ResearchersLinks
Hosts and VirusesA ResearchersLinks journal
Submit manuscriptReview for this journal

Mini-Review · Volume 3, Issue 2, 28 May 2016 · Open Access

Use of Suppression Subtractive Hybridization for Viral Diagnostics

· ·

Affiliations & Notes ⌄
  1. 1Fiona Elsey Cancer Research Institute, 106-110 Lydiard Street South, Ballarat Technology Park Central, Ballarat, VIC 3353, Australia
  2. 2School of Applied and Biomedical Sciences, Federation University, Victoria, 3350, Australia. Email: monirul@fecri.org.au
  3. 3Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, Victoria 3010, Australia
Article Info ⌄
Journal:
Hosts and Viruses
Published:
28 May 2016
Download PDF ePub eFLIP Set Alert Get Rights Reprints

From the article

  • In direct detection, the clinical specimen is examined directly for the presence of virus particles, virus antigen or viral nucleic acids.
  • Hence, to our understanding, SSH is the only unbiased method, which can be effectively used for the detection of a completely unknown virus in infected tissue or specimens.
  • Detection of herpes simplex virus in clinical specimens by cytospin-enhanced direct immunofluorescence.
  • Enhanced expression of interferon-regulated genes in the liver of patients with chronic hepatitis C virus infection: detection by suppression-subtractive hybridization.

Selected from the article's own text.

Abstract

Efficient identification of viruses is paramount to not only define the cause of uncharacterized infections but also to estimate the dynamics of viral population in the infected hosts. Several recent methodologies and chemistries have been proposed for viral diagnostics with variable outcomes and successes. In this review, we discuss a novel approach to identify the least transcripts of viral genomes using suppression subtractive hybridization that can be adapted to unanimously identify viruses of diverse genetic backgrounds. 

†Table has been reproduced from our earlier publication (Islam et al., 2015)

SSH is an unbiased method and can be used to identify any viral infection. All viruses produce their transcripts (mRNAs) in the host cells irrespective of their genetic makeup (DNA or RNA virus). Subtraction of total mRNA of non-infected control cells from that of infected host cells (forward subtraction) produces differential mRNA population that contains mRNA population from viral origin. Cloning and sequencing that differential mRNA population gives information about the genetic make-up of the infecting virus. The virus could be a known virus or a completely unknown virus. Full-length genome sequencing of the known virus is often unnecessary but can be done by using virus specific primers. On the other hands, full-length genome sequencing of unknown virus is crucial for the complete identification of the virus and it is a difficult task. A sequence identity search of the cloned cDNA (partial genome) in the DNA databank gives an impression about the putative virus or its origin (genus). A virus with the highest identity may be a close relative of the putative virus. If the putative virus is a DNA virus (single stranded or double stranded), genetic material can be isolated from the host cell and then directly used for sequencing. If the putative virus is a RNA virus, cDNA of the virus can be synthesized from the total RNA of the host cell and then sequenced using designed sequencing primers, which may not necessarily be 100% identical to the target virus sequences. In our SSH findings, putative virus had the highest homology (95 -97%) with an RNA virus, murine hepatitis virus (MHV) and we designed the sequencing primers from the genomic sequence of MHV-A59 virus. Since MHV is single stranded RNA virus, we prepared cDNA from the host cell (infected liver cells) and then sequenced the whole genome using a total of 45 pairs of designed primer. Nearly 70% of the full-length viral genome was sequenced using 23 pairs of designed primer from MHV-A59 genomic sequence and rest of the sequence was identified using nested primers and gene walking strategy. 3’-end of the viral genome was amplified using 3’-RACE and then when sequenced. 5’-RACE did not work, so we used 5’-end consensus primer for sequencing 5’- end.

Although the basic principle of the SSH method looks very simple, it is actually a lengthy process. More than one month is necessary to complete the whole process. Steps involved in the method are: (1) isolation of total RNA from infected and non-infected tissue samples, (2) preparation of cDNA from both RNAs, (3) Digestion of cDNA using Rsa I restriction enzyme, (4) purification of the digested cDNA, (5) ligation of adaptors to driver cDNA, (6) hybridization of tester and driver cDNA, (7) Suppression PCR, (8) purification of PCR products, (9) cloning PCR products into host vector system, (10) preparation of the subtracted cDNA library, (11) isolation of cDNA clones, and (12) sequencing number of clones needed for statistical significance. Some of the above mentioned methods are very crucial and need to be quality tested before going to the next step. Hence additional time is necessary to complete the process. The largest amount of time (nearly one month) is taken by the steps 9 – 12, whereas only one week is necessary to carry out the actual subtraction procedure (steps 1-8). If, somehow, we could avoid or bypass steps 9-12, we could substantially reduce the time needed. Adopting next generation sequencing at step 9 might give similar results but we haven’t tested the possibility yet.

Differential RNA sequencing between the infected and non-infected subjects could also be used as alternative for SSH, but the risk of losing rare transcripts is very high as no suppression PCR is involved in RNA sequencing. In fact, viral transcripts would be rare compared to the infected host cell transcripts and may not be identifiable by RNA sequencing. Besides this, differential RNA sequencing produces relative (comparative) abundance of transcripts but the absence of transcript in the non-infected cells could be problematic for bioinformatics and may not be suitable for detecting viral infection. Hence, to our understanding, SSH is the only unbiased method, which can be effectively used for the detection of a completely unknown virus in infected tissue or specimens. Another advantage is that this process also selects host genes that are expressed in response to the viral infection. The only drawback is the length of time required to complete the technique. However, introducing some automation in different steps or replacing steps 9-12 with next generation sequencing can easily reduce the time required to complete the process to nearly one week. As a result, we are proposing that SSH could become an ideal technique for detection of rare viral infection.

A recent variation on the SSH technique is the VirCapSeq method that uses millions of synthetic oligonucleotides that are prepared against every known vertebrate viral sequence, including polymorphic nucleotides . This technique can also greatly enrich viral sequences 10,000 times for direct deep sequence analysis. However, again it is limited to identifying only the known viruses whose sequence has contributed to the design of the selection probes. Perhaps using these probes to deplete SSH libraries will enhance the identification of completely new viral entities.

References

  1. Adam, N.M., Sorooshain, S., Zhang, X., Tezara, C., Azizi, P., Shahebi, M., Bande, Y., Nurhaiza, S., Kaiser, A., 2012. Comparison of suppression subtractive hybridization with other methods used to identify differentially expressed genes in plants. In: Adam, N. M., Sorooshian, S. (ed). Engineering Research Methods. Pp. 15-24. Lulu.com, Serdang, Malaysia.
  2. A-Nuegoonpipat, A., Panthuyosri, N., Anantapreecha, S., Chanama, S., Sa-Ngasang, A., Sawanpanyalert, P. and Kurane, I. (2008). Cross-reactive IgM responses in patients with dengue or Japanese encephalitis. J Clin Virol 42: 75-77. http://dx.doi.org/10.1016/j.jcv.2007.10.030[CrossRef]
  3. Bej, A.K., Mahbubani, M.H, Atlas, R.M. (1991). Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications. Crit Rev Biochem Mol Biol 26: 301-334.
  4. Briese, T., Kapoor, A., Mishra, N., Jain, K., Kumar, A., Jabado, O.J., Lipkin, W.I. (2015). Virome Capture Sequencing Enables Sensitive Viral Diagnosis and Comprehensive Virome Analysis. MBio 6: e01491-15. http://dx.doi.org/10.1128/mBio.01491-15[CrossRef]
  5. Diatchenko, L., Lau, Y.F., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., Siebert, P.D. (1996). Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci U S A 93: 6025-6030. http://dx.doi.org/10.1073/pnas.93.12.6025[CrossRef]
  6. Elnifro, E.M., Ashshi, A.M., Cooper, R.J., Klapper, P.E. (2000). Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 13: 559-570. http://dx.doi.org/10.1128/CMR.13.4.559-570.2000[CrossRef]
  7. Emmanuel, P.J. (1993). Polymerase chain reaction from bench to bedside. Applications for infectious disease. J Fla Med Assoc 80: 627-630.
  8. Fan, J., Henrickson, K.J., Savatski, L.L. (1998). Rapid simultaneous diagnosis of infections with respiratory syncytial viruses A and B, influenza viruses A and B, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (Hexaplex). Clin Infect Dis 26: 1397-1402. http://dx.doi.org/10.1086/516357[CrossRef]
  9. Grondahl, B., Puppe, W., Hoppe, A., Kuhne, I., Weigl, J.A., Schmitt, H.J. (1999). Rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-PCR: feasibility study. J Clin Microbiol 37: 1-7.
  10. Harrison, T.J. (1998). The polymerase chain reaction--a time of transition from research to routine. J Clin Pathol 51: 491-492. http://dx.doi.org/10.1136/jcp.51.7.491[CrossRef]
  11. Hsiung, G.D. (1984). Diagnostic virology: from animals to automation. Yale J Biol Med 57: 727-733.
  12. Islam, M.M., Toohey, B., Purcell, D.F., Kannourakis, G. (2015). Suppression subtractive hybridization method for the identification of a new strain of murine hepatitis virus from xenografted SCID mice. Arch Virol 160: 2945-2955. http://dx.doi.org/10.1007/s00705-015-2592-y[CrossRef]
  13. Kuang, W.W., Thompson, D.A., Hoch, R.V., Weigel, R.J. (1998). Differential screening and suppression subtractive hybridization identified genes differentially expressed in an estrogen receptor-positive breast carcinoma cell line. Nucleic Acids Res 26: 1116-1123. http://dx.doi.org/10.1093/nar/26.4.1116[CrossRef]
  14. Landry, M.L., Cohen, S., Ferguson, D. (2000). Impact of sample type on rapid detection of influenza virus A by cytospin-enhanced immunofluorescence and membrane enzyme-linked immunosorbent assay. J Clin Microbiol 38: 429-430.
  15. Landry, M.L. and Ferguson, D. (2000). SimulFluor respiratory screen for rapid detection of multiple respiratory viruses in clinical specimens by immunofluorescence staining. J Clin Microbiol 38: 708-711.
  16. Landry, M.L., Ferguson, D., Wlochowski, J. (1997). Detection of herpes simplex virus in clinical specimens by cytospin-enhanced direct immunofluorescence. J Clin Microbiol 35: 302-304.
  17. Leland, D.S. and Ginocchio, C.C. (2007). Role of cell culture for virus detection in the age of technology. Clin Microbiol Rev 20: 49-78. http://dx.doi.org/10.1128/CMR.00002-06[CrossRef]
  18. Lukyanov, S.A., Rebrikov, D., Buzdin, A.A. (2007). Suppression subtractive hybridization. In: Anonymous , Nucleic Acids Hybridization Modern Applications. Pp. 53-84. Springer, Berlin. http://dx.doi.org/10.1007/978-1-4020-6040-3_3[CrossRef]
  19. Niesters, H.G. (2004). Molecular and diagnostic clinical virology in real time. Clin Microbiol Infect 10: 5-11. http://dx.doi.org/10.1111/j.1469-0691.2004.00699.x[CrossRef]
  20. Patzwahl, R., Meier, V., Ramadori, G., Mihm, S. (2001). Enhanced expression of interferon-regulated genes in the liver of patients with chronic hepatitis C virus infection: detection by suppression-subtractive hybridization. J Virol 75: 1332-1338. http://dx.doi.org/10.1128/JVI.75.3.1332-1338.2001[CrossRef]
  21. Read, S.J., Burnett, D., Fink, C.G. (2000). Molecular techniques for clinical diagnostic virology. J Clin Pathol 53: 502-506. http://dx.doi.org/10.1136/jcp.53.7.502[CrossRef]
  22. Ristevski, S., Purcell, D.F., Marshall, J., Campagna, D., Nouri, S., Fenton, S.P., McPhee, D.A., Kannourakis, G. (1999). Novel endogenous type D retroviral particles expressed at high levels in a SCID mouse thymic lymphoma. J Virol 73: 4662-4669.
  23. Sahebi, M., Hanafi, M.M., Abdullah, S.N., Rafii, M.Y., Azizi, P., Nejat, N., Idris, A.S. (2014). Isolation and expression analysis of novel silicon absorption gene from roots of mangrove (Rhizophora apiculata) via suppression subtractive hybridization. Biomed Res Int 2014: 971985. http://dx.doi.org/10.1155/2014/971985[CrossRef]
  24. Shackel, N.A., McGuinness, P.H., Abbott, C.A., Gorrell, M.D., McCaughan, G.W. (2003). Novel differential gene expression in human cirrhosis detected by suppression subtractive hybridization. Hepatology 38: 577-588. http://dx.doi.org/10.1053/jhep.2003.50376[CrossRef]
  25. Storch, G.A. (2000). Diagnostic virology. Clin Infect Dis 31: 739-751. http://dx.doi.org/10.1086/314015[CrossRef]
  26. Takayama, M. (1994). Reactivity of varicella-zoster virus subunit antigens in enzyme-linked immunosorbent assay to sera from varicella, zoster, and herpes simplex virus infections. Med Microbiol Immunol 183: 1-11. http://dx.doi.org/10.1007/BF00193626[CrossRef]
  27. Tang, Y.W. and Ou, C.Y. (2012). Past, present and future molecular diagnosis and characterization of human immunodeficiency virus infections. Emerg Microbes Infect 1: e19. http://dx.doi.org/10.1038/emi.2012.15[CrossRef]
  28. Vafai, A., Wroblewska, Z., Graf, L. (1990). Antigenic cross-reaction between a varicella-zoster virus nucleocapsid protein encoded by gene 40 and a herpes simplex virus nucleocapsid protein. Virus Res 15: 163-174. http://dx.doi.org/10.1016/0168-1702(90)90006-W[CrossRef]
  29. Van Regenmortel, M.H. (1993). Synthetic peptides versus natural antigens in immunoassays. Ann Biol Clin (Paris) 51: 39-41.
  30. Wagar, E.A. (1996). Direct hybridization and amplification applications for the diagnosis of infectious diseases. J Clin Lab Anal 10: 312-325. http://dx.doi.org/10.1002/(SICI)1098-2825(1996)10:6<312::AID-JCLA2>3.0.CO;2-8[CrossRef]
  31. Wolcott, M.J. (1992). Advances in nucleic acid-based detection methods. Clin Microbiol Rev 5: 370-386.
  32. Wu, M., Tu, T., Huang, Y., Cao, Y. (2013). Suppression subtractive hybridization identified differentially expressed genes in lung adenocarcinoma: ERGIC3 as a novel lung cancer-related gene. BMC Cancer 13: 44-2407-13-44.
  33. Yamada, K., Takasaki, T., Nawa, M., Yabe, S., Kurane, I. (2003). Antibody responses determined for Japanese dengue fever patients by neutralization and hemagglutination inhibition assays demonstrate cross-reactivity between dengue and Japanese encephalitis viruses. Clin Diagn Lab Immunol 10: 725-728. http://dx.doi.org/10.1128/cdli.10.4.725-728.2003[CrossRef]

Related articles

Services

Article usage statistics

Loading…

Use of Suppression Subtractive Hybridization for Viral Diagnostics · ResearchersLinks