Viral Diagnostics
Jonathan Gubbay
Ontario Agency for Health Protection and Promotion
Public Health Laboratory-Toronto
Overview
Clinical virology lab can provide significant
benefit to patient care
Traditionally epidemiologic and academic
role
Current rapid assays impact on
therapeutic and public health decisions.
– Change largely due to molecular methods
Impact of PCR on virology
Recent identification of several respiratory
viruses
– Metapneumovirus
– Multiple coronaviruses: SARS, 229E, NL63,
OC43, HKU1.
– Human bocavirus
– Polyomaviruses KI, WU
Why Expanding Role for
Diagnostic Virology Lab
Increased pool of immunocompromised
Increasing antiviral agents
Results in increasing demand for rapid
methods, viral load testing, antiviral
susceptibility, genotyping.
Methods in use in virology.
Detecting Active Infection:
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Electron Microscopy
Viral culture
Detection of viral antigens
Detection of viral nucleic acid.
Histopathology
Assessing virus-specific immune response
– Serologic testing (won’t cover today)
Specimen choice and collection
Specimen quality limits test quality
Pathogen detection depends on:
– Appropriate collection site.
– Proper timing of specimen collection.
– Effective and timely processing of sufficient
specimen.
Specimen storage and transport
Keep specimens other than blood at 4ºC
If delay >24hrs, freeze at 70ºC or below.
Avoid any storage at -20ºC: greater loss in
infectivity
Nonenveloped viruses (adenovirus,
enteroviruses) more stable than enveloped
(e.g. RSV, VZV, CMV).
Viral Transport Medium
Salt solution – ensures proper ionic
concentrations
Buffer - maintains pH
Protein - for virus stability
Antibiotics or antifungals – to prevent
contamination
Cell Culture
Viruses are obligate intracellular organisms –
require living cells for virus isolation
Advantages:
– Relatively sensitive and specific
– Can detect many different viruses
– Provides a viral isolate for further characterization
(serotyping, genotyping, susceptibility)
Cell culture –limitations
Certain viruses don’t grow or grow slowly
Other techniques for detecting viral
infection more cost effective
Successful culture depends on viability of
virus in specimen
Standard Cell Cultures
Originally used animals and embryonated
eggs.
Monolayer cell culture techniques (1933)
Roller tube cell cultures (1940)
Standard Cell Cultures
Primary cells
– 1-2 passages
Diploid (semicontinuous) cells
– 20-50 passages
Heteroploid cells.
– Indefinite passages
Cytopathic Effect
Monitor tube cultures daily initially
Monitor for 10-21 days
Compare to uninoculated controls from
same batch
Rounding, refractile cells, syncytium
formation, cell destruction
Cell culture – clues to virus
causing CPE
Type of specimen
Cell line displaying CPE
Type of CPE
Hemadsorbing viruses
Orthomyxoviruses (influenza) and some
paramyxoviruses (parainfluenza, measles,
mumps)
Insert viral glycoproteins (haemaglutinin)
into host cell membrane.
Promotes attachment of RBC of certain species
(e.g guinea pig) to cell membrane.
Interference
Rubella virus growing in monkey kidney
cells inhibits infection with echovirus 2.
Adenovirus CPE
RSV - syncytia
Cell culture – definitive
identification
Reaction with monoclonal antibodies.
Antibodies chemically conjugated to a
fluorochrome.
Indirect or direct immunofluorescence.
Neutralization – use monospecific or
pooled antisera to prevent infection of
susceptible cells (enterovirus serotyping)
Shell Vial Culture
System which detects viral infection prior to CPE
developing.
Low speed centrifugation enhances infection.
First used for CMV
– MRC5 cells, stain for early antigen protein after 1848hr incubation
Also developed for other viruses: VZV, HSV,
adenovirus, respiratory viruses
CMV early antigen staining
Mixed Cell Culture
Shell vial culture with mixed cell lines
Allows detection of wider range of viruses.
R-MixTM: mink lung + A549
– RSV, parainfluenza 1-3, influenza,
adenoviruses
R-Mix Too: MDCK + A549
– Above plus metapneumovirus
E-Mix: BGMK + A549
Recent R-Mix Too vs Rhesus
Monkey Kidney at PHL-Toronto
257 stored primary samples; 194 positive
R-Mix shell vials stained at 1,2 and 5d
Tube culture monitored for 10d
R-Mix detected 67.5% of and tube culture 45.9%
of previously positives
Of pos R-Mix, 73.3% and 95.5% detected after 1
and 2 days.
Of pos TC, 2%, 25% and 60% isolated by day
1,2 and 7; 36% took 10 days to be isolated.
Genetically Engineered Cell Lines
HSV
– Baby hamster kidney (BHK-21) transformed with an
HSV-inducible promoter (UL39 gene)
– UL39 attached to functional E Coli β-galactosidase
gene.
– β-galactosidase activity induced by HSV 1 or 2
infection.
– Addition of substrate (X-Gal) for this enzyme results in
coloured product in HSV-infected cells.
– Commercially available as enzyme-linked virusinducible system (ELVIS HSV ID).
Genetically Engineered Cell Lines
Rapid detection after overnight incubation.
Antiviral Susceptibility Testing
May do for herpesviruses when:
– HSV or VZV cutaneous lesions fail to resolve
or appearance of new ones while on oral
therapy.
– Progressive retinal or visceral CMV disease
while patient on therapy.
Ganciclovir-resistant CMV
First reported ganciclovir R CMV among
HIV-infected with CD4 <50 x 109/
Emerging problem in HSCT and SOT
patients.
R testing now recommended for HIV
treatment failure; many do routinely at
diagnosis
Resistance testing now important
in influenza therapy
CDC Issues Interim Recommendations for the Use
of Influenza Antiviral Medications in the Setting of
Oseltamivir Resistance among Circulating Influenza
A (H1N1) Viruses,
2008-09 Influenza Season
Friday, December 19, 2008, 11:50 EST (11:50 AM EST)
http://www2a.cdc.gov/HAN/ArchiveSys/ViewMsgV.asp?AlertNum=00279
Phenotypic assays
Measure effect of antiviral drug on growth
of a virus.
Measured by infectivity (plaque reduction),
viral antigen or viral nucleic acid
production, enzyme activity.
Directly measure and quantify effects of
antivirals on growth.
Slow, labour intensive, difficult to
standardise
Phenotypic assays
Expressed as drug concentration that
inhibits 50% or 90% of viral growth (IC50
and IC90) relative to a no drug control.
Phenotypic susceptibility assays in use for
herpesviruses (CMV, HSV, VZV), influenza
and HIV-1.
Plaque reduction assay for HSV
susceptibility approved by CLSI.
Plaque Reduction Assay
Proposed breakpoints for HSV and CMV
S, I, R based on IC50.
Many variables effect results
– Cell line
– Viral inoculum
– Incubation period
Plaque Reduction Assay
http://pathmicro.med.sc.edu/mhunt/plaque.jpg
Phenotypic susceptibility to
neuraminidase inhibitors
Directly measure NA activity
Viral NA incubated with different
concentrations of NA inhibitors.
Fluorescent or chemiluminescent
substrate added and quantitated.
Phenotypic assays for HIV
susceptibility
Traditionally measured p-24 antigen in cell
culture by EIA in presence of antiviral
drug.
Recombinant virus phenotypic assays
– Insertion of RT and polymerase from patient
into a vector consisting of a rapidly replicating
viral strain and a reporter gene (luciferase).
– Measure viral growth in presence of drug
compared to wild type virus. (Phenosense,
Antivirogram)
Genotypic Assays
Allow rapid detection of genetic mutations
associated with antiviral drug resistance.
1. Nucleic acid amplification
2. Sequencing of amplified product
3. Compare amplicon sequence to reference
strain
Genotypic Assays
Most useful when a discrete number of
known resistance mutations
– CMV mutations causing ganciclovir
resistance.
UL 97 (protein kinase) – ganciclovir
UL54 (DNA polymerase) – ganciclovir, foscarnet,
cidofivir
– Antiretroviral-refractory HIV infections.
Amplification and sequencing of RT and
polymerase genes
Genotypic Assays
– Lamivudine R in Hep B
– M2 mutations in amantadane R
Influenza A.
– NA (H275Y) and/or hemagluttinin
mutations in Influenza A associated
with NA inhibitor R.
Direct Detection of Virus or Viral
Antigen: Electron Microscopy
Superseded by other methods in most
labs
Still important for rapid detection of viruses
in clinical samples.
Able to detect multiple pathogens
Direct Detection of Virus or Viral
Antigen: Electron Microscopy
Specimen absorbed onto thin
plastic/carbon film.
Applied to surface of EM grid before
staining.
Can use positive or negative stain (most
common – phosphotungstic acid)
– Penetrates virion and provides contrast for
visualization of cell surface.
Direct Detection of Virus or Viral
Antigen: Electron Microscopy
Quick
Looks for many viruses
Useful if unknown pathogen
Less prone to cross contamination vs molecular.
Expensive equipment, need expertise to read
Not well suited to screening large numbers of
samples.
Low sensitivity – need 105-108 viral particles/ml
to detect.
Adenovirus
http://www.ncbi.nlm.nih.gov/ICTVdb/Images/Safrica/adeno3.htm
Human Rotavirus
Paramyxovirus (Parainfluenza)
Histopathology/Cytology
Some viruses produce characteristic
cytologic/histologic changes
Not enough time to go into detail now….
Direct Detection of Viruses:
Immunoassays
Utilize antibodies (monoclonal or
polyclonal) against specific viral
antigen/antigens.
Ag/Ab complexes detected by:
– Direct visualization
– Solid Phase Enzyme immunoassays
– Enzyme Linked immunoassays
Direct Visualization: direct method
Direct or indirect staining methods
Direct: antibody conjugated with either an
enzyme (e.g horseradish peroxidase) or
fluorescent label (e.g FITC).
Substrated added to Ag/Ab-Conjugate on
glass slide, resulting in colour.
Ag/Ab-FITC visualized using fluorescent
microscope.
Direct visualization: indirect method
An unlabelled (e.g mouse) antibody is
added to bind to the antigen of interest
A second antibody labelled with conjugate
is added.
Visualized using substrate as with direct
method
Indirect allows amplification of signal –
many more labelled antibodies can bind to
the intermediate unlabelled one.
Direct and indirect
immunofluorescence
DFA – direct immunofluoresence.
IFA – indirect immunofluoresence.
Widely used in virology
Cheap, easy to do.
Monoclonal abodies give high specificity.
Pooling of monoclonals can detect multiple
viruses – e.g resp virus DFA
Used for CMV pp65 antigenemia quantification
DFA RSV
CMV pp65 antigen
http://ibmi.mf.uni-lj.si/acta-apa/acta-apa-00-3/Marin.html
ELISAs/EIAs
Surface of solid phase (microtitre plate)
coated with antibody
Antigen of interest binds if present.
Second enzyme-conjugated antibody
added
Substrate added and colour
generated/read by spectrophotometer.
http://www.dshs.state.tx.us/lab/images/eia_1.jpg
ELISAs/EIAs
Sometimes second antibody is unlabelled and
use 3rd labelled antibody.
Most give qualitative result, some quantitative.
Newer EIAs use microbeads as the solid phase
to increase surface area of contact.
– Decreases reaction time to 30min.
Sensitive – detect viral antigens at pico to
nanomolar (10-12 to 10-9mol/litre)
ELISAs
Can be noncompetitive “sandwich” as described
previously
Competitive – Solid phase is coated with antigen
to be detected.
– Patients sample is mixed with detecting antibody,
which is then applied to solid phase.
– If sample contained antigen, less antibody is free to
bind to the solid phase with prebound antigen.
– Rest of test run as before. Intensity of colour
formation inversely proportional to amount of antigen
in sample.
Nucleic Acid Detection
Short length of viral genome makes them
ideal candidate for nucleic-acid based
diagnosis
PCR
– conventional PCR – agarose gel detection of
product
– Real-time PCR- products detected using
probes or intercalating dyes within the
reaction.
– Microarrays – chip or bead based.
PCR
Each cycle of PCR doubles the number of
copies (amplicons)
Over 1 million amplicons after 20 cycles.
Primers determine sensitivity and
specificity of PCR reactions.
5’ Exonuclease Probes
©Clinical and Laboratory Standards Institute. All rights reserved.MM3-A2
©Clinical and Laboratory Standards Institute. All rights reserved.MM3-A2
Molecular Beacons
FRET probes
Multiplex PCR
Multiple viruses can cause same clinical
syndrome
– Respiratory infections
Can perform multiplex PCR assays to
detect multiple viruses in one reaction.
Commercial assays to detect up to 18
respiratory viruses in 1 test.
Seeplex®
RV/PB18 ASE Detection
Multiplex–PCR System for the detection of 13
Respiratory Viruses (Influenza A/B virus, RSV
A/B, Rhinovirus, Coronavirus OC43/HKU1,
coronavirus 229E/NL63, adenovirus,
parainfluenza virus 1-3m bocavirus,
enterovirus
5 Pneumonia causing bacteria (Mycoplasma
pneumoniae, Haemophilus influenzae,
Streptococcus pneumoniae, Chlamydophila
pneumoniae, Legionella pneumophila).
Development of a Respiratory Virus Panel Test for
Detection of Twenty Human Respiratory Viruses by Use of
Multiplex PCR and a Fluid Microbead-Based Assay
J. Mahony, et al.
Department of Pathology and Molecular Medicine, McMaster University, and St. Joseph’s
Healthcare, Hamilton,
Ontario, Canada,1 and TmBioscience Corporation, Toronto, Ontario, Canada2
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2007, p. 2965–2970 Vol. 45,
No. 9
Detects 20 different respiratory viruses
Development of a Respiratory Virus Panel Test for
Detection of Twenty Human Respiratory Viruses by Use of
Multiplex PCR and a Fluid Microbead-Based Assay
Reverse transcriptase
PCR
Target specific primer extension
– containing both a virus-specific
oligonucleotide sequence and a tag
oligonucleotide that hybridizes to a
complementary anti-tag oligonucleotide bound
to 21 spectrofluorometrically labeled
microspheres
Development of a Respiratory Virus Panel Test for
Detection of Twenty Human Respiratory Viruses by Use of
Multiplex PCR and a Fluid Microbead-Based Assay
TSPE: Target specific primer
extension
J. Mahony, et al. J Clin Micro; Sept. 2007, p. 2965–2970 Vol. 45, No. 9
Development of a Respiratory Virus Panel Test for
Detection of Twenty Human Respiratory Viruses by Use of
Multiplex PCR and a Fluid Microbead-Based Assay
Biotin labelled TSPE product reacts with
streptavadin/phycoerythrin
Flow cell with 2 lasers
– Red laser detects specific bead (1 per test
target)
– If virus was present and TSPE occurred,
green laser detects presence of phycoerythrin
and gives positive signal.
Non-PCR-based Nucleic Acid
Amplification Systems
Strand Displacement Amplification
Ligase Chain Reaction
Nucleic Acid Sequence-Based
Amplification
Hybridization –Based Assays
Quantitative NATs
Summary
Viral diagnostics is a dynamic field
New applications of molecular technology
being introduced continuously
Increased therapeutic options for viral
infections have increased the clinical
relevance of making a viral diagnosis.