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”
•
•
•
•
•
•
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
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. ($1520/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
•
•
•
•
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
RRT-PCR
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
Matrix
H5
H7
Isolate
Matrix
H5
Duck/NJ/7717-70/95 H1N1
+
-
-
Chicken/Netherlands/03
+
-
-
Mallard/NY/6750/78 H2N2
+
-
-
Turkey/Ontario/6118/67 H8N4
+
-
-
Env/NY/19019-6-98 H3N8
+
-
-
Chicken/NJ/1220/97 H9N2
+
-
-
Duck/Victoria/9211-18-1400/92 H3N8
+
-
-
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
+
-
-
Isolate
Subtype
Subtype
H7
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
+
+
+
+
+
–
+
–
+
+
–
–
–
+
+
–
+
–
–
*MA+
–
and H7+ only
+
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%)
Statistical Analysis
By Specimen
Paired Comparisons
Tests
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
PCR/VI
Statistical Analysis
By Specimen
PCR/VI
Dir/VI
Sensitivity
88.2
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
Speed
Cost
Virus Isolation
Best
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
–
–
–
–
Individual samples/ centrifuge
Vacuum manifolds
96 well plates
Robots/automated (96, 48, 32 sample)
Sample types and processing methods
Species/
Sample Type
Processing
Method
Notes
Tracheal or
oropharyngeal
swab
RNeasy or Ambion
Magnetic bead
RNA extraction,
then RRT-PCR
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
For HPAI viruses high levels of
virus may be in tissues.
Environmental
samples
(Swab)
Macerate with
glass beads in trizol
and then Magnetic
beads
Virus isolation to
detect live virus
Gallinaceous
Poultry
(chickens,
turkeys, quail)
Recommended
Specimen
Waterfowlducks
RRT-PCR can detect
inactivated virus, so may be
inappropriate
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 RRTPCR 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
16K-radius 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 RRTPCR 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