The starting points of any diagnostic approach are the patients and their symptoms, as well as anatomical location and sample matrix. Ideally, virus diagnostics could have a broader approach not fixed to a specific sequence, allowing the identification of several and also unexpected pathogens.
An optimal microbiological test would identify all pathogens, whether bacterial, fungal, parasitic or viral, causing similar symptoms, preferrably from an easily collectable sample matrix. An optimal test could be one that identifies a broad range of pathogens or disease markers for example from blood or urine. The different nature of microorganisms makes this task very challenging but sequencing the whole nucleic acid content of a sample has proven successful in identification of pathogens independent of their location in the tree of life. Several NGS-based applications for identifying the whole microbial content including minor populations and new pathogens in a given sample have already been developed (Bhaduri et al. 2012; Kostic et al. 2011; Naccache et al. 2014). Novel sequencing techniques have enabled the discovery of many new viruses and revealed the variety of already identified virus species, as is exemplified by the discovery of new polyomaviruses (Buck et al. 2012; Feng et al. 2008; Scuda et al. 2011; Siebrasse et al. 2012).
Various applications have also been established specifically for identifying viruses (Daly et al.
2011; de Vries et al. 2011; Kohl et al. 2015), their integration into the host genome (Chen et al. 2013; Li et al. 2013; Schelhorn et al. 2013; Wang, Jia, Zhao 2013; Wang, Jia, Zhao 2015), as well as for identifying particularly RNA viruses (Manso, Bibby, Mbisa 2017) from different sample matrices. This elucidates the effort and interest directed towards applying NGS techniques for virus identification. Despite the intense study involved in this field, NGS methods are not yet commonly applied in routine virus diagnostics and patient management, mainly because of the tools, skills and turnaround time required for analysing NGS data. The biggest challenge of NGS is the bioinformatics needed to retrieve the desired information amongst the enormous data typically produced by NGS, and to interpret the relevance of the acquired information. Despite these challenges, the breakthrough of NGS as method for routine virus diagnostics is rapidly approaching.
The broader the approach comes often at the cost of decreased sensitivity. Techniques exploiting specific virus sequences, such as PCR, are widely used in virus diagnostics because of their sensitivity and ability to detect specific virus strains. However, in the diagnostics of chronic virus infections highly sensitive test is not always needed and may not be optimal for the purpose. In
addition to the mere detection of a virus, more detailed information about virus replication or virus strain is required. This is exemplified by HPV, BKPyV and JCVPyV infections which are all common chronic virus infections that do not cause problems in the vast majority of people. Thus, the goal in diagnosing these infections is to detect persistent infection associated with increaced risk of severe disease, reactivation, and/or emergence of highly pathogenic strains. PCR-based monitoring is applied in patient management to detect increase in viral load indicating active virus replication. This can be a risk factor associated to disease development, such as enhanced BKPyV replication in renal transplant patients. Also, specific strains of interest can be extracted from a pool of irrelevant or related sequences by using sequence-specific PCR methods. Yet, highly specific PCR is also vulnerable to mutations, and sequence variation within primer or probe sequences may lead to false negative results. Sequence-specificity relates particularly to the rapidly evolving and divergent RNA viruses, such as HCV, which require highly specific capture sequences for the identification of different strains and subtypes. To decrease the prevalence and disease burden of HCV, all infections should be identified to prevent HCV-induced cirrhosis, hepatocellular carcinoma, and the spread of this virus.
CONCLUDING REMARKS
As in life generally, things are hard to find if you do not know what you are looking for. Current diagnostics of chronic virus infections is based on a suspicion of a specific pathogen(s) associated to the symptoms of the patient. Diagnostics has focused on the viruses and their detection. However, the main reason for performing virus diagnostics is patient management and to prevent or treat a disease. Thus, prognostic markers indicating the development of a disease would be needed. In addition to the detection of a virus or a viral component, also new surrogate markers associated to disease development would be beneficial. An ideal diagnostic test would not need discrimination between classes of pathogens before testing, but instead identify all possible pathogens causing similar symptoms and affecting the same anatomical region. As more knowledge accumulates about cellular processes, viral pathogenesis and virus-host interactions, also new potential disease markers may be discovered. As demonstrated by the present study, a variety of new molecular methods and markers are already available. The field of virus diagnostics is rapidly evolving as the knowledge involving the biology and pathogenesis of viruses expands. Along with new molecular methods new disease patterns may emerge, and new viruses and disease association will be discovered. In the future we are likely to see highly effective diagnostic tests identifying several viral as well as other microbial pathogens. Hopefully these diagnostic tools can increasingly be applied as prognostic tools for the wellbeing of patients.
ACKNOWLEDGEMENTS
This thesis work was carried out at the Department of Virology and Immunology in HUSLAB, Helsinki University Hospital Laboratories, and at the Department of Virology in University of Helsinki, Faculty of Medicine. I want to acknowledge Director Maija Lappalainen (Division of Clinical Microbiology, HUSLAB) and the head of department, Professor Kalle Saksela (Department of Virology, Faculty of Medicine), for providing excellent working facilities to conduct this research work.
This thesis was supervised by Docent Eeva Auvinen, and I want to thank her for all the help and guidance she has given me in conducting this research work and in writing this thesis. I appreciate her vast knowledge and passion for science, and without her this thesis work would not have even started. I also want to thank Docent Tytti Vuorinen ja Docent Matti Waris for all the effort they put into reviewing this thesis and for their valuable comments. Moreover, I am deeply grateful to Docent Janne Aittoniemi for agreeing to act as my opponent. I also want to express my gratitude to Docent Laura Mannonen, Docent Laura Kakkola and Docent Leena Maunula for taking part in my thesis committee and for all the support and advice given during this process.
I am grateful to all the people who have contributed to this research work. I especially want to thank all the co-authors of the original publications as well as the past and present workers at the Department of Virology and Immunology in HUSLAB. A special thank you goes to the lovely people working in the Nucleic Acid Detection Laboratory, with whom I have shared many fun and educative moments in the lab. My appreciation goes also to my fellow co-workers in our little joint laboratory: Hanna Seppälä, Kati Hokynar and Anu Kaitonen. Thank you for your support, and for making the ordinary workdays lighter and brighter! Similarly, I want to thank the members of the Viral Zoonosis Research Unit with whom I have shared many fun moments. Especially Suvi Kuivanen, Satu Kurkela and Anne Jääskeläinen are acknowledged for all the helpful conversations and deep laughs shared in the coffee room. Furthermore, I want to express my gratitude to Tanja Holma and Jaana Valve for the fruitful collaboration which developed into friendship.
I want to thank all my wonderful friends for all the fun, crazy, delicious, recreational, sweaty and musical moments spent together during this process. I like to thank each and everyone of you for bringing energy, perspective, motivation and strength into my life during those moments when uplift was needed. A special thank you for all the additional peer-support goes to my fellow students and microbiologists Paula Typpö, Iines Salonen, Emmi Kuva and Samira Ripatti. One of my oldest friends, Else Kosonen, is also deeply thanked for the invaluable help she has provided me in creating the layout and figures of this thesis.
My deepest gratitude goes to my beloved family. My parents Eija and Jorma have always been there for me, showing their love and support and believing in me, and I am sincerely grateful to have such amazing parents! Last but not least, I want to express my gratitude to my wonderful husband Tommi. You are the love of my life, my best friend and soulmate, and the most wonderful, caring and loving person I have ever met. Words are not enough to thank you for all your love, support and patience during this process.
Orionin Tutkimussäätiö, Kliinisen Kemian Tutkimussäätiö, Laboratoriolääketieteen Edistämis-säätiö, Sukupuolitautien vastustamisyhdistys and Virustautien Tutkimussäätiö are acknowledged for the financial support of this doctoral research work.
REFERENCES
AASLD/IDSA HCV Guidance Panel. 2015. Hepatitis C guidance: AASLD-IDSA recommendations for testing, managing, and treating adults infected with hepatitis C virus.
Hepatology 62(3):932-54.
Abend JR, Low JA, Imperiale MJ. 2007. Inhibitory effect of gamma interferon on BK virus gene expression and replication. J Virol 81(1):272-9.
Aberg JA, Gallant JE, Ghanem KG, Emmanuel P, Zingman BS, Horberg MA, Infectious Diseases Society of America. 2014. Primary care guidelines for the management of persons infected with HIV: 2013 update by the HIV medicine association of the infectious diseases society of America. Clin Infect Dis 58(1):1-10.
Agrawal PG, Mahajan SA, Khopkar US, Kharkar VD. 2013. Epidermodysplasia verruciformis:
An unusual malignant transformation. Indian J Dermatol Venereol Leprol 79(1):97-9.
Alcami A and Koszinowski UH. 2000. Viral mechanisms of immune evasion. Immunol Today 21(9):447-55.
Ambalathingal GR, Francis RS, Smyth MJ, Smith C, Khanna R. 2017. BK polyomavirus: Clinical aspects, immune regulation, and emerging therapies. Clin Microbiol Rev 30(2):503-28.
Ang KK, Harris J, Wheeler R, Weber R, Rosenthal DI, Nguyen-Tan PF, Westra WH, Chung CH, Jordan RC, Lu C, et al. 2010. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med 363(1):24-35.
Antonelli A, Ferrari SM, Ruffilli I, Fallahi P. 2014. Cytokines and HCV-related autoimmune disorders. Immunol Res 60(2-3):311-9.
Antonsson A, Karanfilovska S, Lindqvist PG, Hansson BG. 2003. General acquisition of human papillomavirus infections of skin occurs in early infancy. J Clin Microbiol 41(6):2509-14.
Antonsson A, Green AC, Mallitt KA, O'Rourke PK, Pawlita M, Waterboer T, Neale RE. 2010.
Prevalence and stability of antibodies to the BK and JC polyomaviruses: A long-term longitudinal study of australians. J Gen Virol 91(Pt 7):1849-53.
Antonsson A, Green AC, Mallitt KA, O'Rourke PK, Pandeya N, Pawlita M, Waterboer T, Neale RE. 2010. Prevalence and stability of antibodies to 37 human papillomavirus types - A population-based longitudinal study. Virology 407(1):26-32.
Ashrafi GH, Brown DR, Fife KH, Campo MS. 2006. Down-regulation of MHC class I is a property common to papillomavirus E5 proteins. Virus Res 120(1-2):208-11.
Auvinen E. 2017. Diagnostic and prognostic value of MicroRNA in viral diseases. Mol Diagn Ther 21(1):45-57.
Axley P, Ahmed Z, Ravi S, Singal AK. 2018. Hepatitis C virus and hepatocellular carcinoma:
A narrative review. J Clin Transl Hepatol 6(1):79-84.
BaAlawi F, Robertson PW, Lahra M, Rawlinson WD. 2012. Comparison of five CMV IgM immunoassays with CMV IgG avidity for diagnosis of primary CMV infection. Pathology 44(4):381-3.
Bartenschlager R, Cosset FL, Lohmann V. 2010. Hepatitis C virus replication cycle. J Hepatol 53(3):583-5.
Basnyat P, Virtanen E, Elovaara I, Hagman S, Auvinen E. 2017. JCPyV microRNA in plasma inversely correlates with JCPyV seropositivity among long-term natalizumab-treated relapsing- remitting multiple sclerosis patients. J Neurovirol 23(5):734-41.
Basu P, Banerjee D, Mittal S, Dutta S, Ghosh I, Chowdhury N, Abraham P, Chandna P, Ratnam S.
2016. Sensitivity of APTIMA HPV E6/E7 mRNA test in comparison with hybrid capture 2 HPV DNA test for detection of high risk oncogenic human papillomavirus in 396 biopsy confirmed cervical cancers. J Med Virol 88(7):1271-8.
Bauman Y, Nachmani D, Vitenshtein A, Tsukerman P, Drayman N, Stern-Ginossar N, Lankry D, Gruda R, Mandelboim O. 2011. An identical miRNA of the human JC and BK polyoma viruses targets the stress-induced ligand ULBP3 to escape immune elimination. Cell Host Microbe 9(2):93-102.
Beaudenon S and Huibregtse JM. 2008. HPV E6, E6AP and cervical cancer. BMC Biochem 9 Suppl 1:S4,2091-9-S1-S4.
Behrens SE, Tomei L, De Francesco R. 1996a. Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. Embo J 15(1):12-22.
Behrens SE, Tomei L, De Francesco R. 1996b. Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. Embo J 15(1):12-22.
Benedicto I, Gondar V, Molina-Jimenez F, Garcia-Buey L, Lopez-Cabrera M, Gastaminza P, Majano PL. 2015. Clathrin mediates infectious hepatitis C virus particle egress. J Virol 89(8):4180-90.
Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, Hall KP, Evers DJ, Barnes CL, Bignell HR, et al. 2008. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456(7218):53-9.
Berger JR, Levy RM, Flomenhoft D, Dobbs M. 1998. Predictive factors for prolonged survival in acquired immunodeficiency syndrome-associated progressive multifocal leukoencephalopathy. Ann Neurol 44(3):341-9.
Bhaduri A, Qu K, Lee CS, Ungewickell A, Khavari PA. 2012. Rapid identification of non-human sequences in high-throughput sequencing datasets. Bioinformatics 28(8):1174-5.
Bialasiewicz S, Cho Y, Rockett R, Preston J, Wood S, Fleming S, Shepherd B, Barraclough K, Sloots TP, Isbel N. 2013. Association of micropapillary urothelial carcinoma of the bladder and BK viruria in kidney transplant recipients. Transpl Infect Dis 15(3):283-9.
Binggeli S, Egli A, Schaub S, Binet I, Mayr M, Steiger J, Hirsch HH. 2007. Polyomavirus BK- specific cellular immune response to VP1 and large T-antigen in kidney transplant recipients.
Am J Transplant 7(5):1131-9.
Bishop JA, Lewis JS,Jr, Rocco JW, Faquin WC. 2015. HPV-related squamous cell carcinoma of the head and neck: An update on testing in routine pathology practice. Semin Diagn Pathol 32(5):344-51.
Blanchard E, Belouzard S, Goueslain L, Wakita T, Dubuisson J, Wychowski C, Rouille Y. 2006.
Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol 80(14):6964-72.
Bochud PY, Magaret AS, Koelle DM, Aderem A, Wald A. 2007. Polymorphisms in TLR2 are associated with increased viral shedding and lesional rate in patients with genital herpes simplex virus type 2 infection. J Infect Dis 196(4):505-9.
Bochud PY, Hersberger M, Taffe P, Bochud M, Stein CM, Rodrigues SD, Calandra T, Francioli P, Telenti A, Speck RF, et al. 2007. Polymorphisms in toll-like receptor 9 influence the clinical course of HIV-1 infection. Aids 21(4):441-6.
Bonagura VR, Hatam LJ, Rosenthal DW, de Voti JA, Lam F, Steinberg BM, Abramson AL. 2010.
Recurrent respiratory papillomatosis: A complex defect in immune responsiveness to human papillomavirus-6 and -11. Apmis 118(6-7):455-70.
Bonagura VR, Du Z, Ashouri E, Luo L, Hatam LJ, DeVoti JA, Rosenthal DW, Steinberg BM, Abramson AL, Gjertson DW, et al. 2010. Activating killer cell immunoglobulin-like receptors 3DS1 and 2DS1 protect against developing the severe form of recurrent respiratory papillomatosis. Hum Immunol 71(2):212-9.
Bouvard V, Baan RA, Grosse Y, Lauby-Secretan B, El Ghissassi F, Benbrahim-Tallaa L, Guha N, Straif K, WHO International Agency for Research on Cancer Monograph Working Group.
2012. Carcinogenicity of malaria and of some polyomaviruses. Lancet Oncol 13(4):339-40.
Brady MT, MacDonald AJ, Rowan AG, Mills KH. 2003. Hepatitis C virus non-structural protein 4 suppresses Th1 responses by stimulating IL-10 production from monocytes. Eur J Immunol 33(12):3448-57.
Bredholt G, Olaussen E, Moens U, Rekvig OP. 1999. Linked production of antibodies to mammalian DNA and to human polyomavirus large T antigen: Footprints of a common molecular and cellular process? Arthritis Rheum 42(12):2583-92.
Broekema NM and Imperiale MJ. 2013. miRNA regulation of BK polyomavirus replication during early infection. Proc Natl Acad Sci USA 110(20):8200-5.
Buck CB, Phan GQ, Raiji MT, Murphy PM, McDermott DH, McBride AA. 2012. Complete genome sequence of a tenth human polyomavirus. J Virol 86(19):10887-12.
Buckwalter SP, Sloan LM, Cunningham SA, Espy MJ, Uhl JR, Jones MF, Vetter EA, Mandrekar J, Cockerill FR,3rd, Pritt BS, et al. 2014. Inhibition controls for qualitative real-time PCR assays:
Are they necessary for all specimen matrices? J Clin Microbiol 52(6):2139-43.
Bzhalava D, Eklund C, Dillner J. 2015. International standardization and classification of human papillomavirus types. Virology 476:341-4.
Cacoub P, Comarmond C, Domont F, Savey L, Desbois AC, Saadoun D. 2016. Extrahepatic manifestations of chronic hepatitis C virus infection. Ther Adv Infect Dis 3(1):3-14.
Castellsague X, Drudis T, Canadas MP, Gonce A, Ros R, Perez JM, Quintana MJ, Munoz J, Albero G, de Sanjose S, et al. 2009. Human papillomavirus (HPV) infection in pregnant women and mother-to-child transmission of genital HPV genotypes: A prospective study in spain. BMC Infect Dis 9:74,2334-9-74.
Cesaro S, Dalianis T, Hanssen Rinaldo C, Koskenvuo M, Pegoraro A, Einsele H, Cordonnier C, Hirsch HH, ECIL-6 Group. 2018. ECIL guidelines for the prevention, diagnosis and treatment of BK polyomavirus-associated haemorrhagic cystitis in haematopoietic stem cell transplant recipients. J Antimicrob Chemother 73(1):12-21.
Chaudhuri S, Symons JA, Deval J. 2018. Innovation and trends in the development and approval of antiviral medicines: 1987-2017 and beyond. Antiviral Res 155:76-88.
Chen BJ and Lamb RA. 2008. Mechanisms for enveloped virus budding: Can some viruses do without an ESCRT? Virology 372(2):221-32.
Chen Y, Yao H, Thompson EJ, Tannir NM, Weinstein JN, Su X. 2013. VirusSeq: Software to identify viruses and their integration sites using next-generation sequencing of human cancer tissue. Bioinformatics 29(2):266-7.
Chesters PM, Heritage J, McCance DJ. 1983. Persistence of DNA sequences of BK virus and JC virus in normal human tissues and in diseased tissues. J Infect Dis 147(4):676-84.
Choo QL, Richman KH, Han JH, Berger K, Lee C, Dong C, Gallegos C, Coit D, Medina-Selby R, Barr PJ. 1991. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A 88(6):2451-5.
Christen U, Hintermann E, Holdener M, von Herrath MG. 2010. Viral triggers for autoimmunity:
Is the 'glass of molecular mimicry' half full or half empty? J Autoimmun 34(1):38-44.
Clad A, Reuschenbach M, Weinschenk J, Grote R, Rahmsdorf J, Freudenberg N. 2011.
Performance of the aptima high-risk human papillomavirus mRNA assay in a referral population in comparison with hybrid capture 2 and cytology. J Clin Microbiol 49(3):1071-6.
Clerici M, Balotta C, Salvaggio A, Riva C, Trabattoni D, Papagno L, Berlusconi A, Rusconi S, Villa ML, Moroni M, et al. 1996. Human immunodeficiency virus (HIV) phenotype and interleukin-2/ interleukin-10 ratio are associated markers of protection and progression in HIV infection. Blood 88(2):574-9.
Clifford GM, Gallus S, Herrero R, Munoz N, Snijders PJ, Vaccarella S, Anh PT, Ferreccio C, Hieu NT, Matos E, et al. 2005. Worldwide distribution of human papillomavirus types in cytologically normal women in the international agency for research on cancer HPV prevalence surveys: A pooled analysis. Lancet 366(9490):991-8.
Cohen J. 1960. A coefficient of agreement for nominal scales. Educ Psychol Meas 20(1):37-46.
Cohen N, Gupta M, Doerwald-Munoz L, Jang D, Young JE, Archibald S, Jackson B, Lee J, Chernesky M. 2017. Developing a new diagnostic algorithm for human papilloma virus associated oropharyngeal carcinoma: An investigation of HPV DNA assays. J Otolaryngol Head Neck Surg 46(1):11.
Coller KE, Heaton NS, Berger KL, Cooper JD, Saunders JL, Randall G. 2012. Molecular determinants and dynamics of hepatitis C virus secretion. PLoS Pathog 8(1):e1002466.
Collins ML, Irvine B, Tyner D, Fine E, Zayati C, Chang C, Horn T, Ahle D, Detmer J, Shen LP, et al. 1997. A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml. Nucleic Acids Res 25(15):2979-84.
Compton J. 1991. Nucleic acid sequence-based amplification. Nature 350(6313):91-2.
Conger KL, Liu JS, Kuo SR, Chow LT, Wang TS. 1999. Human papillomavirus DNA replication.
interactions between the viral E1 protein and two subunits of human dna polymerase alpha/
primase. J Biol Chem 274(5):2696-705.
Crotta S, Stilla A, Wack A, D'Andrea A, Nuti S, D'Oro U, Mosca M, Filliponi F, Brunetto RM, Bonino F, et al. 2002. Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein. J Exp Med 195(1):35-41.
Currer R, Van Duyne R, Jaworski E, Guendel I, Sampey G, Das R, Narayanan A, Kashanchi F.
2012. HTLV tax: A fascinating multifunctional co-regulator of viral and cellular pathways.
Front Microbiol 3:406.
Dalianis T and Hirsch HH. 2013. Human polyomaviruses in disease and cancer. Virology 437(2):63-72.
Daly GM, Bexfield N, Heaney J, Stubbs S, Mayer AP, Palser A, Kellam P, Drou N, Caccamo M, Tiley L, et al. 2011. A viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing. PLoS One 6(12):e28879.
de Jong A, van der Burg SH, Kwappenberg KM, van der Hulst JM, Franken KL, Geluk A, van Meijgaarden KE, Drijfhout JW, Kenter G, Vermeij P, et al. 2002. Frequent detection of human papillomavirus 16 E2-specific T-helper immunity in healthy subjects. Cancer Res 62(2):472-9.
de Martel C, Maucort-Boulch D, Plummer M, Franceschi S. 2015. World-wide relative contribution of hepatitis B and C viruses in hepatocellular carcinoma. Hepatology 62(4):1190- 200.
De Strooper LM, Meijer CJ, Berkhof J, Hesselink AT, Snijders PJ, Steenbergen RD, Heideman DA.
De Strooper LM, Meijer CJ, Berkhof J, Hesselink AT, Snijders PJ, Steenbergen RD, Heideman DA.