Optimization of RHDV type 1 and 2 inactivation modes

The purpose of these studies was to optimize RHDV type 1 and 2 (RHDV1 and RHDV2) inactivation modes to use the obtained antigens in inactivated vaccines and diagnosticums. The inactivating effect of aminoethylethylenimine and β-propiolactone was studied in different concentrations in correlation with the exposure time and temperature. The correlation between the inactivating effect of the compound used and the accepted test conditions (concentration, temperature, and exposure time) was studied on a group of rabbits, each of which was injected intramuscularly with 1 cm3 of the inactivated material sample. At the end of the maximum exposure interval, a control sample of the viral material, kept under the same conditions without any inactivant added was similarly tested. Lethality was considered to evaluate the damaging action in the test and control groups: L = m/n, where m is the number of dead animals; n is the total number of rabbits in the group for testing of the inactivated material sample. The postmortem diagnosis was confirmed by testing the rabbit liver tissue homogenate for relative antigens using ELISA. It was found that aminoethylethylenimine and β-propiolactone did not have the same effect on the studied variants of the virus. In order to preserve at maximum the antigenic structures of the virus, the following inactivation modes were considered to be optimal: for RHDV1-aminoethylethylenimine at a concentration of 0.3% at 37 °C, exposure time – 72 hours, or β-propiolactone at a concentration of 0.1–0.3% at 25–37 °С, exposure time – 24–48 hours; for RHDV2 – aminoethylethylenimine at a concentration of 1% at 37 °C, exposure time – 72 hours, or β-propiolactone at a concentration 0.3% at 25 °С, exposure time – 24 hours.

1. Rabbit haemorrhagic disease. In: OIE. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2018; Chap. 3.6.2: 1389–1406. Available at: https://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.06.02_RHD.pdf (date of access: 09.10.2020).

2. Moss S. R., Turner S. L., Trout R. C., White P. J., Hudson P. J., Desai A., et al. Molecular epidemiology of Rabbit haemorrhagic disease virus. J. Gen. Virol. 2002; 83 (Pt 10): 2461–2467. DOI: 10.1099/0022-1317-83-10-2461. PMID: 12237428.

3. Makarov V. V., Vishnyakov I. F., Vlasov N. A., Malakhova M. S. Rabbit hemorrhagic disease virus: physical and structural characteristics. [Virus gemorragicheskoj bolezni krolikov: fizicheskie i strukturnye harakteristiki]. Vestnik of the Russian Agricultural Sciences. 1992; 4: 49–52. eLIBRARY ID: 21484449. (in Russian)

4. Vlasov N. A. Physico-chemical properties and antigenic structure of rabbit hemorrhagic disease virus [Fiziko-himicheskie svojstva i antigennaya struktura virusa gemorragicheskoj bolezni krolikov]: Author´s abstract of Doctor’s thesis (Biological Sciences). Pokrov; 1998. 46 p. Available at: https://dlib.rsl.ru/viewer/01000254860#?page=1. (in Russian)

5. Shevchenko A. A., Shevchenko L. V., Zerkalev D. Yu., Shevkoplyas V. N., Chernykh O. Yu. Viral haemorrhagic disease of rabbits. Veterinaria Kubani. 2011; 2: 3–6. Available at: http://vetkuban.com/num2_20111.html. (in Russian)

6. Le Gall-Reculé G., Zwingelstein F., Boucher S., Le Normand B., Plassiart G., Portejoie Y., et al. Detection of a new variant of rabbit haemorrhagic disease virus in France. Vet Rec. 2011; 168 (5): 137–138. DOI: 10.1136/vr.d697. PMID: 21493491.

7. The third case of a new type of rabbit hemorrhagic disease virus (RHDV-2) detection in the Russian Federation. [Tretij sluchaj obnaruzheniya virusa gemorragicheskoj bolezni krolikov novogo tipa – VGBK-2 v Rossijskoj Federacii]. News FRCVM. Available at: https://ficvim.ru/2019/02/tretij-sluchaj-obnaruzheniya-virusa-gemorragicheskoj-bolezni-krolikov-novogo-tipa-vgbk-2-v-rossijskoj-federacii/ (date of access: 09.10.2020). (in Russian)

8. FAQ. Hämorrhagische Krankheit der Kaninchen (RHDV, RHDV-2). Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit. Stand: 02.05.2017. Available at: https://www.openagrar.de/servlets/MCRFileNodeServlet/openagrar_derivate_00002514/FLI-Information-FAQ-RHDV-2017-05-02.pdf (date of access: 07.10.2020). (in German)

9. State Register of medicines for veterinary use. [Gosudarstvennyj reestr lekarstvennyh sredstv dlya veterinarnogo primeneniya]. Rosselkhoznadzor. Component “Irena”. Available at: https://galen.vetrf.ru/#/registry/pharm/registry?page=1 (date of access: 09.10.2020). (in Russian)

10. INgezim RHDV DAS. ELISA for the detection of RHDV in biological samples [Immunofermentnyj analiz dlya vyyavleniya virusa gemorragicheskoj bolezni krolikov v biologicheskih obrazcah] (Ingenasa, Spain). Available at: http://vetprofilab.ru/f/rus_17rhdk2.pdf. (in Russian)

11. Muzalevskaya A. V. Pathomorphology of viral haemorrhagic rabbit disease [Patomorfologiya virusnoj gemorragicheskoj bolezni krolikov]: Author´s abstract of Candidate’s thesis (Veterinary Sciences). Saratov; 2008. 21 p. Available at: https://dlib.rsl.ru/viewer/01003457192#?page=1. (in Russian)

12. Mahar J. E., Hall R. N., Peacock D., Kovaliski J., Piper M., Mourant R., et al. Rabbit hemorrhagic disease virus 2 (RHDV2; GI.2) is replacing endemic strains of RHDV in the Australian landscape within 18 months of its arrival. J. Virol. 2018; 92 (2):e01374-17. DOI: 10.1128/JVI.01374-17.

13. Peacock D., Kovaliski J., Sinclair R., Mutze G., Iannella A., Capucci L. RHDV2 overcoming RHDV immunity in wild rabbits (Oryctolagus cuniculus) in Australia. Vet. Rec. 2017; 180 (11): 280. DOI: 10.1136/vr.104135.

14. Sergeev V. A., Nepoklonov E. A., Aliper T. I. Viruses and virus vaccines. [Virusy i virusnye vakciny]. Moscow: Biblionika; 2007. 524 р. (in Russian)

15. Pharmacological group - alkylating agents. Russian Register of Medicinal Products (RLS) [Farmakologicheskaya gruppa – alkiliruyushchie sredstva. Registr lekarstvennyh sredstv Rossii (RLS)]. Available at: https://www.rlsnet.ru/fg_index_id_268.htm (date of access: 09.10.2020). (in Russian)

16. Bahnemann H. G. Inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethyleinimine. Vaccine. 1990; 8 (4): 299–303. DOI: 10.1016/0264-410x(90)90083-x.

17. Brown F. Inactivation of viruses by aziridines. Vaccine. 2001; 20 (3–4): 322–327. DOI: 10.1016/s0264-410x(01)00342-5.

18. Bahnemann H. G. The inactivation of foot-and-mouth disease virus by ethylenimine and propilenimine. Zentralbl. Veterinärmed. B. 1973; 20 (5): 356–360. DOI: 10.1111/j.1439-0450.1973.tb01136.x.

19. Rweyemamu M. M., Unehara O., Giorgi W., Medeiros R., Lucca D., Baltazar M. Effect of formaldehyde and binary ethyleneimine (BEI) on the integrity of foot and mouth disease virus capsid. Rev. Sci. Tech. 1989; 8 (3): 747–764. DOI: 10.20506/rst.8.3.425. PMID: 32344961.

20. Barhoom S., Forgacs A., Solyom F. Development of an inactivated vaccine against infectious laryngotracheitis (ILT) – serological and protection studies. Avian Pathol. 1986; 15 (2): 213–221. DOI: 10.1080/03079458608436282.

21. Sarachai C., Sasipreeyajan J., Chansiripornchai N. Avian influenza virus (H5N1) inactivation by binary ethylenimine. Thai J. Vet. Med. 2010; 40 (1): 41–46. Available at: https://www.researchgate.net/publication/233231064_Avian_Influenza_Virus_H5N1_Inactivation_by_Binary_Ethylenimine.

22. Cunliffe H. R. Inactivation of foot-and-mouth disease virus with ethylenimine. Appl. Microbiol. 1973; 26 (5): 747–750. PMID: 4357652; PMCID: PMC379895.

23. Graves J. H., Arlinghaus R. B. Acetylethylenimine in the preparation of inactivated foot-and-mouth disease vaccines. In: Proceedings, Annual Meeting of the United States Animal Health Association. 1967; 71: 396–403. PMID: 5257323.

24. Shevchenko A. A., Vishnjakov I. F., Dymin M. A., Zubairov M. M., Karpov G. M., Neverovskij A. I., et al. Process for manufacturing inactivated vaccine used against viral hemorrhagic diseases of rabbits. Patent No. 2039570 Russian Federation, Int. Cl. А61К39/125 (05.1992). Vserossijskij nauchno-issledovatel’skij institut veterinarnoj virusologii i mikrobiologii. No. 5043431/13. Application: 26.05.1992. Date of publication: 20.07.1995. (in Russian)

25. Shevchenko A. A., Shevchenko L. V., Gnezdilov I. D., Golenskij A. G., Kruzhnov N. N., Zerkalev D. Ju. Vaccine against viral hemorrhagic disease in rabbits. Patent No. 2229895 Russian Federation, Int. Cl. А61К39/125 (11.2002). Kubanskij gosudarstvennyj agrarnyj universitet. No. 2002130881/13. Application: 18.11.2002. Date of publication: 10.06.2004. (in Russian)

26. Instructions for veterinary use of tissue inactivated hydroxyaluminium vaccine against viral haemorrhagic rabbit disease. [Instrukciya po veterinarnomu primeneniyu vakciny protiv virusnoj gemorragicheskoj bolezni krolikov tkanevoj inaktivirovannoj gidrookis’alyuminievoj]. FRCVM; 2019. Available at: https://ficvim.ru/wp-content/uploads/2019/03/%D0%98%D0%BD%D1%81%D1%82%D1%80-%D0%92%D0%B0%D0%BA%D1%86%D0%B8%D0%BD%D0%B0-%D0%92%D0%93%D0%91%D0%9A-%D0%B6%D0%B8%D0%B4%D0%BA%D0%B0-%D1%8F-17.01.19.pdf (date of access: 09.10.2020). (in Russian)

27. Inactivated vaccine against rabbit hemorrhagic disease (“Lapimun Gem-2”, BioTestLab). Available at: https://www.biotestlab.ua/products/lapimun-gem-2/ (date of access: 09.10.2020). (in Russian)

28. Vaccine against rabbit haemorrhagic disease (“Pestorin”, Bioveta). Available at: https://www.bioveta.cz/ru/preparaty/zdorove-zhivotnyheu/en/products/veterinary-products/pestorin-suspension-for-injection-for-rabbit.html (date of access: 09.10.2020). (in Russian)

29. Vaccine against viral hemorrhagic rabbit disease (“Rabbivak-V” (LLC “TD “Biagro”). VetLek. Online veterinary pharmacy. Available at: https://www.vetlek.ru/directions/?id=787 (date of access: 09.10.2020). (in Russian)

30. Live dry vaccine against myxomatosis and viral hemorrhagic rabbit disease with solvent (“Nobivac® Myxo-RHD”, Intervet International B.V.). Available at: https://www.msd-animal-health.ru/product/nobivacmuho-rhd/ (date of access: 09.10.2020). (in Russian)