Application of Real Time RT-PCR to
Detect Avian Influenza Virus
David L. Suarez D.V.M., Ph.D.
Research Leader Exotic and Emerging
Avian Viral Disease Research Unit
Agricultural Research Service
United States Department of Agriculture
Diagnosis of Avian Influenza Virus
• Clinical signs
– Low pathogenic avian influenza
– Highly Pathogenic avian influenza
• Diagnostic Tests
– Antibody detection
• AGID, HI
• ELISA
– Virus detection
•
•
•
•
•
Virus isolation
Directigen
Realtime RT-PCR (RRT-PCR)
Traditional RT-PCR
NASBA
– Other Diagnostic Tests
Influenza A Virus
Negative sense RNA
Single stranded
Segmented
Neuraminidase
Hemagglutinin
M2
PB1
PB2
PA
HA
NP
NA
MA
NS
M1
16 Hemagglutinin subtypes
9 Neuraminidase Subtypes
Clinical Signs
• Highly Pathogenic Avian Influenza
– Symptoms depend on species
– Systemic lesions with high mortality
• Low Pathogenic Avian Influenza
–
–
–
–
–
–
Can be subclinical
Increased daily mortality
Respiratory disease symptoms
Drops in egg production
Increased condemnation at slaughter
Other symptoms can be strain dependant
Serology
• Detection of type and subtype specific antibodies
to previous influenza infection (AGID, ELISA,
and HI)
• Typically have antibody response 7-10 days after
bird is infected
• Birds stop shedding virus soon after antibodies are
detectable
• Widely used for routine surveillance for LPAI
viruses because of cost and birds stay seropositive
for months
• Serology less valuable for HPAI
Direct Detection of Virus
“Critical Component of Outbreak Control”
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•
•
•
•
•
Virus Isolation
Real-time RT-PCR
Traditional RT-PCR
NASBA
Antigen Capture Elisa tests
Other tests
Virus Isolation in SPF
embryonated chicken eggs
• Sensitive
• Necessary for viral characterization
• Embryonated chicken eggs are perishable and
supply may be limited
• Requires days to weeks for results
• Concerns about cross contamination
• Requires high levels of biosecurity
Real-time RT-PCR (RRT-PCR)
• One-Step RT-PCR test was developed for typing
and subtyping of influenza viruses using
fluorescent Taqman probes
• Sensitivity similar to virus isolation
• The test doesn’t require running the PCR product
on a gel and the probe confirms specificity
• The complete test, including the RNA isolation
step, can be completed in less than three hours
• Requires expensive equipment, but can be done
faster and cheaper than conventional virus
isolation
Positive
Negative
Traditional RT-PCR
• Perform RT-PCR in a one-step or two-step format
• Analyze DNA product on an ethidium bromide
stained agarose gel
• Sensitivity can be similar to virus isolation
• Working with large amounts of amplified DNA
creates a cross contamination risk!
• Numerous recommendations to reduce
contamination
• Requires less expensive equipment, but greater
manpower to perform test
NASBA Technology
• Nucleic acid sequence based amplification
(NASBA)
• Isothermal amplification with several enzymes
• NASBA-ECL similar sensitivity to virus isolation
• Commercial kit-requires additional equipment
• Not quantitative
• Expensive
Antigen Capture ELISA Tests
•
•
•
•
•
Antigen capture test-most produced for human use only
Type A test only- does not subtype
Lower sensitivity than virus isolation or RRT-PCR
Low level of false positives from clean samples
Test is easy to use and requires no additional
equipment
• Not always available in large quantities
• Directigen most commonly used in U.S. ($15-20/test)
Why RRT-PCR for AIV?
• Advantages
– Rapid test results (3 hours for rush samples)
– Reduced cross contamination
– Reagent Cost
• RNA extraction $2-4
• RRT-PCR $4
– Scalable- large numbers of samples may be
processed
– Can type and subtype AIVs
– Viable virus not necessary
Development and Evaluation of
an RRT-PCR Test for AIV
•
•
•
•
•
•
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Test Design
Bench Validation
Field Validation
Transfer of technology to other laboratories
Proficiency testing
Performance during an outbreak
Future goals
Critical Control Points For
Successful RRT-PCR
• RNA extraction procedure
– Efficient and reproducible extraction procedure
– Works with a variety of samples
– High throughput and cost are issues
• RT-PCR Amplification reagents
– Commercial kits preferred
– Sensitivity, cost and ease of use are issues
• PCR primers and probes
– Provides specificity
– Affects sensitivity
Real-time PCR chemistries
• Sequence specific
–
–
–
–
–
Taqman/ Dual-labeled probe/ Hydrolysis probe
Fluorescence resonance energy transfer (FRET)
Molecular Beacons
Lux primers
Scorpion probes
• Non-sequence specific
– dsDNA binding dyes (SYBR green)
Hydrolysis/Taqman probes
Reporter
Primer 1
Quencher
Taq
Primer 2
Reporter
Quencher
Taq
RRT-PCR for Avian Influenza
A Two Test Procedure
• Type A influenza Test
– Detects any Type A
influenza virus
– Detects conserved
Matrix gene
– Works with samples
from any species
– Used as screening test
– Sensitivity similar to
virus isolation
• Subtype specific Test
– Different test for every
subtype
– Provides confirmation
of Matrix (MA) test
– Less sensitive than MA
– Available H5, H6, H7
and H9 tests
– More affected by strain
variation
Sample
Negative
No further
testing
AIV
RNA extraction
Type A flu
RRT-PCR
Negative
Positive
HA subtype RRTPCR
Positive
Virus Isolation
Report to NVSL
Virus isolation
Bench Validation Procedures
• Develop specific primers and probes for pathogen
based on available sequence
• Optimize for Mg, cycling parameters,
probe/primer concentrations
• Determine sensitivity and limit of detection on
laboratory samples
• Determine specificity with a panel of
characterized viruses
• Compare samples from experimentally infected
animals to performance standard
Real-time RT-PCR for AIV Sensitivity
• Compared to Egg Infectious Dose
– Type A influenza – 10-1 EID50
– H5 and H7 subtype- 101 EID50
• Determined with in vitro transcribed RNA
– Type A influenza - 103 copies
– H5 and H7 subtypes– 104 copies
Specificity Panel
Isolate
Subtype
Matrix
H5
H7
Isolate
Duck/NJ/7717-70/95 H1N1
+
-
-
Mallard/NY/6750/78 H2N2
+
-
Env/NY/19019-6-98 H3N8
+
Duck/Victoria/9211-18-1400/92 H3N8
Subtype
Matrix
H5
H7
Chicken/Netherlands/03
+
-
-
-
Turkey/Ontario/6118/67 H8N4
+
-
-
-
-
Chicken/NJ/1220/97 H9N2
+
-
-
+
-
-
Chicken/Korea/96006/96 H9N2
+
-
-
Duck/Alberta/286/78 H4N8
+
-
-
Chicken/Germany/N/49 H10N7
+
-
-
Chicken/Puebla/8629-602/94 H5N2
+
+
-
Turkey/VA/31409/91 H10N7
+
-
-
Chicken/MA/11801/86 H5N2
+
+
-
Chicken/NJ/15906-6/96 H11N1
+
-
-
Avian/NY/31588-2/00 H5N2
+
+
-
Duck/England/56 H11N1
+
-
-
Chicken/NJ/17169/93 H5N2
+
+
-
Duck/LA/188B/87 H12N5
+
-
-
Chicken/Hong Kong/220/97 H5N1
+
+
-
Gull/MD/704/77 H13N6
+
-
-
Duck/Malaysia/97 H5N3
+
+
-
Mallard/Gurjev/263/82 H14N5
+
-
-
Chicken/NY/14677-13/98 H6N2
+
-
-
Shearwater/W.Australia/2576/79 H15N6
+
-
-
Turkey/PA/7975/97 H7N2
+
-
+
Aichi/68 H3N2
+
-
-
Chicken/PA/13552-1/98 H7N2
+
-
+
Equine/KY/211/87 H3N8
+
-
-
Quail/AR/16309/94 H7N3
+
-
+
Swine/MN/9088/99 H3N2
+
-
-
Chicken/NY/8030-2/96 H7N2
+
-
+
Swine/IN/1726/89 H1N1
+
-
-
Initial Comparison of Tests
• Virus isolation and RRT-PCR were compared
during the summer of 2001 on 1550 samples
from the LBMs of NY and NJ
• H7N2 was commonly isolated from birds in
the markets
• Good correlation of tests at market level
• Virus isolation appeared to be more sensitive
than RRT-PCR although both tests appeared to
miss positive samples
Field Validation of Diagnostic
Tests
• Literature on validation primarily targeted to
serologic tests
• Goal of 1000 negative samples and 300 positive
samples
• Compare diagnostic sensitivity (Dsn) and
diagnostic specificity (Dsp) with performance
(“Gold”) standard
• Ideally compare 3 different geographic regions
H7 Low Pathogenic Outbreak in
Virginia, USA
• March 2002, a low pathogenic H7N2 was diagnosed
in turkeys in Virginia
• Outbreak quickly spread and eventually 197 infected
flocks were identified
• Control was by eradication (stamping out)
• 4.5 million turkeys and chickens were destroyed
• Direct government costs for eradication was 65
million dollars and total cost was over 160 million
dollars
• First time a real-time RT-PCR test was used
significantly in an animal disease outbreak
Results of VA Study
• >3,600 samples tested between 4/30 – 5/15
– Directigen – Harrisonburg, VA
– VI – NVSL (DVL)
– RRT-PCR – NVSL (CVB-L)
• USDA-ARS, SEPRL, Athens, GA (6), DVL
(1), CVB-L (1)
• Samples included dead bird surveillance and suspect
samples
• Evaluated at specimen and submission level
Results
By Specimen
PCR* Directigen
VI
Interpretation
+
+
+
Total agreement (48/95 = 50.5%)
+
+
–
VI missed (4/95 = 4.2%)
+
–
+
D missed (19/95 = 20.0%)
+
–
–
D & VI missed (13/95 = 13.7%)
–
+
+
PCR missed (3/95 = 3.2%)
–
+
–
D false positive (2/95 = 2.1%)
–
–
+
PCR and D missed (6/95 = 6.3%)
*MA+
and H7+ only
Statistical Analysis
By Specimen
Paired Comparisons
Tests
PCR/VI
Dir/VI
PCR/Dir
# of Positive Specimens
88/80
60/80
88/60
Chi-Square Statistic
1.88
10.45
18.27
p-value
0.170
0.002
0.001
Statistical Analysis
By Specimen
Sensitivity
PCR/VI
88.2
Dir/VI
67.1
Specificity
99.5
99.8
Total agreement on all samples (+, – ) = 98.7%
n = 3,628
Conclusions
• RRT-PCR was equal to or more sensitive than VI
– Could replace VI without adversely affecting control
program
• Directigen test was valuable because of rapid
detection
• Virus isolation used to confirm RRT-PCR and
Directigen positives and characterize isolates
• RRT-PCR was eventually used in the VA state lab
to provide faster diagnosis
Conclusions:
Sensitivity, Speed, and Cost
Sensitivity
Virus IsolationBest
Speed Cost
Worst
Intermediate
Directigen
Worst
Best
Worst
RRT-PCR
Best
Intermediate Best
All 3 types of tests have a role in future disease
outbreaks
Real-time PCR platforms
• Many real-time PCR platforms
available
• Some optimization of test required
• Platforms have different
characteristics
–
–
–
–
Speed
Capacity
Optical channels (multiplex)
Cost
Lab Equipment Logistics
• Bio-safety cabinet space
– Ideally 3 cabinets
• 1. RNA extraction (full exhaust if Trizol is used)
• 2. RNA Transfer to reaction tubes
• 3. Clean reagents, master mix preparation (Cell
culture hood)
– Two cabinets (more realistic)
• 1. RNA extraction
• 2. RNA transfer/ master mix preparation
Between uses hoods need to be wiped out with
disinfectant and gloves should be changed.
RNA extraction
• Methods
– Silica binding columns (ex. RNeasy, Qiagen)
– Magnetic beads (ex. MagMax, Ambion)
– Organic solvents (ex. Trizol, Invitrogen)
• Formats
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–
–
–
Individual samples/ centrifuge
Vacuum manifolds
96 well plates
Robots/automated (96, 48, 32 sample)
Sample types and processing methods
Species/
Sample Type
Gallinaceous
Poultry (chickens,
turkeys, quail)
Recommended
Specimen
Processing
Method
Notes
Tracheal or
oropharyngeal
swab
RNeasy or Ambion
Magnetic bead RNA
extraction, then RRTPCR
Virus primarily replicates in the
respiratory tract (LPAI)
Cloacal Swab
Ambion Magnetic
Bead RNA extraction
then RRT-PCR
Virus primarily replicates in the
intestinal tract. RNA extraction
method must be modified for
cloacal samples
Any species
Tissue samples
Macerate with glass
beads in trizol and
then Magnetic beads
For HPAI viruses high levels of
virus may be in tissues.
Environmental
samples
(Swab)
Virus isolation to
detect live virus
RRT-PCR can detect inactivated
virus, so may be inappropriate
Waterfowlducks
National Animal Health
Laboratory Network (NAHLN)
• NAHLN laboratories are veterinary diagnostic
laboratories (state, veterinary school, private)
throughout the U.S. capable of testing for Foreign
Animal Diseases
• Goal was to provide NAHLN labs a validated
RRT-PCR test for AIV, NDV, FMD and other
select agents
• Provide a rapid diagnostic response during an
outbreak
• Increase surge capacity
• Continued federal (USDA/APHIS) oversight
Authorized Testing Laboratories
• Person, not the laboratory, is authorized to
perform the test
• Person has to go through RRT-PCR training or
have suitable experience
• Must pass, on a yearly basis, a proficiency panel
• Must provide data to USDA/APHIS to receive
reimbursement for testing as part of surveillance
programs
Proficiency Testing
• Panel of 10-14 samples
• Samples are whole virus inactivated by a
phenol disinfectant
• Includes negatives, strong and weak positives
• Includes different HA subtypes
• Successful tests require RNA extraction and
amplification
• Testing started by SEPRL, but transferred to
NVSL/APHIS for AIV
Proficiency Panel Results
Real-time PCR
Instrument
Number
of labs
Samples
(data
sets)
SD
CV
Correct
sample ID’s (%)
SmartCycler
9
336 (24)
1.34
5.19
325 (96.7)
Light Cycler without
BSA
1§
56 (4)
ND*
ND
49 (87.5)
Light Cycler with BSA
1
56 (4)
ND
ND
56 (100)
iCycler
1
28 (2)
ND
ND
28 (100)
ABI 7900
1
14 (1)
ND
ND
14 (100)
Total
12
490 (35)
ND
ND
472 (96.3)
Delmarva Outbreak 2004
• 1st flock identified by passive surveillance (clinical
disease) in Harrington, DE on Feb 5th, 2004
• Presumptive diagnosis the following day by RRT-PCR for
H7 AI
• Quarantines on farm and 2 mile buffer zone established per
MOU the same day
• Agreement in place to cover indemnification,
depopulation, C+D, carcass disposal, etc.
• Extensive surveillance in 2 mile quarantine and 6 mile
buffer zone (11,728 samples in 10 weeks)
• 2nd and 3rd infected flocks identified and depopulated
quickly
H5N2 in Texas
• Feb. 16 – increased mortality in a non-commercial broiler
flock in Gonzales, Texas
• Feb. 17 – diagnostic samples tested positive for H5 AIV at
Texas Veterinary Medical Diagnostic Laboratory
• Texas state officials arrived Feb. 17, placed flock under
Hold Order, began to trace & test epidemiologic links
• Two LBMs in Houston tested AI positive by RRT-PCR
• Baseline testing began on all flocks within 8K and 16Kradius around the index farm
• All flocks within 8K of index placed under Hold Order
• Feb. 20 – NVSL confirmed virus as H5N2
• Feb. 21 – Index farm depopulated (6,608 birds)
Texas Outbreak Continued
• Feb. 23 – NVSL determined H5N2 virus had amino acid
sequence compatible with HPAI
• Infected LBMs and their holding facilities were
depopulated on Feb. 23
• All other Houston LBMs (3) depopulated as dangerous
contacts
• C&D of LBMs completed Feb. 29
• March 1 – NVSL reported results of chicken/embryo
innoculation tests – no illness or deaths (pathogenicity
index=0)
• Surveillance-Over 2000 RRT-PCR tests and 3000 serologic
tests performed in first month.
HPAI H5N2 – Gonzales, Texas
5-mile “infected zone”
39 non-commercial
5 commercial
10-mile “surveillance zone”
178 non-commercial
35 commercial
Keys to Rapid Control
• Availability of sensitive and specific diagnostic
test at a local or regional laboratory
• Once a positive sample is identified initiate
quarantine and confirm sample identity (virus
isolation required)
• Plans for control, including indemnity, carcass
disposal, and movement controls, must be in place
before outbreak occurs
Maintenance of Test
• Sequence variation can cause test failure
• Monitoring of new outbreaks to assure test
performance is necessary
• New commercial reagents should be
evaluated for improved performance
• Issues of application need to be evaluated
Evaluation of H5 Subtype RRT-PCR
Test for Asian H5N1
• H5 test was originally designed primarily for
North American isolates
• Could identify Asian H5N1 viruses with lower
sensitivity
• Sequence analysis of Asian isolates showed good
conservation with reverse primer and probe, but 4
mismatches with forward primer
• Redesigned H5 test to include forward primers
optimized for both Asian and North American
viruses
– NA H5F TGACTATCCACAATACTCA
– EA H5F TGACTACCCGCAGTATTCA
Future Developments
• Dried down reagent beads
– Includes Primer, probes, buffers, and internal
control
– Internal control should prevent false negatives
– Multiplex test (requires multiple channels)
– Longer shelf-life
– Better reagent consistency among labs and tests
– Simpler protocol
Thank you
Acknowledgements
Suzanne DeBlois