Diagnostic Methods in Virology

Mgr. Luděk Eyer, Ph.D.Veterinary Research Institute, Brno

Department of VirologyEmerging Viral Diseases

1. Direct Examination Electron Microscopy (morphology) Light microscopy (histological appearance)Immunofluorescence (antigen detection)Molecular techniques (detection of viral genomes)

2. Indirect ExaminationCell Culture methods (plaque formation, cytopathic effect)Embryonated eggs (haemagglutination, inclusion bodies)Laboratory animals (disease or death confirmation by neutralization)

3. SerologyClassical Techniques Advanced Techniques*Complement fixation tests *Immunoassays (RIA, ELISA)*Haemagglutination inhibition tests * Western Blot*Neutralization tests*Single Radial Haemolysis

General overview of diagnostic methods in virology

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

• 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

• simple operation, rapid reaction, easy detection• not require thermal cycler • performed in a heating block or water bath• single uniform temperature

• 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%)

Amplification methods: advantages and drawbacks

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

capsid membrane envelope

proteasenucleotide triphosphatase

helicasemethyltransferaseRNA-polymerase

protease cofactor

+ssRNA (11 kb)single ORF – single polyprotein

Ixodes ricinus ticks in different developmental stages

adult female

nymph

larvae

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 tick-borne encephalitis diagnostics

• 252-bp long portion of highly conserved NS5 region of TBEV genome

• AMV reverse transcriptase

PCR-method for early differential diagnosis of tick-borne encephalitis

(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

virus RNA isolationQIAamp RNA kit

(Qiagen)

tick samples

tick homogenisation

PCR amplification of sequence encoding E protein

identification of TBEV positive samples

(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)

Miniature RT–PCR system for diagnosis of RNA-

based viruses

Molecular diagnosis of TBE: future perspectives

(Liao, 2005)

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

TBEV protein E

IgM / IgG from serum or CSF

Secondary anti-IgM or anti-IgG antibody

Enzyme

Substrate

Product Signal

Commercial IgG/IgM-ELISA kits for the detection of anti-TBEV 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

PanBio Dengue Duo IgM and IgG Rapid Strip Test (PanBio, Brisbane, Australia)

Serological diagnosis of TBE: future perspectives

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

Other methods for TBE diagnosisCell 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

TBEV protein E

rabbit anti-TBEV protein E antibody (primary ab)

goat anti-rabbit antibody (secondary ab)

Fluorophore

cell structures

Fluorescence/confocal microscopy

Immunofluorescence staining of TBEV-infected PS cells

Immunofluorescence staining: antiviral activity testing

no antivirals

7-deaza-2´-C-methyladenosine(Eyer et al., 2015)

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