date of issue: July 31, 2020

Test report
Inactivation of classical swine fever virus by ALTANT

Graduate School of Veterinary Medicine, Hokkaido University Microbiology classroom
Yoshihiro Sakoda[E-Tech Co., Ltd. translated this report from "Japanese version" to "English version"]1

[Purpose]
Evaluate the inactivating effect of “ALTANT” on classical swine fever virus.
[Materials and methods]
1. Virus
Classical swine fever virus GPE-strain (infection value 107.5TCID50/ml)
2. examined goods“ALTANT”
3. Infectious titer measurement cells
Pig kidney-derived cell line (SK-L cell)
4. Protein detection reagent for infectious titer measurement
Nano Glo HiBiT Lytic Detection System (Promega)
5. Virus inactivation test
900μl of the test product was placed in a test tube, 100μl of GPE-strain diluted 5-foldwith sterile distilled water (DW) was added thereto, and the mixture was mixed with vortex
and reacted at 25℃.
After 15 seconds, 1 minute, and 5 minutes, 100μl of each was collected, and 0.3 sodium
thiosulfate and 400μl of horse serum were added as a stop solution to prepare a sample for
infectious titer measurement.
In addition, sterile DW was used instead of the test product as a control.
The infectious titer measurement sample and the control sample were each diluted 10-foldwith MEM (including 10% horse serum), and 50μl of the 10-fold serial dilution was added
to 4 holes of the 96-well plate, and the cells derived from porcine kidney (strained cells derivedfrom pig kidney) SK-L cells) were simultaneously injected in 100μl.
After 4 days, the reaction was carried out with the Nano Glo HiBiT Lytic Detection System(Promega), the luciferase activity was measured, and then the virus infectivity titer (TCID 50/ml) was calculated.
6. Confirmation after stop
90μl of the test product or sterile DW was placed in a test tube, and 10μl of 0.3% sodium
thiosulfate and 400μl of horse serum were added as a stop solution and mixed with a vortex.
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Add 10μl of GPE-strain 5 times diluted with sterile DW to the reaction solution after
shutdown, dilute each 10 times with MEM (including 10% horse serum), and add 50μl of 10times diluted solution to 4 holes of 96-well plate , 100μl of porcine kidney-derived cell (SKL cells) was simultaneously injected therein.
After 4 days, the reaction was carried out with the Nano Glo HiBiT Lytic Detection System(Promega), the luciferase activity was measured, and then the virus infectivity titer (TCID 50/ml) was calculated.
[Result]
1. Virus inactivation test (Fig. 1, left and center)In the test product, the virus fell below the detection limit in 15 seconds.
The virus infection rate has been reduced to 1/1000 compared to DW.
2. Check the stop solution (Fig. 1, left and right)
As a result of adding the virus after the reaction with the stop solution, the results weresimilar to those when DW was used in the virus inactivation test.
Therefore, it was confirmed that the reaction of the test product was stopped by the stopsolution.
[Summary and consideration]“ALTANT” was found to instantly inactivate the classical swine fever virus.
When using pigs in the field, it is desirable to additionally investigate the effects of organicmatter.

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Fig. 1

Inactivation of classical swine fever virus GDE-/ Hibit strain by "ALTANT"(Log TCID50/mL)

Infection value

Mean of N = 2(Belowthe detectionlimit)
15sec.

1min.

5min.

DW：Virus fluid（9：1）

15sec.

1min.

5min.

ALTANT：Virus fluid（9：1）#A#B
Control#A: After mixing DW, sodium thiosulfate and serum, virus solution was added and the infectious titer was measured.
#B: After mixing altanto, sodium thiosulfate and serum, virus was added and the infectious titer was measured.
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