STANDARD OPERATING Detection of antibodies against vesicular and

PROCEDURE:
related viruses by the virus neutralisation test
(VNT)

1. MATERIALS
1.1. Equipment

Bench centrifuge Any make

Bottles Sterile, plastic bijoux, universals, bottles or similar

Class 2 microbiological safety cabinet Any make

Cryotubes Approved make

Any make kept between -30°C and -5°C and

Freezers
-50°C and -80°C.

Incubator Any make kept at +35°C to +39°C.

Microplates Nunc TC microwell, 96 well

Micro-plate sealers Any make

Microscope Any make, objectives as appropriate

Single and multiple channel Finnpipettes (or

Pipettes
equivalent), Finnpipette multistepper and
Eppendorf multipette.

Finntips or similar, some with aerosol filter plug

Pipette Tips
and Eppendorf combitips

Reagent reservoirs Reusable or disposable, any make

Refrigerator Any make, kept between +1°C and +8°C

Any make, for heat inactivation - kept between

Water bath
54°C and 60°C

1.2. Reagents and consumables

Cells IB-RS-2, at an approximate concentration of 1 X106 per ml

Diluent Eagle`s MEM + HEPES (Sigma M-7278 or similar), add 2mls 1.0M
NaOH and 1ml antibiotics

Disinfectant solutions FAM (Evans) diluted 1/240 in water for FMDV and VSV and diluted
1/100 for SVD, made daily – or see current Disease Security
regulations

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 Field antibiotics Amphotericin B, Penicillin 10MU, Neomycin 25,000µg/ml, Polymixin
B 100,000U/ml, Sterile Water

Glycerol Any make, dispensed into aliquots and autoclaved

Isolates of the viruses Foot-and-Mouth Disease Virus (FMDV) Swine Vesicular Disease
Virus (SVDV), Vesicular Stomatitis Virus (VSV)

1M NaOH solution Any make

Reference Sera (RS) Convalescent or vaccinal serum (homologous or heterologous)

against standard virus isolates can be used

NOTE: A new control is tested and validated before it is introduced into routine diagnostic testing. The
control will be accepted only if the test passes the criteria as described in section 3.1.

NOTE: Future known positive VSV samples will be tested separately from diagnostic samples, to avoid
contamination with positive samples.

2. PROCEDURE

Preparing Sera

2.1. Dilute the serum samples received. This is usually 1/4 making the first dilution on the plate 1/8. If
only small amounts of serum are available a first well dilution of 1/16 is acceptable. If higher
dilutions are used it will not be possible to ascertain negative results.

Testing new controls

2.2. A new virus control is titrated to obtain a titre and then tested with potential reference serum
controls to obtain serum titres. It is not necessary for the serum control to be homologous to the
virus control as long as it gives an acceptable titre i.e. ≥ log101.8. Once a reference serum control
has been selected three sets of test plates should be set up, (2 Virus Titration plates and 2
Reference Serum plates per set) i.e. if a virus had a titre of 4.5 one set would be set up at 4.5 and
a further two sets, either side of this titre 4.4 and 4.6, 12 plates in total (see appendix 1b and 1c).

Virus Neutralisation Test (VNT) Procedure

NOTE: Appendix 1a highlights the different plate layouts that can be used:
Layout A - Full plate titration for positive or unknown samples
Layout B – Spot test to screen sera
Other layouts are acceptable.

NOTE: If the virus titre is known this assay can be performed as a single dose VNT. In this case the
reference serum plates are still run using three virus doses; however, the test serum is only run against one
virus dose.

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2.3. Mark plates for including virus, test date and operators’ initial (see Appendix 1): Colour code the
test plates and bottles of diluent. Three groups of plates need to be set-up:
2.3.1. Test Plates – Appendix 1a
2.3.2. Virus Titration – Appendix 1b; two plates are required; however, controls only need to be
added to the first plate
2.3.3. Reference Serum – Appendix 1c; two plates are required
2.4. Dispensing Diluent:
2.4.1. Reference Serum Plates - 50μl of diluent into every well
2.4.2. Virus titration Plate - 50μl of diluent to all wells in columns 1-9
2.4.3. In the first virus titration plate that contains the controls put 150μl into A10-12 and 100μl into
C10-12. On the same plate put 150μl serum free media (if using) in B10-12 and 100μl into
D10-12. A10-12 and B10-12 are diluent controls. C10-12 and D10-12 are for cell controls.
2.4.4. Test plates – for plate layout A add 50μl to rows B-H. For plate layout B add 25μl to rows B,
D, F and H.
NOTE: Serum free media can be used if and when available.
2.5. On the two reference serum plates add 50μl of reference serum to every well in row H. Mix the
contents of each well in row H and transfer 50µls from each well into row G. Mix as above then
transfer 50µl into row F. Continue this procedure and discard 50µl from row A.
NOTE: Reference serum should be appropriately pre-diluted, inactivated and stored frozen, such
that upon reading, the 50% endpoint occurs about halfway up the plate when 100 TCID50 are used.
2.6. On the test plates add the pre-diluted test serum (2.1). For plate layout A add 50μl of sera to rows
A and B. Mix the contents of each well in row B and transfer 50μl from each well into row C. Mix as
above and then transfer into row D. Continue all the way down the plate and discard 50μl from the
last row. For plate layout B add 50μl of the pre-diluted test sera to rows A, C, E and G and 25μl of
sera to rows B, D, F and H.
2.7. Check the expected virus titre. If, for example, if the virus titre is log104.8 (10-4.8) subtract log102.0
(100 TCID virus dose) to give the dilution (log102.8). Together with a fourfold (log100.6) step on
either side, this will result in the three virus challenge dilutions: log102.2, log102.8 and log103.4.
2.8. Calculate the volume required for each virus dilution. All 4 control plates need approximately 4mls.
Test plates require approximately 5mls each.
2.9. Dilute the glycerinated virus by making a 10-1 step, unless the antigen is of such low titre that
different volumes must be used. To ascertain other dilutions, use the “Dilution Factor Conversion
Table” (see 10.2 -Appendix 2) and dispense the correct volumes of diluent into bottles.
2.10. The second and third virus dilutions (log102.8 and log103.4) are made by making two further
fourfold steps. After the third virus dilution, make six fourfold steps to dilute the virus past its 50%
endpoint.
2.11. Dispense 50μls of the highest virus dilution into every well of column 9 of both virus titration plates.
Similarly, add 50μls of the next highest dilution to column 8 and so on until the third dilution, which
should be dispensed into column 3. Also add this virus dilution to section A-H 9-12 of both
reference serum plates then to any test sera as appropriate Likewise, add the second virus dilution

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 to column 2 of the virus titration plate and section A-H 5-8 in the reference serum plates and to any
test sera. Finish by adding the first virus dilution to column 1 in both virus titration plates, then
section A-H 1-4 of both reference serum plates and to the test sera if applicable, and then cover
with a plastic lid.
2.12. Incubate plates for 1 hour +/-30 minutes, at room temperature.
2.13. Dispense 50μl of cell suspension to all wells used in the test, except for the A and B10-12 (if using
serum free) on the virus titration plate. Agitate the cell suspension occasionally to prevent
sedimentation.
2.14. Cover the plates with micro-plate sealers, wipe with disinfectant and place in the incubator.
2.15. Read all plates in the following days microscopically.
2.16. For the virus control plates, wells showing any cytopathic effect are scored positive. Take final
readings at any time when all of the acceptance criteria below are met. Ideally the virus titre should
have achieved a steady maximum value, preferably after the minimum period of incubation of
about 48 hours. However, readings taken at day 1 are acceptable if the control values are valid.
SVD test plates should always be checked at day 1. Record the control values.
2.16.1. Virus Titration plates: add the number of wells which show CPE for both virus control plates
together and enter into the “Sum” box for each dilution. For higher virus dilutions (lower
concentration), add all totals less than 16 together to give as many full columns as possible, and
accumulate the remainder in the next box along.
For example:

Log virus dilutions 2.2 2.8 3.4 4.0 4.6 5.2 5.8 6.4 7.0
CPE+ wells (sum of 2 plates) 16 16 16 16 14 8 5 0 0
Totals for calculation 16 16 16 16 16 11 0 0 0

2.16.2. Refer to the “calculation of virus control titres when diluted fourfold” (see 10.3- Appendix 3) to find
the increment to be added to the last dilution in which all 16 wells showed CPE. In the case above
this would be the log104.6 dilution.
2.16.3. For the example above, add 0.71 to the virus dilution (4.6) at which all 16 wells showed CPE =
5.31 TCID50.
2.16.4. To obtain the virus doses used for any given day’s results, subtract the lowest virus dilution (2.2
from the virus titre (5.31) and record this figure (3.11) against ‘Read 1 virus dose’. Repeat for the
remaining two virus dilutions and record as appropriate. Thus, only the highest virus dilution giving
a virus dose of 1.91 has given an acceptable virus dose (see 3.0).
2.17. For the reference serum control plates add the number of wells showing no CPE for both plates
together enter into the ‘Sum’ box for each dilution (maximum in full column = 8/8 -v). For highest
serum dilutions (lower concentration), add all totals less than 8 to give as many full columns as
possible, and accumulate the remainder in the next box along.

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 For example:
Log reference serum dilutions 1.8 2.1 2.4 2.7 3.0 3.3 3.6 3.9
No CPE wells (sum of 2 plates) 8 8 8 8 6 3 1 0
Totals for calculation 8 8 8 8 8 2 0 0

2.17.1. Refer to the “calculation of reference serum control tires when diluted twofold” (see 10.4 -
Appendix 4) to find the increment to be added to the last dilution in which all 16 wells showed no
CPE. In the case above this would be the log103.0 dilution
2.17.2. For the example above, add 0.23 to the reference serum dilution (3.0) at which the last full column
where no CPE was observed (i.e. protected) = 3.23. Carry out this procedure for each virus
dilution. Using the 3 virus doses and control serum titres calculate the serum titre at the log 102
dose of virus and record.
2.18. Record the results. A “-“sign denotes a well not showing any CPE. Those showing any CPE are
denoted by a “+” sign. Calculate each serum titre with reference to the table in Appendix 5 (see
10.5) and select the most appropriate set of readings with reference to the acceptance criteria
below. If toxicity is observed for any test serum, record “T” in the boxes affected; “C” indicates
contamination and “U” any test serum well which is unreadable.
2.19. Plates containing re-bleed samples are double checked by another competent operator. Record
control data.
2.20. Without removing their seals, discard used VNT plates directly into double autoclave bags in an
infectious tin.

3. RESULTS
3.1. Acceptance criteria

Tests are considered valid provided that:

a) The virus dose delivered (final titre minus Challenge Dilution) is within the range 1.5 to 2.5
TCID50.
b) The reference serum titre is within log10 0.3 of the running mean, at an acceptable virus dose (see
above).
c) The cell sheet in the cell controls is intact and not showing CPE-like effects.

Results for individual test sera are considered acceptable provided that:
a) The virus dose delivered (see above) is within the range 1.5 to 2.5 TCID50.
b) Contamination or toxicity does not prevent the test sera being accurately read.

3.2. Interpretation of results

3.2.1. Assuming the criteria for acceptance of results are met, test sera are considered negative if they
have a titre of 1/11 or less. If, due to a small volume of test sera, the starting dilution is 1/16 rather
than 1/8, then <16 is given as a negative result. Sera may also be passed as negative with such
titres if the virus dose is too low, and/or if the reference serum titre is too high. This is based on the
conditions having been biased in favour of neutralisation occurring. Sera with titres greater than

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 1/11 but less than or equal to 1/32 are considered doubtful and recorded as “inconclusive”
(import/export animals must be re-bled and retested or a veterinary inspection must be carried
out). Serum titres greater than or equal to 1/45 are considered positive.

4. TROUBLESHOOTING
4.1. Repeat the test if the cells in the cell controls are of such poor quality that they cannot readily
distinguished from those where CPE is present. If cells are suspected to be causing test failures,
then a different revival or passage should be requested. It may be necessary to request a re-bleed
if test sera prove to be extremely toxic or show CPE-like effects. Latter effects can result from
incorrect inactivation. If one of either the virus control or reference serum control plates is
unreadable it is possible to calculate control titres with one plate using the Kärber formula:

Kärber Calculation:

Virus Titre = L + d(S – 0.5) Serum Titre = L + d(S – 0.5)

L = 1st dilution on the plate L = 1st dilution on the plate
D = difference between log dilution steps (0.6) D = difference between log dilution steps (0.3)
S = sum of wells showing CPE / total no. of wells S = sum of wells showing no CPE / total no. of wells
per dilution per dilution
0.5 = constant 0.5 = constant

5. APPENDIX/ APPENDICES
5.1. Appendix 1 – Plate Layouts

Appendix 1a – Plate layout for test samples

Layout A – full plate titration for positive or unknown samples

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Layout B - spot test to screen sera

Appendix 1b – Virus Titrations

Appendix 1c – Reference Serum

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5.2. Appendix 2 – Dilution Factor Conversion table (logarithmic to arithmetic)

p = mLs of previous dilution

x = mLs of diluent needed to make the next dilution

LOG ARITH- LOG ARITH-
STEP METIC STEP METIC
p x p x
0.3 1+1 1.2 1 + 15
0.4 1 + 1.5 1.3 1 + 19
0.5 1 + 2.2 1.4 1 + 24
0.6 1+3 1.5 1 + 31
0.7 1+4 1.6 1 + 39
0.8 1 + 5.3 1.7 1 + 49
0.9 1+7 1.8 1 + 62
1.0 1+9 1.9 1 + 79
1.1 1 + 11.5 2.0 1 + 99

10.3 Appendix 3 – Calculation of virus control titres when diluted fourfold (log100.6)

WELLS ADD TO LAST WELLS ADD TO LAST

WITH CPE FULL COLUMN WITH CPE FULL COLUMN

0/16 0.30 8/16 0.60

1/16 0.34 9/16 0.64
2/16 0.38 10/16 0.68
3/16 0.41 11/16 0.71
4/16 0.45 12/16 0.75
5/16 0.49 13/16 0.79
6/16 0.53 14/16 0.83
7/16 0.56 15/16 0.86

10.4 Appendix 4 – Calculation of reference serum control titres when diluted twofold (log100.3)
WELLS ADD TO LAST WELLS ADD TO LAST
PROTECTED FULL COLUMN PROTECTED FULL COLUMN
0/8 0.15 5/8 0.34
1/8 0.19 6/8 0.38
2/8 0.23 7/8 0.41
3/8 0.26 8/8 0.45
4/8 0.30

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10.5 Appendix 5 – Titres for sera tested in duplicate, based on numbers of protected well

- = no CPE (neutralisation)

+ = CPE (no neutralisation)

8 16 32 64 128 256 512 1024 = reciprocal dilutions

- + + + + + + + 1/8 (arith) 0.9 (log10)

+ + + + + + + +

- + + + + + + + 1/11 1.05
- + + + + + + +

- - + + + + + + 1/16 1.2
- + + + + + + +

- - + + + + + + 1/22 1.35
- - + + + + + +

- - - + + + + + 1/32 1.5
- - + + + + + +

- - - + + + + + 1/45 1.65
- - - + + + + +

- - - - + + + + 1/64 1.8
- - - + + + + +

- - - - + + + + 1/90 1.95
- - - - + + + +

- - - - - + + + 1/128 2.1
- - - - + + + +

- - - - - + + + 1/178 2.25
- - - - - + + +

- - - - - - + + 1/256 2.4
- - - - - + + +

- - - - - - + + 1/355 2.55
- - - - - - + +

- - - - - - - + 1/512 2.7
- - - - - - + +

- - - - - - - + 1/708 2.85
- - - - - - - +

- - - - - - - - 1/1024 3.0
- - - - - - - +

- - - - - - - - 1/1413 3.15
- - - - - - - -

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