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)
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
*Complement fixation tests
*Haemagglutination inhibition tests
*Neutralization tests
*Single Radial Haemolysis
Advanced Techniques
*Immunoassays (RIA, ELISA)
* Western Blot
Main molecular testing techniques for virus determination
Cobo, 2012
Why?
• Virus identification
• Virus subtyping
• Virus genotypization
• Identification of drug-resistant mutants
more effective therapy
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
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).
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
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
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
PCR techniques
Reverse transcriptase-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
Quantitative real-time PCR
Fluorescence resonance
evergy transfer, FRET
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
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)
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)
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
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%)
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)
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.
Methods for Diagnosis
of Tick-borne Encephalitis
Tick-borne encephalitis virus belongs to the Flaviviridae family
(+ssRNA viruses)
Structure of the tick-borne encephalitis virus
Genome of the tick-borne encephalitis virus
+ssRNA (11 kb)
single ORF – single polyprotein
capsid
membrane
protease
nucleotide triphosphatase methyltransferase
helicase
envelope
RNA-polymerase
protease cofactor
Ixodes ricinus ticks in different
developmental stages
adult female
larvae
nymph
The geographical distribution of Ixodes spp.
Tick-borne encephalitis virus subtypes
Transmission of TBE virus within the life cycle of ixodid ticks
Tick-borne encephalitis diagnosis
ELISA, FIA, neutraliz. tests
Fatal cases:
el. microscopy / RT-PCR
from brain and other organs
Revese transcriptase-PCR methods for tickborne encephalitis diagnostics
PCR-method for early differential diagnosis of tickborne encephalitis
• 252-bp long portion of
highly conserved NS5 region
of TBEV genome
• AMV reverse transcriptase
(Saksida et al., 2005)
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)
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)
Diagnosis of TBEV in ticks
tick samples
identification of TBEV
positive samples
tick homogenisation
virus RNA isolation
QIAamp RNA kit
(Qiagen)
PCR amplification of
sequence encoding E
protein
(Růžek et al., 2007)
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)
(DeBiasi and Tyler, 2004)
Molecular diagnosis of TBE: future perspectives
Miniature RT–PCR system
for diagnosis of RNAbased viruses
(Liao, 2005)
Serological (ELISA-based) methods for tick-borne
encephalitis diagnostics
Product
Signal
Enzyme
Secondary antiIgM or anti-IgG
antibody
IgM / IgG from
serum or CSF
Substrate
TBEV
protein E
Commercial IgG/IgM-ELISA kits for the detection of antiTBEV antibodies
(Niedrig, 2000)
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
Serological diagnosis of
TBE: future perspectives
PanBio Dengue Duo IgM and IgG Rapid
Strip Test (PanBio, Brisbane, Australia)
Virological interpretations of serological test
results in case of a clinically suspected TBE
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 )
TBEV-induced cytopathic effect quantification
(Eyer et al. 2015)
Histopathological changes
(Růžek et al., 2010)
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
Immunofluorescence staining
of TBEV-infected PS cells
Immunofluorescence staining: antiviral activity testing
no antivirals
(Eyer et al., 2015)
7-deaza-2´-C-methyladenosine
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