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SAM 116.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 116

Supplemental Assay Method for the Phenotypic Examination of Pseudorabies 

Virus for Thymidine Kinase Activity by a Plaque Selection Method

Date: October 16, 2014

Number: SAM 116.05

Supersedes: SAM 116.04, February 9, 2011

Standard Requirement: 9 CFR 113.318

Contact: Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 08Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 16Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 16Dec14 

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Supplemental Assay Method for the Phenotypic Examination of Pseudorabies Virus for Thymidine Kinase 

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the Test Vaccine

4. Performance of the Assay

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions
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Supplemental Assay Method for the Phenotypic Examination of Pseudorabies Virus for Thymidine Kinase 

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro assay method which uses a 

selective media in a cell culture system to detect the presence or absence of extraneous 

thymidine kinase (TK)-positive pseudorabies virus (PRV) in a thymidine kinase-negative (TK-), 

modified-live PRV vaccine.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Water bath

2.1.2 Incubator, 36°± 2°C, high humidity, 5 ± 1% CO2

2.1.3 Microscope, inverted light

2.1.4 Microscope, ultraviolet (UV) light 

2.1.5 Freezer, ultra-low

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

reagents and supplies must be sterile.

2.2.1 Connective tissue, mouse, TK mutant (L-M[TK-]) cells

2.2.2 Madin-Darby bovine kidney (MDBK) cells or other susceptible cells

2.2.3 PRV Reference, Shope strain

2.2.4 Minimum essential medium (MEM) (National Centers for Animal 

Health (NCAH) Media #20030)

1. 9.61 g MEM with Earles salts without bicarbonate

2. 1.1 g sodium bicarbonate (NaHCO3)
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3. Dissolve with 900 mL deionized water (DI).

4. Add 5.0 g lactalbumin hydrolysate or edamine to 10 mL DI. Heat to 

60°± 2°C until dissolved. Add to Step 3 with constant mixing.

5. Q.S. to 1000 mL with DI; adjust pH to 6.8-6.9 with 1N hydrochloric 

acid (HCl).

6. Sterilize through a 0.22-μm filter.

7. Aseptically add:

a. 50 μg/mL gentamicin sulfate

b. 10 mL L-glutamine (200 mM)

8. Store at 2°- 7°C.

2.2.5 Growth Medium

1. 900 mL of MEM

2. Aseptically add 100 mL gamma-irradiated fetal bovine serum (FBS)

3. Store at 2°- 7°C.

2.2.6 Hypoxanthine, aminopterin, thymine (HAT) Medium

1. 200 mL Growth Medium

2. 2 mL HAT media supplement (50X)

2.2.7 0.01 M Phosphate buffered saline (PBS) (NCAH Media #30054)

1. 1.19 g sodium phosphate, dibasic, anhydrous (Na2HPO4)

2. 0.22 g sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)

3. 8.5 g sodium chloride (NaCl)

4. Q.S. to 1000 mL with DI.

5. Adjust pH to 7.2-7.6 with 0.1 N sodium hydroxide (NaOH) or 1 N 

HCl.
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6. Sterilize by autoclaving at 15 psi, 121°± 2°C for 35 ± 5 minutes.

7. Store at 2°- 7°C.

2.2.8 80% acetone

1. 80 mL acetone

2. 20 mL DI

3. Store at room temperature.

2.2.9 Tissue culture flask, 25-cm

2

2.2.10 Pipette, 10-mL

2.2.11 Graduated cylinder, 25-mL, 50-mL, 100-mL, and 250-mL, sterile

2.2.12 Swine anti-PRV fluorescein isothiocyanate- labeled conjugate (PRV 

Conjugate)

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience in aseptic techniques and cell culture growth and 

maintenance.

3.2 Preparation of equipment/instrumentation

On the day of testing, set a water bath at 36°± 2°C.

3.3 Preparation of reagents/control procedures

3.3.1 A day prior to test initiation and a day prior to each of the 3 additional 

L-M(TK-) passages, seed 25-cm

2

flasks with L-M(TK-) cells, in Growth Medium, 

at a cell count that will produce a monolayer after 1 day of incubation. Cells used 

for seeding should be from monolayers that had been previously passaged every 3 

to 4 days. Three L-M(TK-) control flasks and 1 L-M(TK-) flask are required for 

each Test Vaccine. Incubate at 36°± 2°C in a CO2 incubator.
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3.3.2 A day before the last passage, seed 25-cm2flasks with MDBK cells, in 

Growth Medium, at a cell count that will produce a monolayer after 1 day of 

incubation. Three MDBK control flasks and 1 MDBK flask are required for each 

Test Vaccine. Incubate at 36°± 2°C in a CO2 incubator.

3.3.3 PRV Positive Control. On the day of test initiation, rapidly thaw a vial of 

PRV Reference in a 36°± 2°C water bath. Dilute the PRV Reference in MEM to 

contain 10450% tissue culture infective dose (TCID50)/100 μL.

3.3.4 Working PRV Conjugate. On the day of the fluorescent antibody (FA) 

Confirmatory Test, dilute the PRV Conjugate in PBS according to the Center for 

Veterinary Biologics (CVB) supplied Reagent Data Sheet.

3.4 Preparation of the Test Vaccine

On the day of test initiation, using a graduated cylinder, rehydrate a vial of the Test 

Vaccine with the supplied diluent. Incubate for 15 ± 5 minutes at room temperature.

4. Performance of the Assay

4.1 First passage on L-M(TK-) cells

4.1.1 On the day of test initiation, decant Growth Media from all the L-M(TK-) 

flasks except 1, which remains unopened as a cell control. Label the unopened 

flask “Cell Control.”

4.1.2 The initial test of a Test Vaccine will be with a single vial (a single sample 

from 1 vial). Inoculate 1.0 mL of the Test Vaccine into an L-M(TK-) flask labeled 

with the Test Vaccine identification.

4.1.3 One mL of PRV Positive Control is inoculated into an L-M(TK-) flask 

labeled “PRV Positive Control.”

4.1.4 One mL of HAT Medium is inoculated into an L-M(TK-) flask labeled 

“Medium Control.” 

4.1.5 Allow inocula to absorb by incubating flasks at 36°± 2°C for 60 ± 10 

minutes in a CO2 incubator.

4.1.6 After incubation, add 9.0 mL of HAT Medium to all flasks except the Cell 

Control (remains unopened). Incubate all flasks at 36°± 2°C for 4 days in a CO2

incubator.
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4.1.7 During incubation, periodically observe all flasks for bacterial or fungal 

contamination. It is not necessary for microscopic observation of the L-M(TK-) 

cells. However, on examination, the L-M(TK-) cells will be rounded due to the 

HAT Medium. The Cell Control flask should remain normal.

4.1.8 After incubation, all flasks are frozen at -70°± 5°C for at least 2 hours. 

Flasks may be held at -70°± 5°C until the next passage. The Cell Control flask is 

discarded, as a new Cell Control flask will be used with each passage.

4.1.9 Thaw all the remaining flasks, with frequent shaking, at room temperature

until completely thawed.

4.2 Repeat Steps 1 through 9 for a total of 4 passages, inoculating each flask with 

1.0 ml of the thawed cell and media suspension of the appropriate control or Test Vaccine

from the previous passage instead of the initial inocula.

4.3 Passage on MDBK cells

4.3.1 From the last thawed passage (4th passage) on L-M(TK-) cells, repeat 

Sections 4.1.1 through 4.1.3, except substitute MDBK cells for the L-M(TK-) 

cells and inoculate each flask with 1.0 mL of the 4th passage cell and media 

suspension of the appropriate control or Test Vaccine instead of the initial 

inocula.

4.3.2 Allow inocula to absorb by incubating at 36°± 2°C for 60 ± 10 minutes in 

a CO2 incubator. 

Note: Do not use HAT Medium.

4.3.3 After incubation, add 9.0 mL of Growth Medium to all flasks except the 

Cell Control (remains unopened). Incubate at 36°± 2°C for 4 days in a CO2

incubator.

4.3.4 Observe all flasks daily with the inverted light microscope for typical PRV 

CPE. The CPE of PRV is visible as grape-like clusters of rounded cells in the cell 

monolayer. The PRV Positive Control flask should show typical PRV CPE after 4 

days. Any Test Vaccine observed to have typical PRV CPE is considered a 

Suspect Test Vaccine.

4.4 Confirmation of PRV CPE in Suspect MDBK Test Vaccine flasks (FA 

Confirmatory Test). Conduct an FA Confirmatory Test using specific PRV conjugate on 

any Suspect Test Vaccine. The PRV Positive Control flask, the HAT Medium flask, and 

the Cell Control flask are similarly examined and used as controls. If CPE is not 

observed in a Test Vaccine, this procedure is not required.
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4.4.1 Decant Growth Medium from the MDBK flasks for the Suspect Test 

Vaccine(s), the PRV Positive Control, the HAT Medium, and the Cell Control 

(FA Test) flasks.

4.4.2 Rinse the FA Test flasks with PBS; decant the liquid.

4.4.3 Fill the FA Test flasks with 80% acetone and incubate at room 

temperature for 15 ± 5 minutes.

4.4.4 Decant the 80% acetone from the FA Test flasks and air dry at room 

temperature.

4.4.5 Pipette 2 mL of working PRV Conjugate into the FA Test Flasks and 

incubate for 45 ± 15 minutes at 36°± 2°C in a CO2 incubator.

4.4.6 Decant and add 20 mL of PBS to the FA Test flasks and rotate back and 

forth to rinse the cells.

4.4.7 Repeat Step 6 for a total of 5 washes.

4.4.8 Rinse the FA Test flasks in DI, decant, and allow to air dry or dry at 36°± 

2°C.

4.4.9 Examine the FA Test flasks with a UV microscope.

4.4.10 A flask is considered positive for PRV if typical, nuclear, apple-green 

fluorescence is observed in any cell.

4.4.11 For a valid FA test, the PRV Positive Control must show typical, applegreen fluorescent infected cells.

4.4.12 For a valid FA test, the HAT Medium flask and the Cell Control flask 

must not show fluorescence.

4.4.13 If either Section 4.4.11 or Section 4.4.12 criteria are not met, the test is 

considered a NO TEST and the entire test, starting with a new vial of the Test 

Vaccine, is repeated.
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5. Interpretation of the Test Results

5.1 For a valid test, the HAT Medium flask and MDBK Cell Control must remain 

free of CPE and all flasks must remain free of bacterial or fungal contamination.

5.2 The PRV Positive Control must show CPE after passage in the MDBK cells.

5.3 For a SATISFACTORY test, a Test Vaccine must not show CPE after passage in 

MDBK cells. 

5.4 Any Test Vaccine exhibiting typical PRV CPE in the initial test and found 

positive in a valid FA Confirmatory Test is retested in duplicate using 2 new vials of the 

Test Vaccine.

5.4.1 If either of the 2 retests of the Test Vaccine exhibits typical PRV CPE and 

is found positive in a valid FA Confirmatory Test, the Test Vaccine is 

UNSATISFACTORY.

5.4.2 If no typical PRV CPE is observed in either of the 2 retests, the Test 

Vaccine is SATISFACTORY.

5.5 Any Test Vaccine exhibiting typical PRV CPE in the initial test and found 

negative in a valid FA Confirmatory Test is considered INCONCLUSIVE for TKactivity. Repeat Section 4.3.1 and examine for possible viral contamination. 

6. Report of Test Results

Record all test results on the test record.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.318, U.S. Government Printing 

Office, Washington, DC.

7.2 Kit, S and Qavi, H. Thymidine kinase (TK) induction after infection of TKdeficient rabbit cell mutants with bovine herpesvirus type I (BHV-1): isolation of TKBHV-1 mutants. Virol 1983; 130(2):381-389.
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8. Summary of Revisions

Version .05

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .04

 The Contact information has been updated.

Version .03

 The Contact has been changed from Kenneth Eernisse to Joseph Hermann.

 2.1: The pipettor has been removed from the list of equipment/instrumentation used 

for this test.

Version .02

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document:

 2.2.4.2 The amount of sodium bicarbonate (NaHCO3) has been changed from 2.2 g 

to 1.1 g.

 2.2.4.7 L-glutamine has been added. Penicillin and streptomycin have been removed.

 2.2.5.2 L-glutamine has been removed.

 2.2.11 “Syringe and needle” has been changed to “graduate cylinders”

 “Reference and Reagent Sheet” has been changed to “Reagent Data Sheet” 

throughout the document.

 “Test Serial” has been changed to “Test Vaccine” throughout the document.

 The refrigeration temperatures have been changed from 4° ± 2°C to 2°- 7°C. This 

reflects the parameters established and monitored by the Rees system.