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 ServiceUnited 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

HA

PB1PB2PA

NPNAMANS

M2

Hemagglutinin

Neuraminidase

M1

Influenza A VirusNegative sense RNA Single strandedSegmented

16 Hemagglutinin subtypes9 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. ($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

• 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

Taq

Taq

Reporter

Reporter

Quencher

Quencher

Primer 1

Primer 2

RRT-PCR for Avian InfluenzaA 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 RNA extraction

Type A fluRRT-PCR

HA subtype RRT-PCR

Positive

No further testing

Negative

NegativePositive

Virus Isolation Report to NVSL

Virus isolation

AIV

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

Chicken/Netherlands/03 + - -

Turkey/Ontario/6118/67 H8N4 + - -

Chicken/NJ/1220/97 H9N2 + - -

Chicken/Korea/96006/96 H9N2 + - -

Chicken/Germany/N/49 H10N7 + - -

Turkey/VA/31409/91 H10N7 + - -

Chicken/NJ/15906-6/96 H11N1 + - -

Duck/England/56 H11N1 + - -

Duck/LA/188B/87 H12N5 + - -

Gull/MD/704/77 H13N6 + - -

Mallard/Gurjev/263/82 H14N5 + - -

Shearwater/W.Australia/2576/79 H15N6 + - -

Aichi/68 H3N2 + - -

Equine/KY/211/87 H3N8 + - -

Swine/MN/9088/99 H3N2 + - -

Swine/IN/1726/89 H1N1 + - -

Isolate Subtype Matrix H5 H7

Duck/NJ/7717-70/95 H1N1 + - -

Mallard/NY/6750/78 H2N2 + - -

Env/NY/19019-6-98 H3N8 + - -

Duck/Victoria/9211-18-1400/92 H3N8 + - -

Duck/Alberta/286/78 H4N8 + - -

Chicken/Puebla/8629-602/94 H5N2 + + -

Chicken/MA/11801/86 H5N2 + + -

Avian/NY/31588-2/00 H5N2 + + -

Chicken/NJ/17169/93 H5N2 + + -

Chicken/Hong Kong/220/97 H5N1 + + -

Duck/Malaysia/97 H5N3 + + -

Chicken/NY/14677-13/98 H6N2 + - -

Turkey/PA/7975/97 H7N2 + - +

Chicken/PA/13552-1/98 H7N2 + - +

Quail/AR/16309/94 H7N3 + - +

Chicken/NY/8030-2/96 H7N2 + - +

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

ResultsBy Specimen

InterpretationDirectigen VI

+

+

+

+

–

–

–

+

+

–

–

+

+

–

PCR*

+

–

+

–

+

–

+

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 AnalysisBy Specimen

Paired ComparisonsPCR/VI

88/80

1.88

0.170

Dir/VI

60/80

10.45

0.002

PCR/Dir

88/60

18.27

0.001

Tests

# of Positive Specimens

Chi-Square Statistic

p-value

Statistical AnalysisBy Specimen

Sensitivity 88.2 67.1

Specificity 99.5 99.8

PCR/VI Dir/VI

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

RRT-PCR can detect inactivated virus, so may be inappropriate

Virus isolation to detect live virus

(Swab)Environmental samples

For HPAI viruses high levels of virus may be in tissues.

Macerate with glass beads in trizoland then Magnetic beads

Tissue samplesAny species

Virus primarily replicates in the intestinal tract. RNA extraction method must be modified for cloacal samples

Ambion Magnetic Bead RNA extraction then RRT-PCR

Cloacal SwabWaterfowl-ducks

Virus primarily replicates in the respiratory tract (LPAI)

RNeasy or Ambion Magnetic bead RNA extraction, then RRT-PCR

Tracheal or oropharyngealswab

Gallinaceous Poultry (chickens, turkeys, quail)

NotesProcessing Method

Recommended Specimen

Species/ Sample Type

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 ResultsReal-time PCR

InstrumentNumberof labs

Samples (data sets)

SD CVCorrect

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

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-commercial5 commercial

10-mile “surveillance zone”178 non-commercial35 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

Acknowledgements

Suzanne DeBlois

Thank you