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 Techniques Advanced 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? more effective therapy 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 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: ). In both panels, nuclei are stained with DAPI (blue).

7. Amplified nucleic acid techniques
Signal amplification techniques
bDNA assays
hybrid capture assays
Target amplification techniques
PCR techniques
transcription-based amplification methods
strand displacement amplification
Probe amplification techniques
cleavase-invander technology
ligase chain reaction
cycling probe 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 Nested-PCR Multiplex PCR
RNA transcription into cDNA
retroviral reverse transcriptases or thermostable Tth DNA-polymerase
commercially available kits: HCV, HIV-1 in clinical specimens
Nested-PCR
2 pairs of amplification primers
increased sensitivity and specificity
Multiplex PCR
2 or more primer sets
more than one target sequence co-amplified
commercial kits: viruses of respiratory and central nervous system
Real time PCR/Quantitative real time PCR
starget amplification and detection steps are simultaneous
software-based monitoring the data at every cycle - 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-Mediated Isothermal Amplification (LAMP)
Helicase-Dependent Amplification (HDA)
LAMP: Dengue, SARS, influenza A/B, CMV, HSV, VZV...
HDA: HIV-1 in human plasma, HSV 1 and 2 from genital lesions

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
capsid
membrane
envelope
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. Miniature RT–PCR system for diagnosis of RNA-based viruses
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

47. Immunofluorescence staining of TBEV-infected PS cells

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

49. 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