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Diagnostic Methods in Virology

Diagnostic Methods in Virology. Mgr. Luděk Eyer, Ph.D. Veterinary Research Institute, Brno Department of Virology Emerging Viral Diseases. General overview of diagnostic methods in virology. 1. Direct Examination Electron Microscopy (morphology)

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Diagnostic Methods in Virology

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  1. Diagnostic Methods in Virology Mgr. Luděk Eyer, Ph.D. Veterinary Research Institute, Brno Department of Virology Emerging Viral Diseases

  2. General overview of diagnostic methods in virology 1. Direct Examination Electron Microscopy (morphology) Light microscopy (histological appearance) Immunofluorescence (antigen detection) Molecular techniques (detection of viral genomes) 2. Indirect Examination Cell Culture methods (plaque formation, cytopathic effect) Embryonated eggs (haemagglutination, inclusion bodies) Laboratory animals (disease or death confirmation by neutralization) 3. Serology Classical TechniquesAdvanced Techniques *Complement fixation tests *Immunoassays (RIA, ELISA) *Haemagglutination inhibition tests * Western Blot *Neutralization tests *Single Radial Haemolysis

  3. Main molecular testing techniques for virus determination Cobo, 2012

  4. Why? • Virus identification • Virus subtyping • Virus genotypization • Identification of drug-resistant mutants more effective therapy

  5. Non-amplified nucleic acid probes (hybridization techniques) • hybridization of target (viral) sequence with nucleic acid probes (DNA/RNA) labeled with radioisotopes, enzymes or fluorescent or molecules • Liquid-phase, solid-phase (Southern‘ s, Northern blott), in situ hybridization, FISH, reverse hybridization • FISH: cytopathic effect deficiences, internal viral processing, real-time monitoring, localization of viral DNA/RNA within cells, studying the life cycles • Epstein-Barr, Dengue, HIV, poliovirus

  6. Multiplex detection in Huh-7 cells using the RNA FISH assay. Multiplex fluorescence RNA in situ detection of HCV viral genomic RNA (green) and 18S RNA (red) in Huh-7 cells lacking (-HCV) or containing (+HCV) an HCV replicon (Ikeda et al., 2002, J. Virol., 76: 2997-3006). In both panels, nuclei are stained with DAPI (blue).

  7. Amplified nucleic acid techniques • Signalamplificationtechniques • bDNAassays • hybrid captureassays • Target amplificationtechniques • PCR techniques • transcription-basedamplificationmethods • stranddisplacementamplification • Probeamplificationtechniques • cleavase-invander technology • ligasechainreaction • cyclingprobe technology

  8. bDNA (branched DNA) assays • The signal is proportional to the number of labeled probes • Commercially available assayes (Bayer HealthCare, Diagnostic Division, Tarrytown, N.Y.) • HCV, HBV, HIV-1

  9. Hybrid Capture Assays • RNA/DNA hybrid molecule • Anti-hybrid antibody (capture), anti-hybrid detection antibody (labeled) • Commercially available assays: Digene Corp. (Gaithersburg, Maryland, USA): HPV, CMV, Chlamydia, Neisseeria

  10. PCR techniques • Reverse transcriptase-PCR • RNA transcriptionintocDNA • retroviral reverse transcriptasesorthermostableTth DNA-polymerase • commerciallyavailablekits: HCV, HIV-1 in clinicalspecimens • Nested-PCR • 2 pairsofamplificationprimers • increased sensitivity and specificity • Multiplex PCR • 2 or more primersets • more thanonetargetsequence co-amplified • commercialkits: virusesofrespiratory and centralnervoussystem • Real time PCR/Quantitativerealtime PCR • stargetamplification and detectionsteps are simultaneous • software-based monitoring the data ateverycycle - quantification

  11. Quantitative real-time PCR Fluorescence resonance evergy transfer, FRET

  12. Nucleic acid sequence-based amplification (NASBA) • isothermal RNA amplification method • avian myeloblastosis virus RT, RNase-H, T7-RNA polymerase • no requirement for a thermal cycler, rapid kinetics, ssRNA-no denaturation prior deteiction • bioMérieux: HIV-1, CMV, enterovirus, respiratory syncytial virus West Nile virus, St. Louis encephalitis, Dengue virus

  13. Cycling probe technology • detection of low amount of target DNA • chimeric DNA-RNA-DNA probe labeled with fluorofore and quencher • fast, linear, isothermal, simple, low background (target DNA is not amplified)

  14. Ligase Chain Reaction (LCR) • Ligation of oligonucleotide probes • Cycles: denaturation, hybridization of probes, ligation • 4 LCR primers labeled with fluorophores • thermostable DNA-ligase from Thermus aquaticus • detection of point mutations (antiviral resistance mutants)

  15. Isothermal amplification methods • simple operation, rapid reaction, easy detection • not require thermal cycler • performed in a heating block or water bath • single uniform temperature • Loop-MediatedIsothermalAmplification (LAMP) • Helicase-DependentAmplification (HDA) • LAMP: Dengue, SARS, influenza A/B, CMV, HSV, VZV... • HDA: HIV-1 in human plasma, HSV 1 and 2 fromgenitallesions

  16. Amplification methods: advantages and drawbacks • high sensitivity • rapid methods • cost-effective diagnostic technques • can not be used for identification of new viruses • not convenient for viruses showing huge genetic variability • reverse transcription step of forming cDNA is not efficient enough (efficacy ~ 20%)

  17. Postamplification analyses Sequencing (identification of unknown viruses, identification of resistance mutations, HCV/HBV genotyping, HIV drug resistance testing for monitoring treatment...) Luminex analysis (Multiplexed microsphere-based array, combination of multiplex PCR and flow cytometry) Nucleic acid arrays (DNA microarrays) Mass spectrometry (protein expression/proteome analysis)

  18. DNA-microarrays • Labeled PCR product is hybridized to the probes, and hybridization signals are mapped to sevral positions within the array. • Sequencing by hybridization • Confocal microscopy is used to can the chip. • First application: rapid sequencing to detect HIV mutations associated with drug resistance.

  19. Methods for Diagnosis of Tick-borne Encephalitis

  20. Tick-borne encephalitis virus belongs to the Flaviviridae family (+ssRNA viruses)

  21. Structure of the tick-borne encephalitis virus

  22. Genome of the tick-borne encephalitis virus +ssRNA (11 kb) single ORF – single polyprotein protease nucleotide triphosphatase helicase methyltransferase RNA-polymerase envelope capsid membrane protease cofactor

  23. Ixodes ricinus ticks in different developmental stages adult female larvae nymph

  24. The geographical distribution of Ixodes spp.

  25. Tick-borne encephalitis virus subtypes

  26. Transmission of TBE virus within the life cycle of ixodid ticks

  27. Tick-borne encephalitis diagnosis ELISA, FIA, neutraliz. tests Fatal cases: el. microscopy / RT-PCR from brain and other organs

  28. Revese transcriptase-PCR methods for tick-borne encephalitis diagnostics

  29. PCR-method for early differential diagnosis of tick-borne encephalitis • 252-bp long portion of highly conserved NS5 region of TBEV genome • AMV reverse transcriptase (Saksida et al., 2005)

  30. Multiplex RT-PCR for subtyping of tick-borne encephalitis virus isolates Unique conmbination of oligonucleotide primers hybridizing with subtype-specific signature positions of the sequence encoding viral E protein. (Růžek et al., 2007)

  31. Multiplex RT-PCR for subtyping of tick-borne encephalitis virus isolates Subtyping of TBEV strains using the multiplex RT-PCR. Members of separate subtypes were analyzed individually (a) and in all possible combinations (b). (Růžek et al., 2007)

  32. Diagnosis of TBEV in ticks tick samples virus RNA isolation QIAamp RNA kit (Qiagen) identification of TBEV positive samples tick homogenisation PCR amplification of sequence encoding E protein (Růžek et al., 2007)

  33. Quantitative real-time RT-PCR for the laboratory detection of tick-borne encephalitis virus RNA • targeting the 3´-noncoding region of the TBEV genome • highly sensitive and specific method to quantify of even low viral loeads in serum/CSF/tick homogenate samples. (Schwaiger and Cassinotti, 2003)

  34. (DeBiasi and Tyler, 2004)

  35. Molecular diagnosis of TBE: future perspectives Miniature RT–PCR system for diagnosis of RNA-based viruses (Liao, 2005)

  36. Serological (ELISA-based) methods for tick-borne encephalitis diagnostics

  37. Product Signal Secondary anti-IgM or anti-IgG antibody Enzyme IgM / IgG from serum or CSF Substrate TBEV protein E

  38. Commercial IgG/IgM-ELISA kits for the detection of anti-TBEV antibodies (Niedrig, 2000)

  39. Subviral particle-based ELISA • co-expression of recombinant prM/E protein of TBEV in mammalian cells • realease of subviral particles (SPs) into the culture medium • using the SPs as antigen for development of TBE-specific ELISA • SP-IgG and SP-IgM ELISA systems • No cross-reactivity with antibodies against other flaviviruses

  40. Serological diagnosis of TBE: future perspectives PanBio Dengue Duo IgM and IgG Rapid Strip Test (PanBio, Brisbane, Australia)

  41. Virological interpretations of serological test results in case of a clinically suspected TBE

  42. Other methods for TBE diagnosis Cell culture methods: cytopathic effect examination Mock-infected cells: (no CPE) TBE infected cells: (strong CPE) Cell lines for TBE: porcine kidney cells (PS), human neuroblastoma cells (UKF-NB-4 )

  43. TBEV-induced cytopathic effect quantification (Eyer et al. 2015)

  44. Histopathological changes (Růžek et al., 2010)

  45. Immunofluorescence staining Fluorescence/confocal microscopy Fluorophore goat anti-rabbit antibody (secondary ab) rabbit anti-TBEV protein E antibody (primary ab) TBEV protein E cell structures

  46. Immunofluorescence staining of TBEV-infected PS cells

  47. Immunofluorescence staining: antiviral activity testing no antivirals (Eyer et al., 2015) 7-deaza-2´-C-methyladenosine

  48. Tick-borne encephalitis diagnosis: conclusion • Currently the diagnosis of TBE is based on serological methods (detection of specific antibodies in serum/whole blood)/CSF from the second week of the disease onwards • Disadvantages: antibody cross-reactivity with other flaviviruses • Molecular detection by PCR could be valuable for the early diagnosis of a specific etiological agent. • Early detection: improving prognosis, introduction of the earlier appropriate therapy

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