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STANDARD OPERATING PROCEDURE

Title:

West Nile Virus Plaque Reduction Neutralisation Test (PRNT)


Author: L.P. Phipps 12/10/2009

SOP Reference: Edition:

VI.556 2

Implementation date:

Authorisation Approver Name: Sarah Eagle

Crown Copyright Not for publication without permission. SOPs must not be released outside the VLA without approval by a Head of Department.

Printed Copy Control Printed copies are non-maintained unless on blue paper and signed by the Local Quality Manager. Unsigned copies on white paper are only valid for use on the day of printing. Local Quality Manager Signature:

Date: Location:

VI.556 Edition 2
SOP LL template version 6: (issued 16 August 07)

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Veterinary Laboratories Agency Standard Operating Procedure West Nile Virus Plaque Reduction Neutralisation test (PRNT) for West Nile Virus

Contents
1. INTRODUCTION ......................................................................................................................3 1.1 1.2 2. 3. PURPOSE/SCOPE OF THIS SOP ...........................................................................................3 BACKGROUND INFORMATION ................................................................................................3

SAFETY...................................................................................................................................3 MATERIALS ............................................................................................................................4 3.1 3.2 3.3 3.4 3.5 DOCUMENTATION AND SOFTWARE ........................................................................................4 CHEMICALS AND REAGENTS .................................................................................................4 MEDIA ...............................................................................................................................5 ANIMALS/MICRO-ORGANISMS/CELLS ....................................................................................5 EQUIPMENT .......................................................................................................................5 TEST RELIABILITY ...............................................................................................................6 TEST PREPARATION ............................................................................................................6 TEST PROCEDURE ..............................................................................................................6

4.

PROCEDURE/METHOD ..........................................................................................................6 4.1 4.2 4.3

5. 6.

RESULTS ................................................................................................................................8 REFERENCES.........................................................................................................................8

APPENDIX 1 ....................................................................................................................................9 APPENDIX 2 ..................................................................................................................................10

VI.556 Edition 2
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Veterinary Laboratories Agency Standard Operating Procedure Plaque Reduction Neutralisation Test (PRNT) for West Nile Virus

1. 1.1 1.1.1

INTRODUCTION Purpose/Scope of this SOP This SOP describes the detection and measurement of anti-WNV neutralising antibodies within serum using a plaque reduction neutralisation method. The plaque reduction neutralisation test (PRNT) is a sensitive method for the detection and quantification of specific virus neutralising antibodies in serum and is carried out in three stages. The first neutralising step is performed by incubating serum dilutions against a standard dose of virus, this is carried out in a 96-well plate. After incubation the virus/serum mixture is transferred to Vero cell monolayers in a 12-well plate and incubated at 37 oC to allow any remaining live virus to adsorb to the cells. After this second incubation, the cells are overlaid with a nutrient gel and incubated for 4 days. The cell monolayers are then fixed and the nutrient gel overlay washed out before staining with crystal violet to allow visualisation and counting of plaques formed (small discreet areas of cell monolayer destroyed by virus). Serum that reduces the number of plaques by 90%, as compared to the virus control, are considered positive for neutralising antibody. Background information West Nile Virus (WNV) is a member of the genus Flavivirus (FamilyFlaviviridae). The virus is primarily transmitted and amplified between avian reservoir hosts by many species of mosquito vector in enzootic transmission cycles. During favourable ecologic and climatic conditions, mosquitoes can transmit WNV to susceptible avian species and mammals causing encephalitis and mortality in horses, humans and some species of both wild and domestic birds. Diagnosis of WNV infection can be confirmed by virus isolation in cell cultures, by identification of specific WNV sequences using reverse transcription polymerase chain reaction (RT-PCR) and indirectly using serological techniques such as virus neutralisation, PRNT or ELISA. SAFETY See current version of VLA Health & Safety Policy. The PRNT for WNV uses infectious virus preparations and can only be carried out in a Class1/3 microbiological safety cabinet (MSC) within a CL3 laboratory. The Rabies High Security Unit [RHSU], building 139 is an enhanced ACDP 3, SAPO 4 suite of laboratories). Staff entering the RHSU must be vaccinated against Rabies with a confirmed antibody titre of > 0.5 international units/ml. All personnel working with WNV must also be vaccinated against Tick borne
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1.1.2

1.2 1.2.1

1.2.2.

2. 2.1. 2.2.

2.3.

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Veterinary Laboratories Agency Standard Operating Procedure Plaque Reduction Neutralisation Test (PRNT) for West Nile Virus

Encephalitis and Japanese Encephalitis with a confirmed protective titre. 2.4 All virus dilution and dispensing procedures must be carried out using plastic tips on approved calibrated pipettes. See risk assessment VIR.RA.018 Sharps and needlestick injuries (Prevention and Response) Staff working with WNV must have read BAAC risk assessments (in particular BAAC 2007/02) and SOPs relevant to working in the RHSU.

2.5

3. 3.1 3.1.1. 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6

MATERIALS Documentation and software Registered log book - West Nile virus serological submissions: PRNT (VIR 1045) Test worksheet FORM VIR 373 Control results sheet FORM VIR 374 Sample results sheets FORM VIR 375 (screen) and/or FORM VIR 376 (titration) Registered PRNT report template (VIR381) Other SOPs referred to: VI.419 VIR0198 Rabies HSU safety cabinet Biomat class I Rabies HSU safety cabinet Envair class I/III Handling zoonotic arboviruses including

3.1.7

Risk Assessments referred to: BAAC 2007/02 experimental infection of mice VIR.RA.018 Sharps and Needlestick injuries (Prevention and Response)

3.1.8 3.1.9

Local File Reference: VIR619 Test Reliability Risk Analysis UM0026 (on Livelink)

3.2 3.2.1 3.2.2

Chemicals and reagents Standard WNV positive reference serum. Standard negative reference serum.

SOP VI.556 Edition 2


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Veterinary Laboratories Agency Standard Operating Procedure Plaque Reduction Neutralisation Test (PRNT) for West Nile Virus

3.2.3 3.2.4 3.2.5 3.3 3.3.1

Carboxymethylcellulose (CMC). 10% Neutral buffered formalin (NBF). 2.3% Crystal violet. Media Eagles minimal essential medium (Earles salts) with antibiotics and antimycotic but without serum (EMEM-WOS). Ordered via Cell and Tissue Culture Unit (CTCU), VLA.

3.3.2

Double strength Eagles minimal essential medium (Earles salts) with antibiotics and antimycotic but without serum (2x EMEM-WOS). CTCU do not supply this reagent refer to Appendix 2 for preparation.

3.4 3.4.1

Animals/Micro-organisms/Cells Vero C1008 cell monolayers in 12 well microplates at 80 95% confluency. Ordered via CTCU, VLA.

3.4.2

Viral isolate WNV Goose Israel 1998 is used at a dilution which produces 50 70 plaques /125l in Vero C1008 cell monolayers in 12 well microplates. Equipment Single channel pipettes to deliver a range of volumes between 25l and 1000l. 12 well multichannel pipette to deliver a range of volumes up to 250l. Sterile tips for all pipettes. Sterile reagent troughs/Petri dishes. 37oC (+/- 2oC) CO2 incubator using 5% (+ or 0.5%). 56oC (+/- 2oC) water bath. Sterile, 96-well microplates, with lids. A range of sterile tissue culture grade plastic tubes, 10ml pipettes, sterile reagent bottles. Pipette pump for 10ml disposable pipette.
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3.5 3.5.1 3.5.2 3.5.3 3.5.4 3.5.5 3.5.6 3.5.7 3.5.8 3.5.9

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Veterinary Laboratories Agency Standard Operating Procedure Plaque Reduction Neutralisation Test (PRNT) for West Nile Virus

4. 4.1 4.1.1 4.1.2

PROCEDURE/METHOD Test Reliability The following procedures are in place in order to assure ongoing test reliability: Method: an External Quality Assurance (EQA) will be set up with QA Sutton Bonington. Also, participation in ring trials is in the process of being set up. Local File Reference: local file VIR619 is maintained within the Virology Department. Test Reliability Risk Assessment: UoM document UM0026 has been completed, and is held on Livelink. Test validation document reference: this test has been validated and allocated test code TC0739. Test preparation Order Vero cell cultures in 12 well microplates and EMEM WOS from VLA, CTCU. Record submitted samples in registered logbook - West Nile virus serological submissions: PRNT (VIR 1045). Also enter submission details into the West Nile Serology submissions database. Samples are allocated a VLA reference number, with the pre-fix WNS (West Nile Serology): for example, WNS08/01 for the first submission in 2008.

4.1.3 4.1.4 4.1.5

4.2 4.2.1 4.2.2

4.2.3 4.2.4 4.2.5 4.2.6 4.2.7

Serum samples should be heat inactivated at ~56oC for 30 minutes in a waterbath before test. Prepare 3% CMC (as detailed in Appendix 1). Prepare 2 x EMEM (as detailed in Appendix 2). Follow current local procedures for entry into, and exit out of, the rabies high security unit. Mix and equilibrate equal volumes of 3% CMC gel and 2 x EMEM under sterile conditions and place in a 5% CO2 incubator immediately prior to test. Prepare MSC for use at class I (refer to SOPs VIR198 or VI.419 for safety cabinet class I use). Test Procedure
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4.2.8

4.3

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Veterinary Laboratories Agency Standard Operating Procedure Plaque Reduction Neutralisation Test (PRNT) for West Nile Virus

4.3.1 4.3.2 4.3.3

All test details are recorded on registered worksheet FORM VIR 373. Check cells are (80-95%) confluent and fit for purpose. Prepare doubling dilutions of each test serum sample in EMEM: For small numbers of samples, perform a full titration (eight serum dilutions, between 1/10 and 1/1280). For large batches of samples, a screening test may be initially performed (1/10 and 1/20 serum dilutions only), with any suspect positives being retested with a full titration

4.3.4

For screening test prepare 1/10 and 1/20 serum dilutions. For positive and negative reference sera (and any samples requiring a full titration) prepare eight serum dilutions between 1/10 and 1/1280 as follows: Add 225l EMEM WOS to the first well and 125l EMEM WOS to each subsequent well of dilution range. Add 25l serum sample to the 225l EMEM WOS in the first well and mix well (=1/10 dilution). Transfer 125l of 1/10 serum dilution to the second well of the dilution range, mix well (=1/20 dilution). Repeat for subsequent dilutions. Discard the final 125l at the end of the titration.

4.3.5 4.3.6 4.3.7

Dilute virus to the appropriate concentration in EMEM WOS to contain 50 70 plaque forming units / 125ul. Add 125l of the diluted virus to all wells containing serum dilutions. Add plate covers, tap gently to mix contents of wells, spray the outside of the plates with 1% Virkon, remove from MSC and incubate for 60 70 minutes at ~37oC in a Flavivirus-dedicated 5% CO2 incubator. Ensure that both EMEM and diluted virus are also sprayed out of MSC and incubated in the same way.

4.3.8

Remove overlying medium from the Vero cell plates (tip into liquid waste container), gently tap plate on tissue paper (open side down) to remove excess medium. Starting with the highest dilution, transfer 250l of the serum/virus mixture onto the corresponding well of the Vero cell plates changing tips between each sample.

4.3.9

Add 250l of EMEM WOS to the negative control well. To all of the three remaining wells add 125l of diluted virus together with 125l EMEM WOS (these are triplicate virus control wells).

4.3.10

Replace 12 well plate cover, spray outside of plates with 1% Virkon, remove from MSC and incubate for 60 70 minutes at ~37oC in 5% CO2
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Veterinary Laboratories Agency Standard Operating Procedure Plaque Reduction Neutralisation Test (PRNT) for West Nile Virus

incubator. 4.3.11 4.3.12 4.3.13 4.3.14 4.3.15 4.3.16 4.3.17 Following incubation, add 1.5ml 3% CMC/2x EMEM WOS gel to each well using 10ml pipette and Pipette pump. Replace culture plate lid, spray outside of plate with 1% Virkon, remove from MSC and incubate for 4 days at ~37oC in 5% CO2 incubator. After incubation add 1ml of 10% NBF to each well and fix for 3 hours. Wash out gel overlay from fixed plates under cold tap water and tap dry on tissue paper (open side down) to remove excess water. Add 200l 2.3% crystal violet to each well and allow to stain for 5 minutes. Wash out crystal violet from wells under cold tap water and tap dry on tissue paper (open side down) to remove excess. Count plaques in wells over a lightbox or over a white background. Record the numbers of plaques in each well using registered results sheets FORM VIR 374 (controls), FORM VIR 375 (screen), FORM VIR 376 (titration). RESULTS Serum samples which produce a 90% reduction in the numbers of WNV plaques in the PRNT are deemed to be positive. Samples that show a 70% reduction in the number of plaques are deemed to be borderline. A titration PRNT should be carried out on serum samples that produce a positive or borderline level of virus neutralisation in the PRNT screen. A positive result titre is expressed as the serum dilution in which 90% plaque reduction is observed, in comparison to the mean of the three virus control wells. The test is valid if the control criteria are met: Negative control sera: no reduction in plaque count. Positive control sera: 90% reduction occurs between serum dilutions of 1/80 to 1/160. Virus control wells: between 50 70 plaques observed. 5.4 A report for the customer is prepared using registered form template VIR381 (PRNT report form). REFERENCES
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5. 5.1

5.2

5.3

6.

SOP VI.556 Edition 2


SOP LL template version 6: (issued 16 Aug 07)

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Date Printed : 18/October/2005

Veterinary Laboratories Agency Standard Operating Procedure Plaque Reduction Neutralisation Test (PRNT) for West Nile Virus

6.1.

Komar, N., Panella, N.A., Burns, J.E., Dusza, S.W., Mascarenhas, T.M. and Talbot, T.O. Serological evidence for the West Nile Virus infection in birds in the New York city vicinity during and outbreak in 1999. Emerging Infectious Diseases, 2001, 7,621-625. Davidson, A.H., Traub-Dargatz, J.L., Rodeheaver, R.M., Ostlund, E.N., Pederson, D.D., Moorhead, R.G., Stricklin, J.B., Dewell, R.D., Roach, S.D., Long, R.E., Albers, S.J., Callan, R.J. and Salman, M.D. Immunologic response to West Nile virus in vaccinated and clinically affected horses. J. Am. Vet. Med. Assoc. 2005, 226, 240-245. Weingartl, H.M., Drebot, M.A., Hubalek, Z., Halouzka, J., Andonova, M., Dibernardo, A., Cottam-Birt, C., Larence, J. and Marszal, P. Comparison of assays for the detection of West Nile virus antibodies in chicken sera. The Canadian J. Vet. Res., 2003, 67, 128-132.

6.2.

6.3

Appendix 1 Preparation of 3% Carboxymethylcellulose (CMC) gel Add 3g CMC per 100ml gel required to 10ml ethanol. Mix well to suspend CMC granules and add to 100ml sterile distilled de-ionised water in a sterile screw capped bottle whilst vigorously swirling. Heat for 8 minutes at medium power in microwave and leave to stand for 5 minutes to drive off all ethanol before replacing screw cap on bottle.

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Veterinary Laboratories Agency Standard Operating Procedure Plaque Reduction Neutralisation Test (PRNT) for West Nile Virus

Appendix 2

Preparation of double strength EMEM WOS

100ml 10X Eagles minimal essential medium (Earle`s salts) 766ml sterile de-ionised water 20ml L-Glutamine (200mM) 20ml Penicillin/Streptomycin/Fungizone 40ml HEPES (1M) 54ml Sodium bicarbonate (7.5%) Make up above ingredients in a sterile 1 litre bottle within a laminar flow cabinet, mix well and store at 4oC (+/ -1oC) until use.

SOP VI.556 Edition 2


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Veterinary Laboratories Agency


Standard Operating Procedure Approval Certification
This is a representation of an electronic record that was signed electronically in Livelink and this page is the manifestation of the electronic signature(s).

UserName: Sarah Eagle (m145684) Title: User Date: Monday, 12 October 2009, 11:50 GMT Daylight Time Meaning: I approve this SOP ================================================

Date Printed : 18/October/2005

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