acad. Sci. USA 102, 12891–12896. Arthur, J.L. et al. (2009) A novel bocavirus associated with acute gastroenteritis in Australian children. PLoS. Pathog. 5, e1000391. doi:10.1371/journal. ppat.1000391 Kapoor, A. et al. (2009) A newly identified bocavirus species in human stool. J. Infect. Dis. 199, 196–200. Allander, T. et al. (2007) Identification of a third human polyomavirus. J. Virol. 81, 4130–4136. Gaynor, A.M. et al. (2007) Identification of a novel polyomavirus from patients with acute respiratory tract infections. PLoS. Pathog. 3, 595–604. Feng, H. et al. (2008) Clonal Integration of a Polyomavirus in Human Merkel Cell Carcinoma. Science. 319,1096–100. Finkbeiner, S.R. et al. (2009) Identification of a novel astrovirus (astrovirus VA1) associated with an outbreak of acute gastroenteritis. J.Virol. 83, 10836– 10839. Finkbeiner, S.R. et al. (2009) Human stool contains a previously unrecognized diversity of novel astroviruses. Virol. J. 6,161. doi:10.1186/1743–422X-6-161 Holtz, L.R. et al. (2008) Identification of a novel picornavirus related to cosaviruses in a child with acute diarrhea. Virol. J. 5,159. doi:10.1186/1743– 422X-5-159. Pyrc, K. et al. (2008) Detection of new viruses by VIDISCA. Virus discovery based on cDNA-amplified fragment length polymorphism. Methods Mol. Biol. 454,73–89. 1 2 6 15. Wang, D. et al. (2003) Viral discovery and sequence recovery using DNA microarrays. PLoS. Biol. 1, 257–260. 16. Ambrose, H.E. & Clewley, J.P. (2006) Virus discovery by sequence-independent genome amplification. Rev. Med. Virol. 16, 365–383. 17. Vos, P. et al. (1995) AFLP: a new technique for DNA fingerprinting. Nuc. Acids Res. 23, 4407–4414. 18. Wang, D. et al. (2002) Microarray-based detection and genotyping of viral pathogens. Proc. Natl. Acad. Sci. USA. 99, 15687–15692. 19. Boriskin, Y.S. et al. (2004) DNA microarrays for virus detection in cases of central nervous system infection. J. Clin. Microbiol. 42, 5811–5818. 20. Lin, B. et al. (2004) Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses. J. Clin. Microbiol. 42, 3232–3239. 21. Korimbocus, J. et al. (2005) DNA probe array for the simultaneous identification of herpesviruses, enteroviruses, and flaviviruses. J. Clin. Microbiol. 43, 3779– 3787. 22. Clewley, J.P. (2004). A role for arrays in clinical virology: fact or fiction? J. Clin. Virol. 29, 2–12. 23. Bachem, C.W.B. et al. (1996) Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J. 9, 745–753. 24. Stang, A. et al. (2005) Characterization of virus isolates by particle-associated nucleic acid PCR. J. Clin. Microbiol. 43, 716–720. 25. Wu, Q. et al. (2010) Virus discovery by deep sequencing and assembly of virusderived small silencing RNAs. Proc. Natl. Acad. Sci. USA. 107, 1606-1611. DOI: 10.1073/pnas.0911353107 26. Kreuze, J.F. et al. (2009) Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: A generic method for diagnosis, discovery and sequencing of viruses. Virology 388,1–7. 27. Maggi, F. et al. (2003) TT virus in the nasal secretions of children with acute respiratory diseases: relations to viremia and disease severity. J. Virol. 77, 2418–2425. 28. Pifferi, M. et al. (2006) High torquetenovirus loads are correlated with bronchiectasis and peripheral airflow limitation in children. Pediatr. Infect. Dis. J. 25, 804–808. 29. Khan, M. et al. (2007) Pneumonia in mice inoculated experimentally with Acanthamoeba polyphaga mimivirus. Microb. Pathog. 42, 56–61. 30. Raoult, D. et al. (2007) The discovery and characterization of Mimivirus, the largest known virus and putative pneumonia agent. Clin. Infect. Dis. 45, 95–102. 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. MICROBIOLOGY AUSTRALIA • SEPTEM B E R 2 0 1 0