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SAM 104.04

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 104

Supplemental Assay Method for Isolation of Parainfluenza-3 Virus from 

Nasal Excretions

Date: October 10, 2014

Number: SAM 104.04

Supersedes: SAM 104.03, February 9, 2011

Standard Requirement: 9 CFR 113.309

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

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 13Nov14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 24Nov14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 25Nov14

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 Isolation of Parainfluenza-3 Virus from Nasal Excretions

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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 sample

4. Performance of the Test

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 Isolation of Parainfluenza-3 Virus from Nasal Excretions

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro assay method using Madin-Darby 

bovine kidney (MDBK) cells for the isolation of parainfluenza 3 virus (PI3V) from nasal 

excretions of calves. PI3V isolation is performed for immunogenicity studies on PI3 live virus 

vaccines in accordance with title 9, Code of Federal Regulations (9 CFR).

2. Materials

2.1 Equipment/instrumentation

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

below.

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

2.1.2 Water bath, 36o± 2oC

2.1.3 Pipettor, 200-μL and 1000-μL, and tips

2.1.4 Vortex mixer

2.1.5 Pipettor, 50- to 300-μL x 12-channel

2.1.6 Microscope, inverted light

2.1.7 Centrifuge and rotor

2.1.8 Pipette-aid

2.2 Reagent/supplies

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

reagents and supplies must be sterile.

2.2.1 PI3V Positive Control

2.2.2 MDBK cell line found to be free of extraneous agents as tested by the 

9 CFR
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2.2.3 Diluent Medium (National Centers for Animal Health (NCAH) Medium 

#20030)

1. 9.61 g minimum essential medium (MEM) with Earles salts without 

bicarbonate

2. 2.2 g sodium bicarbonate (NaHCO3)

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 and add to Step 3 with constant mixing.

5. Q.S. to 1000 mL with DI; adjust pH to 6.8-6.9 with 2N hydrochloric 

acid (HCl).

6. Sterilize through a 0.22-μm filter.

7. Aseptically add:

a. 10 mL L-glutamine

b. 50 μg/mL gentamicin sulfate

8. Store at 4°± 2°C. 

2.2.4 Sample Transport Medium

1. 200 mL Diluent Medium

2. 30 μg/mL gentamicin sulfate

3. 1.5 μg/mL amphotericin B

4. Store at 4°± 2°C.

2.2.5 Growth Medium

1. 900 mL of Diluent Medium

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

3. Store at 4°± 2°C.
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2.2.6 Maintenance Medium

1. 980 mL Diluent Medium

2. Aseptically add 20 mL gamma-irradiated FBS.

3. Store at 4°± 2°C.

2.2.7 Alsevers Solution

1. 20.5 g dextrose (C6H12O6) 

2. 8.0 g sodium citrate (Na3C6H5O7•2H2O)

3. 4.2 g sodium chloride (NaCl)

4. 0.55 g citric acid (C6H8O7•H2O)

5. Dissolve with 100 mL DI. 

6. Filter through a 0.22-μm filter.

7. Store at 4°± 2°C. 

2.2.8 10X Phosphate buffered saline (10X PBS)

1. 80.0 g sodium chloride

2. 2.0 g potassium chloride (KCl)

3. 2.0 g potassium phosphate, monobasic, anhydrous (KH2PO4)

4. Dissolve with 900 mL DI.

5. Add 11.5 g sodium phosphate dibasic, anhydrous (Na2HPO4) to 50 mL

DI, heat to 60°± 2°C until dissolved. Add to Step 4 with constant mixing.

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

7. Autoclave at 15 psi, 121°± 2°C for 35 ± 5 minutes. 

8. Store at 4°± 2°C. 
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2.2.9 1X PBS (PBS)

1. 100 mL 10X PBS

2. 900 mL DI

3. Adjust the pH to 7.0-7.3 with 5N sodium hydroxide (NaOH).

4. Store at 4°± 2°C. 

2.2.10 Guinea pig red blood cells (RBCs) in an equal volume of Alsevers 

Solution

2.2.11 7.5 % Sodium Bicarbonate

1. 7.5 g sodium bicarbonate

2. Q.S. to 100 mL DI.

3. Autoclave at 15 psi, 121°± 2°C for 30 ± 5 minutes.

4. Store at 4°± 2°C.

2.2.12 Trypsin Versene (TV)

1. 8.0 g sodium chloride 

2. 0.40 g potassium chloride 

3. 0.58 g sodium bicarbonate 

4. 0.50 g irradiated trypsin

5. 0.20 g versene or disodium salt ethylenediaminetetraacetic acid 

(EDTA)

6. 1.0 g dextrose

7. 0.4 mL 0.5% phenol red 

8. Dissolve with 1000 mL DI. 

9. Adjust pH to 7.3 with 7.5% sodium bicarbonate.
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10. Filter through a 0.22-μm filter.

11. Aseptically dispense into 100 mL amounts and store at -20°± 4°C. 

2.2.13 Tissue culture plates, 96-well

2.2.14 Polystyrene tubes, 12 x 75-mm and 17 x 100-mm

2.2.15 Conical tube, 50-mL

2.2.16 Serological pipettes, 10-mL

2.2.17 Cotton-tipped applicators

2.2.18 Reagent reservoir

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training in cell culture techniques, virus isolation, the principles of 

aseptic techniques, and animal care and handling techniques.

3.2 Preparation of equipment/instrumentation

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

3.3 Preparation of reagents/control procedures

3.3.1 Sample transport tube 

Prior to Nasal excretion sample collection, fill an appropriate number of 12 x 

75-mm polystyrene tubes with 3 mL of sample transport medium for each calf on 

test. A minimum of 1 tube per calf per day of anticipated collection is prepared.

3.3.2 Nasal excretion sample collections

Take nasal excretion samples from each calf by inserting a sterile cotton-tipped 

applicator several inches into each nasal passage. Immediately immerse the two 

applicators laden with nasal excretion material into a sample transport tube; 

labeled appropriately with calf identifier and date of collection. Freeze and store 

the sample transport tube at -70°± 5°C until virus isolation test is conducted.
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3.3.3 Preparation of MDBK cell culture plate (test plate) 

Cells are prepared from healthy, confluent MDBK cells that are maintained by 

passing weekly. Two days prior to test initiation and 2 days prior to the 2nd 

passage, seed 200 μL/well of 105.4 to 105.6 cells/mL, suspended in Growth 

Medium into all wells of a 96-well cell culture plate with a multichannel pipettor. 

At least 2 nasal excretion samples are tested on 1 plate. Cells are removed from 

the growth containers by using TV solution. The test plate is incubated at 36°± 

2°C in a CO2 incubator for 36 ± 12 hours. Cells should be 80% confluent prior to 

use. 

3.3.4 Preparation of PI3V Positive Control

1. On the day of test initiation, rapidly thaw a vial of PI3V Positive Control 

in a 36°± 2°C water bath and make tenfold dilutions.

a. Using a 10-mL serological pipette, dispense 4.5 mL of Dilution 

Medium in an appropriate number of 17 x 100-mm polystyrene 

tubes to bracket the expected titer endpoint according to the Center 

for Veterinary Biologics (CVB) Reference and Reagent sheet. 

Label tubes (for example: 7 tubes, labeled 10-1to 10-7

respectively).

b. Using a 500 μL pipettor, transfer 500 μL of PI3V Positive 

Control to the first tube labeled 10-1; mix by vortexing.

c. Using a new pipette tip, transfer 500 μL from the 10-1labeled 

tube (Step 1.b) to the 10-2tube; mix by vortexing.

d. Repeat Step 1.c for each of the subsequent dilutions, 

transferring 500 μL of the previous dilution to the next dilution 

tube, until the tenfold dilution series is completed.

3.3.5 Preparation of 0.5% RBCs Suspension for the hemadsorption (HAd) 

test

1. Transfer RBCs to a 50-mL conical tube.

2. Q.S. to 50 mL with Alsevers Solution; mix by inverting several times. 

3. Centrifuge at 400 x g for 15 ± 5 minutes (1500 rpm in a J6B centrifuge 

with a JS-4.0 rotor).
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4. Remove supernatant and buffy coat by aspirating with a 10-mL

serological pipette.

5. Repeat Steps 2 and 3 for a total of 3 washes, removing the supernatant 

each time.

6. Make a 0.5% RBC suspension by pipetting 500 μL of packed RBCs 

into 100 mL of PBS; mix by inverting.

7. Store at 4°± 2°C; use within a week of collection of the RBCs.

 

3.4 Preparation of the sample

3.4.1 On the day of the test initiation, thaw the sample transport tubes 

containing the nasal swabs, express the fluid from the cotton by rotating and 

pressing the applicators against the tube wall, and vortex the media in the tubes to 

release virus from the swab. Discard the used cotton applicators, after expressing 

any fluid.

3.4.2 Centrifuge the sample at 500 x g (2000 rpm in a J6B centrifuge with a JS4.0 rotor) for 20 ± 5 minutes. Transfer 2 mL of the supernatant from each sample 

into a new, labeled 12 x 75-mm polystyrene tube.

3.4.3 Keep the supernatant samples on ice until time of inoculation. 

3.4.4 Store the remaining supernatant sample at -70°± 5°C. 

4. Performance of the Test

4.1 Label the test plate and aseptically decant the Growth Medium into a suitable 

container.

4.2 Inoculate 5 wells with 25 μL/well of each supernatant sample. Change tips 

between each sample.

4.3 Inoculate 5 wells/dilution with 25 μL/well of the PI3V Positive Control (with 

dilutions 10-7through 10-4 from the example in Section 3.3.4(1)). Tip changes are not 

necessary if pipetting from the most dilute (10-7) to the most concentrated (10-4). 

4.4 Maintain 5 wells as uninoculated cell culture controls.
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4.5 Incubate the test plate at 36°± 2°C in a C02 incubator for 60 ± 10 minutes for 

virus adsorption.

4.6 Add 200 μL/well of Maintenance Medium to the test plate with a multichannel 

pipettor. 

4.7 Incubate the test plate at 36°± 2°C in a CO2 incubator for 4 ± 1 days. Observe 

daily for PI3V CPE which is characterized by cell fusion. 

4.8 Harvest samples from each well in which typical PI3V infection is observed into 

labeled 12 x 75-mm polystyrene tubes. Samples may be harvested at time of observation. 

Freeze at -70°± 5°C until next passage.

4.9 At the end of incubation, fluids from wells showing no CPE are passaged onto 

fresh test plates prepared according to Section 3.3.3, by repeating Sections 4.2 through

4.7. In addition, the harvested samples that displayed PI3V infection are thawed and 

passed. A PI3V Positive Control prepared as described in Sections 3.3.4 and 4.3,

respectively, is included. At the end of the 4 ± 1 day incubation, a HAd test is performed 

on all second passage wells to confirm PI3V infection.

4.10 HAd Test

4.10.1 Decant Maintenance Medium from the inoculated test plates (Section 4.9)

in a suitable container.

4.10.2 Rinse all cells of the test plates by filling with PBS; decant.

4.10.3 Dispense 200 μL/well of a 0.5% of the RBC Suspension into all test wells.

4.10.4 Incubate the test plates 15 ± 5 minutes at room temperature (23°± 2°C).

4.10.5 Decant the RBC Suspension from the test plates and repeat

Section 4.10.2 for a total of 2 washes. 

4.10.6 Examine the test plate monolayers on an inverted light microscope at 

100X magnification, and record the results. Wells containing one or more RBC 

clusters adhering to the cell monolayer are considered to be positive for PI3V. 

4.11 Calculate the PI3V endpoint of the PI3V Positive Control using the method of 

Spearman-Kärber as commonly modified. The titers are expressed as log10 50% tissue 

culture infective dose (TCID50) of the test wells.
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Example:

10-4dilution of PI3V Positive = 5/5 wells CPE/HAd positive

10-5dilution of PI3V Positive = 5/5 wells CPE/HAd positive

10-6 dilution of PI3V Positive = 2/5 wells CPE/HAd positive

10-7dilution of PI3V Positive = 0/5 wells CPE/HAd positive

Titer = (X - d/2 + [d * S]) where:

X = log10 of lowest dilution (4)

d = log10 of dilution factor (1)

S = sum of proportion of CPE/HAd positive

(5 + 5 + 2) = 12 = 2.4

 5 5

PI3V Positive Control titer = (4 - 1/2 + (1 * 2.4) = 5.9

Titer of the PI3V Positive Control is 105.9 TCID50/0.025 mL.

5. Interpretation of the Test Results

5.1 The uninoculated cell controls should not exhibit degradation or cloudy media 

that would indicate contamination.

5.2 The calculated HAd50 titer of the PI3V Positive Control must fall within plus or 

minus 2 standard deviations (± 2 SD) of its mean titer, as established from a minimum of 

10 previously determined titers.

5.3 Wells of nasal excretion samples that tested HAd positive (regardless if CPE 

developed) are considered positive for PI3V, whereas those wells of samples that did not 

develop CPE and tested HAd negative after the second serial passage in MDBK cells are 

considered to be negative for PI3V.

5.4 Any nasal excretion sample with 1 or more positive wells is considered to be 

positive for PI3V. A nasal excretion sample is only considered negative if all wells 

inoculated from the nasal excretion sample are negative for PI3V.

6. Report of Test Results

6.1 Results for each nasal excretion sample are reported as positive or negative PI3V 

isolation.
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7. References

7.1 Title 9, Code of Federal Regulations, part 113.309, U.S. Government Printing 

Office, Washington, DC.

7.2 Cottral, G.E., (Ed.). Manual of standardized methods for veterinary microbiology. 

Comstock Publishing Associates, Ithaca and London, 1978, pg.731.

7.3 Finney, D.J. Statistical method in biological assay. Griffin, London. 3rd ed., 

1978, pg. 508.

8. Summary of Revisions

Version .04

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

to keep the same next review date for the document.

Version .03

 The Contact information has been updated.

Version .02

This document was revised to clarify 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: 

 Contacts, Joseph Hermann and Peg Patterson, have been added to the document.

 1: Primary bovine embryonic kidney cell cultures have been replaced by MadinDarby bovine kidney-A cells.

 2.2.3: The description of the antibiotics added to the Diluent Medium has been 

changed to reflect the antibiotics used. Penicillin and streptomycin have been 

replaced by gentamicin sulfate, and Amphotericin B has been eliminated from the 

formula.

 2.2.4: Gentamicin sulfate has replaced penicillin and streptomycin for the transport 

medium.

 2.2.13: The 24-well cell culture plates have been replaced by 96-well plates.
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 The term “PI3V Reference” has been changed to “PI3V Positive Control” throughout 

the document.