Problems of VirologyProblems of Virology0507-40882411-2097Central Research Institute for Epidemiology631510.36233/0507-4088-164Research ArticleAntigenic and immunogenic activity of virus-like particles based on rabbit hemorrhagic disease virus (Caliciviridae: <i>Lagovirus</i>) genotypes GI1 and GI2 recombinant major capsid proteinsMukhinAleksey N.<p>Ph.D. (Biol.), Leading Researcher</p>amuhin@yahoo.comhttps://orcid.org/0000-0001-7008-5212AlekseevKonstantin P.<p>Ph.D. (Biol.), Leading Researcher</p>kalekseev@hotmail.comhttps://orcid.org/0000-0001-9536-3127YuzhakovAnton G.<p>Ph.D. (Biol.), Head of laboratory</p>anton_oskol@mail.ruhttps://orcid.org/0000-0002-0426-9678SeleznevaEkaterina V.<p>microbiologist of department of quality control</p>selja07@rambler.ruhttps://orcid.org/0000-0002-5592-2948MoskvinaAnna S.<p>Ph.D. (Biol.), Leading Researcher</p>annamoskvina17@gmail.comhttps://orcid.org/0000-0002-4542-8196VerkhovskyOleg A.<p>Professor, President</p>info@dpri.comhttps://orcid.org/0000-0003-0784-9341AliperTaras I.<p>Professor, Director of science and innovation</p>coronavirusr@yandex.ruhttps://orcid.org/0000-0003-2696-1363Vetbiochem LLCFederal Scientific Center – All-Russian Research Institute of Experimental Veterinary Medicine named after K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of SciencesResearch Institute for Diagnosis and Prevention of Human and Animal Diseases1805202368213214103032023Copyright © 2023, Mukhin A.N., Alekseev K.P., Yuzhakov A.G., Selezneva E.V., Moskvinа A.S., Verkhovsky O.A., Aliper T.I.2023<p><strong>Introduction.</strong> Rabbit hemorrhagic disease is an acute highly contagious infection associated with two genotypes of pathogenic Lagovirus. Antibodies to major capsid protein (Vp60) are protective.</p>
<p><strong>The aim of the work ‒</strong> is an evaluation of antigenic and immunogenic activity of virus-like particles (VLPs) based on recombinant major capsid proteins of both genotypes of rabbit hemorrhagic disease virus (RHDV) (recVP60-GI1 and recVP60-GI2).</p>
<p><strong>Materials and methods. </strong>Baculovirus-expressed VLPs were evaluated using electron microscopy and administered to clinically healthy 1.53 month old rabbits in a dose of 50 g. Rabbits were challenged with 10<sup>3</sup> LD<sub>50 </sub>of virulent strains Voronezhsky-87 and Tula 21 days post immunization. Serum samples were tested for the presence of RHDV-specific antibodies.</p>
<p><strong>Results.</strong> VLPs with hemagglutination activity forming VLP 3040 nm in size were obtained in Hi-5 cell culture. Specific antibody titers in rabbits measured by ELISA were 1 : 200 to 1 : 800 on 21<sup>th</sup> day post immunization with VLPs. Immunogenic activity of recVP60-GI1 VLPs was 90 and 40%, while it was 30 and 100% for recVP60-GI2 VLPs after the challenge with RHDV genotypes 1 and 2 respectively. The immunogenicity of two VLPs in mixture reached 100%.</p>
<p><strong>Discussion. </strong>VLPs possess hemagglutinating, antigenic and immunogenic activity, suggesting their use as components in substances designed for RHDV specific prophylaxis in rabbits. Results of the control challenge experiment demonstrated the need to include the antigens from both RHDV genotypes in the vaccine.</p>
<p><strong>Conclusion. </strong>Recombinant proteins recVP60-GI1 and recVP60-GI2 form VLPs that possess hemagglutinating an antigenic activity, and provide 90100% level of protection for animals challenged with RHDV GI1 and GI2 virulent strains.</p>RHDVVP60virus-like particlesrecombinant proteinsimmune responseimmunogenicityprotectionВГБКVP60вирусоподобные частицырекомбинантные белкииммунный ответиммуногенностьзащита[Liu S.J., Xue H.P., Pu B.Q., Quian N.H. A new viral disease in rabbits. Anim. Husb. Vet. Med. 1984; 16(6): 253–5.][Mitro S., Krauss H. Rabbit hemorrhagic disease: a review with special reference to its epizootiology. Eur. J. Epidemiol. 1993; 9(1): 70–8. https://doi.org/10.1007/bf00463093][Puggioni G., Cavadini P., Maestrale C., Scivoli R., Botti G., Ligios C., et al. The new French 2010 Rabbit Hemorrhagic Disease Virus causes an RHD-like disease in the Sardinian Cape hare (Lepus capensis mediterraneus). Vet. Res. 2013; 44(1): 96. https://doi.org/10.1186/1297-9716-44-96][Abrantes J., Lopes A.M., Dalton K.P., Melo P., Correia J.J., Ramada M., et al. New variant of rabbit hemorrhagic disease virus, Portugal, 2012-2013. Emerg. Infect. Dis. 2013; 19(11): 1900–1902. https://doi.org/10.3201/eid1911.130908][Dalton K.P., Nicieza I., Abrantes J., Esteves P.J., Parra F. Spread of new variant RHDV in domestic rabbits on the Iberian Peninsula. Vet. Microbiol. 2014; 169(1-2): 67–73. https://doi.org/10.1016/j.vetmic.2013.12.015][Duarte M., Henriques M., Barros S.C., Fagulha T., Ramos F., Luis T., et al. Detection of RHDV variant 2 in domestic rabbits in Azores. Vet. Rec. 2015; 176(19): 499–500. https://doi.org/10.1136/vr.h2402][OIE. Rabbit Haemorrhagic Disease. USA; 2020. Available at: https://www.oie.int/wahis_2/public/wahid.php/Reviewreport/Review?page_refer=MapEventSummary&reportid=34728][Le Pendu J., Abrantes J., Bertagnoli S., Guitton J.S., Le Gall-Reculé G., Lopes A.M., et al. Proposal for a unified classification system and nomenclature of lagoviruses. J. Gen. Virol. 2017; 98(7): 1658–66. https://doi.org/10.1099/jgv.0.000840][Mukhin A.N., Yuzhakov A.G., Selezneva E.V., Drozdova E.I., Verkhovskiy O.A., Aliper T.I. Outbreak of the disease caused by the rabbit hemorrhagic disease virus 2 in the Russian Federation. Agrarnaya nauka. 2021; (4): 25–7. https://doi.org/10.32634/0869-8155-2021-348-4-25-27 EDN: https://www.elibrary.ru/ynmqnh (in Russian)][Burmakina G., Malogolovkina N., Lunitsin A., Titov I., Tsybanov S., Malogolovkin A. Comparative analysis of rabbit hemorrhagic disease virus strains originating from outbreaks in the Russian Federation. Arch. Virol. 2016; 161(7): 1973–9. https://doi.org/10.1007/s00705-016-2864-1][Federal Research Center for Virology and Microbiology. The third case of detection of a new type of rabbit hemorrhagic disease virus – VGBK-2 in the Russian Federation. Available at: https://ficvim.ru/2019/02/tretij-sluchaj-obnaruzheniya-virusa-gemorragicheskoj-bolezni-krolikov-novogo-tipa-vgbk-2-v-rossijskoj-federacii/ (in Russian)][Le Gall-Recule G., Lavazza A., Marchandeau S., Bertagnoli S., Zwingelstein F., Cavadini P., et al. Emergence of a new lagovirus related to Rabbit haemorrhagic disease virus. Vet. Res. 2013; 44(1): 81. https://doi.org/10.1186/1297-9716-44-81][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. https://doi.org/10.1136/vr.104135][Vlasova N.N., Vlasov N.A., Alekseev K.P. Construction of plasmid vector of expressing virus of haemorrhagic disease of rabbits. Veterinariya. 2006; (11): 53–5. https://www.elibrary.ru/hvjxut (in Russian)][Gromadzka B., Szewczyk B., Konopa G., Fitzner A., Kesy A. Recombinant VP60 in the form of virion-like particles as a potential vaccine against rabbit hemorrhagic disease virus. Acta Biochim. Pol. 2006; 53(2): 371–6.][Guo H., Zhu J., Tan Y., Li C., Chen Z., Sun S., et al. Self-assembly of virus-like particles of rabbit hemorrhagic disease virus capsid protein expressed in Escherichia coli and their immunogenicity in rabbits. Antiviral Res. 2016; 131: 85–91. https://doi.org/10.1016/j.antiviral.2016.04.011][López-Vidal J., Gómez-Sebastián S., Bárcena J., Nuñez Mdel C., Martínez-Alonso D., Dudognon B., et al. Improved production efficiency of virus-like particles by the baculovirus expression vector system. PLoS One. 2015; 10(10): e0140039. https://doi.org/10.1371/journal.pone.0140039][Qi R., Miao Q., Zhu J., Tang J., Tang A., Wang X., et al. Construction and immunogenicity of novel bivalent virus-like particles bearing VP60 genes of classic RHDV(GI.1) and RHDV2(GI.2). Vet. Microbiol. 2020; 240: 108529. https://doi.org/10.1016/j.vetmic.2019.108529][Alekseev K.P., Moskvina A.S., Verkhovskiy O.A., Selezneva E.V., Chernykh O.Yu., Mukhin A.N. Obtaining recombinant capsid protein VP60 of rabbit haemorrhagic disease virus and its antigenic and immunogenic activity study. Veterinariya Kubani. 2020; (5): 34–7. https://doi.org/10.33861/2071-8020-2020-5-34-37 EDN: https://www.elibrary.ru/vxzgjm (in Russian)][Selezneva E.V., Mukhin A.N., Ezdakova I.Yu., Verkhovsky O.A., Aliper T.I. The application of recombinant Vp60-based ELISA for haemorrhagic disease virus antibody detection to vaccination against RHD. AIP Conf. Proc. 2022; 2467: 070028-1–070028-5. https://doi.org/10.1063/5.0092529][Pawlikowska M., Hukowska-Szematowicz B., Deptuła W. Phylogenetic analysis of selected strains of Rabbit haemorrhagic disease virus on the basis of N-terminal fragment of the gene encoding structural protein VP60. Bull. Vet. Inst. Pulawy. 2010; 54: 129–33.][Dalton K.P., Arnal J.L., Benito A.A., Chacón G., Martín Alonso J.M., Parra F. Conventional and real time RT-PCR assays for the detection and differentiation of variant rabbit hemorrhagic disease virus (RHDVb) and its recombinants. J. Virol. Methods. 2018; 251: 118–22. https://doi.org/10.1016/j.jviromet.2017.10.009]