biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2021-21-2-76-84biopreparat-333Research ArticleОБЗОРЫREVIEWSМетоды in vitro для выявления вируса бешенства и оценка их использования в производстве антирабического иммуноглобулинаIn vitro methods for rabies virus detection, and evaluation of their use in the production of rabies immunoglobulinhttps://orcid.org/0000-0001-7919-3412ГавриловаЮ. К.GavrilovaYu. K.

Гаврилова Юлия Кирилловна

ул. Университетская, д. 46, Саратов, 410005

Yuliya K. Gavrilova

46 Universitetskaya St., Saratov 410005

ylia-93@list.ruhttps://orcid.org/0000-0003-1461-5383ГенераловС. В.GeneralovS. V.

Генералов Сергей Вячеславович, кандидат биологических наук

ул. Университетская, д. 46, Саратов, 410005

Sergey V. Generalov, Cand. Sci. (Biol.). 

46 Universitetskaya St., Saratov 410005

svgeneraloff@gmail.comhttps://orcid.org/0000-0002-8798-1547АбрамоваЕ. Г.AbramovaE. G.

Абрамова Елена Геннадьевна, доктор биологических наук

ул. Университетская, д. 46, Саратов, 410005;Театральная пл. 1, Саратов, 410012

Elena G. Abramova, Dr. Sci. (Biol.). 

46 Universitetskaya St., Saratov 410005;1 Teatralnaya sq., Saratov 410012

abramova.phd2018@yandex.ruНикифоровА. К.NikiforovA. K.

Никифоров Алексей Константинович, доктор биологических наук

SPIN-код РИНЦ: 3202-3979

ул. Университетская, д. 46, Саратов, 410005;Театральная пл. 1, Саратов, 410012

Aleksey K. Nikiforov, Dr. Sci. (Biol.). 

SPIN-code RINC: 3202-3979

46 Universitetskaya St., Saratov 410005;1 Teatralnaya sq., Saratov 410012

Nikiforov_AK@mircobe.ruФедеральное казенное учреждение здравоохранения «Российский научно-исследовательский противочумный институт «Микроб» Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человекаРоссияRussian Anti-Plague Research Institute “Microbe” of the Federal Service for Surveillance of Consumer Rights Protection and Human WellbeingRussian FederationФедеральное казенное учреждение здравоохранения «Российский научно-исследовательский противочумный институт «Микроб» Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека; Федеральное государственное бюджетное образовательное учреждение высшего образования «Саратовский государственный аграрный университет им. Н. И. Вавилова»РоссияRussian Anti-Plague Research Institute “Microbe” of the Federal Service for Surveillance of Consumer Rights Protection and Human Wellbeing; Saratov State Vavilov Agrarian UniversityRussian Federation2021050420212127684Copyright © Гаврилова Ю.К., Генералов С.В., Абрамова Е.Г., Никифоров А.К., 20212021Гаврилова Ю.К., Генералов С.В., Абрамова Е.Г., Никифоров А.К.Gavrilova Y.K., Generalov S.V., Abramova E.G., Nikiforov A.K.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.biopreparations.ru/jour/article/view/333

Применение современных высокочувствительных методов исследования биологического материала с целью выявления вируса бешенства и антирабических антител актуально не только для диагностики данного заболевания и области экспериментальных исследований, но и для производства антирабических лекарственных препаратов, применяемых для постэкспозиционной профилактики бешенства. Цель работы — анализ существующих в настоящее время методов детекции вируса бешенства и антирабических антител с последующей оценкой возможности применения указанных методов на контрольных этапах производства препарата гетерологичного антирабического иммуноглобулина из сыворотки крови лошади. Поиск современных высокочувствительных методов контроля in vitro, способных конкурировать с биологическим методом — основным методом контроля свойств препарата антирабического иммуноглобулина, является важным аспектом совершенствования технологии производства и повышения качества препарата для профилактики бешенства. В ходе аналитического исследования было выявлено, что в условиях производства антирабического иммуноглобулина в качестве самостоятельных методов, а также в виде альтернативы применяемому в производстве биологическому методу на белых мышах возможно применение метода флуоресцирующих антител, иммуноферментного анализа, методов с применением клеточных культур, атомно-силовой микроскопии, проточной цитометрии. Выбор в пользу указанных методов исследования был обусловлен высокой степенью их чувствительности, специфичностью, скоростью постановки, экономичностью, простотой исполнения и автоматизированным процессом учета результатов.

Current highly sensitive methods for rabies virus and rabies antibodies detection in biological material can be used not only for diagnosis and experimental research, but also for the production of antirabies medicines used for postexposure prophylaxis. The aim of the study was to analyse existing methods for rabies virus and rabies antibodies detection and to assess the potential for using these methods at the control stages during production of heterologous antirabies immunoglobulin obtained from equine serum. The search for cutting-edge highly sensitive in vitro control methods that could compete with the biological method, which is the main method used in antirabies immunoglobulin control, is an important prerequisite for improvement of the production technology and the quality of antirabies medicines. The study demonstrated that the following test methods can be used in the production of antirabies immunoglobulin: fluorescent antibody technique, enzyme-linked immunosorbent assay, cell culture methods, atomic force microscopy, and flow cytometry. These methods could be used alone or as an alternative to the biological method in white mice. These methods were chosen because of their high sensitivity, specificity, rapid and easy implementation, cost-effectiveness, and automatic recording of test results.

вирус бешенстваметоды in vitroИФАFAVN-тестRFFITклеточная культураантирабический иммуноглобулинrabies virusin vitro methodsELISAFAVN testRFFITcell cultureantirabies immunoglobulinИсследование проводилось без спонсорской поддержки.The study was performed without external funding.ReferencesАбрамова ЕГ, Никифоров АК, Лобовикова ОА, Еремин СА, Васин ЮГ, Михеева ТА и др. Производство гетерологичного антирабического иммуноглобулина — итоги первых пяти лет. Проблемы особо опасных инфекций. 2010;(3):58–62. https://doi.org/10.21055/0370-1069-2010-3(105)-58-62Abramova EG, Nikiforov AK, Lobovikova OA, Eremin SA, Vasin YuG, Mikheeva TA, et al. Heterologous anti-rabies immunoglobulin — results of the first five years of production. Problemy osobo opasnykh infektsii = Problems of Particularly Dangerous Infections. 2010;(3):58–62 (In Russ.) https://doi.org/10.21055/0370-1069-2010-3(105)-58-62Онищенко ГГ, Попова АЮ, Ежлова ЕБ, Демина ЮВ, Пакскина НД, Писцов МН и др. Эпидемиологическая обстановка и вопросы идентификации вируса бешенства среди людей на территории Российской Федерации в период 2002–2015 гг. Проблемы особо опасных инфекций. 2017;(3):27–32. https://doi.org/10.21055/0370-1069-2017-3-27-32Onishchenko GG, Popova AYu, Ezhlova EB, Demina YuV, Pakskina ND, Pistsov MN, et al. Epidemiological situation on and problems of identification of rabies virus in humans in the territory of the Russian Federation during the period of 2002–2015. Problemy osobo opasnykh infektsii = Problems of Particularly Dangerous Infections. 2017;(3):27–32 (In Russ.) https://doi.org/10.21055/0370-1069-2017-3-27-32Kuzmin IV. Virus isolation in animals: the mouse inoculation test. In: Rupprecht Ch, Nagarajan Th, eds. Current Laboratory Techniqes in Rabies Diagnosis, Research and Prevention. Vol. 2. San Diego: Elsevier Academic Press; 2015. P. 13–23. https://doi.org/10.1016/B978-0-12-801919-1.00002-6Kuzmin IV. Virus isolation in animals: the mouse inoculation test. In: Rupprecht Ch, Nagarajan Th, eds. Current Laboratory Techniqes in Rabies Diagnosis, Research and Prevention. Vol. 2. San Diego: Elsevier Academic Press; 2015. P. 13–23. https://doi.org/10.1016/B978-0-12-801919-1.00002-6Stantić-Pavlinić M, Hostnik P, Levičnik-Stezinar S, Zaletel-Kragelj L. Vaccination against rabies and protective antibodies — comparison of ELISA and fluorescent antibody virus neutralization (FAVN) assays. Vet Arhiv. 2006;76(4):281–9.Stantić-Pavlinić M, Hostnik P, Levičnik-Stezinar S, Zaletel-Kragelj L. Vaccination against rabies and protective antibodies — comparison of ELISA and fluorescent antibody virus neutralization (FAVN) assays. Vet Arhiv. 2006;76(4):281–9.Lahaye X, Vidy A, Pomier C, Obiang L, Harper F, Gaudin Y, Blondel D. Functional characterization of Negri bodies (NBs) in rabies virus-infected cells: evidence that NBs are sites of viral transcription and replication. J Virol. 2009;83(16):7948–58. https://doi.org/10.1128/JVI.00554-09Lahaye X, Vidy A, Pomier C, Obiang L, Harper F, Gaudin Y, Blondel D. Functional characterization of Negri bodies (NBs) in rabies virus-infected cells: evidence that NBs are sites of viral transcription and replication. J Virol. 2009;83(16):7948–58. https://doi.org/10.1128/JVI.00554-09Niezgoda M, Rupprecht CE. Standard Operating Procedure for the Direct Rapid Immunohistochemistry Test (DRIT) for the Detection of Rabies Virus Antigen. National Laboratory Training Network Course. Atlanta: US Department of Health and Human Services, Centers for Disease Control and Prevention; 2006.Niezgoda M, Rupprecht CE. Standard Operating Procedure for the Direct Rapid Immunohistochemistry Test (DRIT) for the Detection of Rabies Virus Antigen. National Laboratory Training Network Course. Atlanta: US Department of Health and Human Services, Centers for Disease Control and Prevention; 2006.Madhusudana SN, Subha S, Thankappan U, Ashwin YB. Evaluation of a direct rapid immunohistochemical test (dRIT) for rapid diagnosis of rabies in animals and humans. Virol Sin. 2012;27(5):299–302. https://doi.org/10.1007/s12250-012-3265-6Madhusudana SN, Subha S, Thankappan U, Ashwin YB. Evaluation of a direct rapid immunohistochemical test (dRIT) for rapid diagnosis of rabies in animals and humans. Virol Sin. 2012;27(5):299–302. https://doi.org/10.1007/s12250-012-3265-6Mani RS, Madhusudana SN. Laboratory diagnosis of human rabies: recent advances. Sci World J. 2013:569712. https://doi.org/10.1155/2013/569712Mani RS, Madhusudana SN. Laboratory diagnosis of human rabies: recent advances. Sci World J. 2013:569712. https://doi.org/10.1155/2013/569712Horwitz JA, Jenni S, Harrison SC, Whelan SPJ. Structure of a rabies virus polymerase complex from electron cryo-microscopy. Proc Natl Acad Sci USA. 2020;117(4):2099–107. https://doi.org/10.1073/pnas.1918809117Horwitz JA, Jenni S, Harrison SC, Whelan SPJ. Structure of a rabies virus polymerase complex from electron cryo-microscopy. Proc Natl Acad Sci USA. 2020;117(4):2099–107. https://doi.org/10.1073/pnas.1918809117Зайцев БН, Таранов ОС, Рудометова НБ, Щербакова НС, Ильичев АА, Карпенко ЛИ. Оптимизированный метод подсчета количества вирусных частиц с помощью электронной микроскопии. Вавиловский журнал генетики и селекции. 2019;23(3):337–42. https://doi.org/10.18699/VJ19.498Zaitsev BN, Taranov OS, Rudometova NB, Shcherbakova NS, Ilyichev AA, Karpenko LI. An optimized method for counting viral particles using electron microscopy. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2019;23(3):337–42 (In Russ.) https://doi.org/10.18699/VJ19.498Петрова ИД, Зайцев БН, Таранов ОС. Концентрирование вирусов и электронная микроскопия. Вавиловский журнал генетики и селекции. 2020;24(3):276–83. https://doi.org/10.18699/VJ20.620Petrova ID, Zaitsev BN, Taranov OS. Concentration of viruses and electron microscopy. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2020;24(3):276–83 (In Russ.) https://doi.org/10.18699/VJ20.620Грибенча СВ, Козлов АЮ, Костина ЛВ, Елаков АЛ, Лосич МА, Цибезов ВВ и др. Получение моноклональных антител к нуклеопротеину вируса бешенства. Вопросы вирусологии. 2013;58(5):38–43.Gribencha SV, Kozlov AYu, Kostina LV, Elakov AL, Losich MA, Tsibezov VV, et al. Production of the monoclonal antibodies to the rabies virus nucleoprotein. Voprosy virusologii = Problems of Virology. 2013;58(5):38–43 (In Russ.)Rourou S, Ben Zakkour M, Kallel H. Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media. Vaccine. 2019;37(47):6987–95. https://doi.org/10.1016/j.vaccine.2019.05.092Rourou S, Ben Zakkour M, Kallel H. Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media. Vaccine. 2019;37(47):6987–95. https://doi.org/10.1016/j.vaccine.2019.05.092Хисматуллина НА, Гулюкин АМ, Шуралев ЭА, Хаертынов КС, Чернов АН, Филимонова МН и др. Ускоренный метод диагностики бешенства в культуре клеток невриномы Гассерова узла крысы (НГУК-1). Гены и клетки. 2014;9(3):276–80.Khismatullina NA, Gulyukin AM, Shuralev EA, Khaertynov KS, Chernov AN, Filimonova MN, et al. Rapid diagnostic test of rabies using rat Gasser’s ganglion neurinoma cell culture (RGGN-1). Geny i kletki = Genes and Cells. 2014;9(3):276–80 (In Russ.)Cliquet F, Wasniewski M. The fluorescent antibody virus neutralization test. In: Rupprecht Ch, Nagarajan Th, eds. Current Laboratory Techniqes in Rabies Diagnosis, Research and Prevention. Vol. 2. San Diego: Elsevier Academic Press; 2015. P. 217–31. https://doi.org/10.1016/B978-0-12-801919-1.00018-XCliquet F, Wasniewski M. The fluorescent antibody virus neutralization test. In: Rupprecht Ch, Nagarajan Th, eds. Current Laboratory Techniqes in Rabies Diagnosis, Research and Prevention. Vol. 2. San Diego: Elsevier Academic Press; 2015. P. 217–31. https://doi.org/10.1016/B978-0-12-801919-1.00018-XYager ML, Moore SM. The rapid fluorescent focus inhibition test. In: Rupprecht Ch, Nagarajan Th, eds. Current Laboratory Techniqes in Rabies Diagnosis, Research and Prevention. Vol. 2. San Diego: Elsevier Academic Press; 2015. P. 199–215. https://doi.org/10.1016/B978-0-12-801919-1.00017-8Yager ML, Moore SM. The rapid fluorescent focus inhibition test. In: Rupprecht Ch, Nagarajan Th, eds. Current Laboratory Techniqes in Rabies Diagnosis, Research and Prevention. Vol. 2. San Diego: Elsevier Academic Press; 2015. P. 199–215. https://doi.org/10.1016/B978-0-12-801919-1.00017-8Cliquet F, Aubert M, Sagn é L. Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody. J Immunol Methods. 1998;212(1):79–87. https://doi.org/10.1016/s0022-1759(97)00212-3Cliquet F, Aubert M, Sagn é L. Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody. J Immunol Methods. 1998;212(1):79–87. https://doi.org/10.1016/s0022-1759(97)00212-3Баркова ЕП, Нагиева ФГ, Никулина ВГ, Лисаков АН. Быстрый культуральный метод для индикации антигенов вируса бешенства в инфицированных клеточных культурах. Инфекция и иммунитет. 2013;3(4):323–6.Barkova EP, Nagieva FG, Nikulina VG, Lisakov AN. The rapid culture method for the indication of rabies virus antigen in infected cell cultures. Infektsiya i immunitet = Russian Journal of Infection and Immunity. 2013;3(4):323–6 (In Russ.)Bedeković T, Lemo N, Lojkić I, Mihaljević Ž , Jungić A, Cvetnić Ž , et al. Modification of the fluorescent antibody virus neutralization test — elimination of the cytotoxic effect for the detection of rabies virus neutralising antibodies. J Virol Methods. 2013;189(1):204–8. https://doi.org/10.1016/j.jviromet.2013.01.022Bedeković T, Lemo N, Lojkić I, Mihaljević Ž , Jungić A, Cvetnić Ž , et al. Modification of the fluorescent antibody virus neutralization test — elimination of the cytotoxic effect for the detection of rabies virus neutralising antibodies. J Virol Methods. 2013;189(1):204–8. https://doi.org/10.1016/j.jviromet.2013.01.022Moeschler S, Locher S, Conzelmann KK, Krämer B, Zimmer G. Quantification of Lyssavirus-neutralizing antibodies using vesicular stomatitis virus pseudotype particles. Viruses. 2016;8(9):254. https://doi.org/10.3390/v8090254Moeschler S, Locher S, Conzelmann KK, Krämer B, Zimmer G. Quantification of Lyssavirus-neutralizing antibodies using vesicular stomatitis virus pseudotype particles. Viruses. 2016;8(9):254. https://doi.org/10.3390/v8090254Qin S, Volokhov D, Rodionova E, Wirblich C, Schnell MJ, Chizhikov V, Dabrazhynetskaya A. A new recombinant rabies virus expressing a green fluorescent protein: a novel and fast approach to quantify virus neutralizing antibodies. Biologicals. 2019;59:56–61. https://doi.org/10.1016/j.biologicals.2019.03.002Qin S, Volokhov D, Rodionova E, Wirblich C, Schnell MJ, Chizhikov V, Dabrazhynetskaya A. A new recombinant rabies virus expressing a green fluorescent protein: a novel and fast approach to quantify virus neutralizing antibodies. Biologicals. 2019;59:56–61. https://doi.org/10.1016/j.biologicals.2019.03.002Мовсесянц АА, Олефир ЮВ. Современные проблемы вакцинопрофилактики бешенства. БИОпрепараты. Профилактика, диагностика, лечение. 2019;19(1):10–6. https://doi.org/10.30895/2221-996X-2019-19-1-10-16Movsesyants AA, Olefir YuV. Current challenges of preventive vaccination against rabies. BIOpreparaty. Profilaktika, diagnostika, lechenie = BIOpreparations. Prevention, Diagnosis, Treatment. 2019;19(1):10–6 (In Russ.) https://doi.org/10.30895/2221-996X-2019-19-1-10-16Гаврилова ЮК, Генералов СВ, Абрамова ЕГ, Савицкая ЛВ, Галкина МВ, Кочкин АВ. Экспресс-анализ активности антирабических сывороток и иммуноглобулина в клеточных культурах методом иммунофлуоресценции. Биотехнология. 2018;34(4):83–8. https://doi.org/10.21519/0234-2758-2018-34-4-83-88Gavrilova YuK, Generalov SV, Abramova EG, Savitskaya LV, Galkina MV, Kochkin AV. Express analysis of activity of anti-rabies serum and anti-rabies immunoglobulin in cell cultures by immunofluorescence method. Biotekhnologiya = Biotechnology. 2018;34(4):83–8 (In Russ.) https://doi.org/10.21519/0234-2758-2018-34-4-83-88Hanners NW, Eitson JL, Usui N, Richardson RB, Wexler EM, Konopka G, Schoggins JW. Western Zika virus in human fetal neural progenitors persists long term with partial cytopathic and limited immunogenic effects. Cell Reports. 2016;15(11):2315–22. https://doi.org/10.1016/j.celrep.2016.05.075Hanners NW, Eitson JL, Usui N, Richardson RB, Wexler EM, Konopka G, Schoggins JW. Western Zika virus in human fetal neural progenitors persists long term with partial cytopathic and limited immunogenic effects. Cell Reports. 2016;15(11):2315–22. https://doi.org/10.1016/j.celrep.2016.05.075Fontana D, Prieto C, Kratje R, Etcheverrigaray M. Target cells for antibodies detection in rabies vaccine control. Vaccine. 2014;32(24):2805–7. https://doi.org/10.1016/j.vaccine.2014.02.030Fontana D, Prieto C, Kratje R, Etcheverrigaray M. Target cells for antibodies detection in rabies vaccine control. Vaccine. 2014;32(24):2805–7. https://doi.org/10.1016/j.vaccine.2014.02.030Rupprecht ChE, Fooks AR, Abela-Ridder B. Demonstration of Lyssavirus antigens by flow cytometry. In: Rupprecht ChE, Fooks AR, Abela-Ridder B. Laboratory Techniques in Rabies. 5th ed., Vol. 1. Geneva: WHO; 2018. P. 169–75.Rupprecht ChE, Fooks AR, Abela-Ridder B. Demonstration of Lyssavirus antigens by flow cytometry. In: Rupprecht ChE, Fooks AR, Abela-Ridder B. Laboratory Techniques in Rabies. 5th ed., Vol. 1. Geneva: WHO; 2018. P. 169–75.Vengatesan D, Raj GD, Raja A, Ramadass P, Gunaseelan L. Detection of rabies virus antigen or antibody using flow cytometry. Cytometry. 2006;70B(5):335–43. https://doi.org/10.1002/cyto.b.20104Vengatesan D, Raj GD, Raja A, Ramadass P, Gunaseelan L. Detection of rabies virus antigen or antibody using flow cytometry. Cytometry. 2006;70B(5):335–43. https://doi.org/10.1002/cyto.b.20104Генералов СВ, Кравцов АЛ, Кожевников ВА, Гаврилова ЮК, Абрамова ЕГ, Никифоров АК. Проточная цитометрия при анализе вируснейтрализующей активности антирабических сывороток и иммуноглобулина. Инфекция и иммунитет. 2019;9(1):107–14. https://doi.org/10.15789/2220-7619-2019-1-107-114Generalov SV, Kravtsov AL, Kozhevnikov VA, Gavrilova YuK, Abramova EG, Nikiforov AK. Flow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug. Infektsiya i immunitet = Russian Journal of Infection and Immunity. 2019;9(1):107–14 (In Russ.) https://doi.org/10.15789/2220-7619-2019-1-107-114Жилин ЕС, Акматова ЭК, Сансызбай АР. Выбор системы определения биологической активности вируса бешенства штамм «VRC-RZ2». Известия ВУЗов Кыргызстана. 2013;(4):131–8.Zhilin ЕS, Akmatova EК, Sansyzbai AR. The choice of system of determination of biological activity of rabies virus strain “VRC-RZ2”. Izvestiya VUZov Kyrgyzstana = Bulletin of Universities of Kyrgyzstan. 2013;(4):131–8 (In Russ.)Генералов СВ, Ерохин ПС, Красовская ТЮ, Осина НА, Абрамова ЕГ, Никифоров АК, Щербакова СА. Изучение ультраструктуры поверхности клеток линии Vero, инфицированных вирусом бешенства (RABV, Lissavirus, Rhabdoviridae). Вопросы вирусологии. 2017;62(5):227–32. https://doi.org/10.18821/0507-4088-2017-62-5-227-232Generalov SV, Erokhin PS, Krasovskaya TYu, Osina NA, Abramova EG, Nikiforov AK, Shcherbakova SA. A study of the ultrastructure of the surface of the transplantable line Vero cells infected with the rabies virus (RABV, Lissavirus, Rhabdoviridae). Voprosy virusologii = Problems of Virology. 2017;62(5):227–32 (In Russ.) https://doi.org/10.18821/0507-4088-2017-62-5-227-232Генералов СВ, Ерохин ПС, Абрамова ЕГ, Осина НА, Савицкая ЛВ, Кузнецов ОС и др. Способ определения специфической активности антирабического иммуноглобулина на клеточной культуре с применением атомно-силовой микроскопии. Патент Российской Федерации № 2688334; 2019.Generalov SV, Erokhin PS, Abramova EG, Osina NA, Savitskaya LV, Kuznetsov OS, et al. Method for determining specific activity of anti-rabies immunoglobulin on cell culture using atomic force microscopy. Patent of the Russian Federation No. 2688334; 2019 (In Russ.)Гулюкин АМ. Значимость современных методов лабораторной диагностики и идентификации возбудителя бешенства для иммунологического мониторинга данного зооноза. Вопросы вирусологии. 2014;59(3):5–10.Gulyukin AM. Significance of modern methods for laboratory detection of rabies agents and identification of the zoonose immunological survey. Voprosy virusologii = Problems of Virology. 2014;59(3):5–10 (In Russ.)Недосеков ВВ. Сравнительная оценка методов лабораторной диагностики бешенства. Ветеринарная патология. 2002;1:41–7.Nedosekov VV. Comparative evaluation of methods for laboratory diagnosis of rabies. Veterinarnaya patologiya = Veterinary Pathology. 2002;1:41–7 (In Russ.)Silva LHQ da, Bissoto CE, Carvalho C de, Cardoso TC, Pinheiro DM, Perri SHV. Comparison between the counter immunoelectrophoresis test and mouse neutralization test for the detection of antibodies against rabies virus in dog sera. Mem Inst Oswaldo Cruz. 2002;97(2):259–61. https://doi.org/10.1590/s0074-02762002000200020Silva LHQ da, Bissoto CE, Carvalho C de, Cardoso TC, Pinheiro DM, Perri SHV. Comparison between the counter immunoelectrophoresis test and mouse neutralization test for the detection of antibodies against rabies virus in dog sera. Mem Inst Oswaldo Cruz. 2002;97(2):259–61. https://doi.org/10.1590/s0074-02762002000200020Волкова РА, Рунова ВМ, Романова ЛН, Храпова ИС, Эльберт ЛБ, Мальдов ДГ. Применение ракетного иммуноэлектрофореза для определения гликопротеина в концентрированных антирабических вакцинах. Вопросы вирусологии. 1994;39(2):68–71. https://doi.org/10.1007/BF01718243Volkova RA, Runova VM, Romanova LN, Hrapova IS, El’bert LB, Mal’dov DG. The use of rocket immunoelectrophoresis for the determination of glycoprotein in concentrated rabies vaccines. Voprosy virusologii = Problems of Virology. 1994;39(2):68–71 (In Russ.)De Franco M, Massa S, Vassão RC, Siqueira M, Sant’Anna OA. Polygenic control of antibody production and correlation with vaccine induced resistance to rabies virus in high and low antibody responder mice. Arch Virol. 1996;141(8):1397–406. https://doi.org/10.1007/BF01718243De Franco M, Massa S, Vassão RC, Siqueira M, Sant’Anna OA. Polygenic control of antibody production and correlation with vaccine induced resistance to rabies virus in high and low antibody responder mice. Arch Virol. 1996;141(8):1397–406. https://doi.org/10.1007/BF01718243Madhusudana SN, Saraswati S. Development and evaluation of a latex agglutination test for rabies antibodies. J Clin Virol. 2003;27(2):129–35. https://doi.org/10.1016/s1386-6532(02)00135-xMadhusudana SN, Saraswati S. Development and evaluation of a latex agglutination test for rabies antibodies. J Clin Virol. 2003;27(2):129–35. https://doi.org/10.1016/s1386-6532(02)00135-xSaengseesom W, Kasempimolporn S, Akesowan S, Ouisuwan S, Sitprija V. Use of latex agglutination test to determine rabies antibodies in production of rabies antisera in horses. Southeast Asian J Trop Med Public Health. 2010;41(6):1387–92.Saengseesom W, Kasempimolporn S, Akesowan S, Ouisuwan S, Sitprija V. Use of latex agglutination test to determine rabies antibodies in production of rabies antisera in horses. Southeast Asian J Trop Med Public Health. 2010;41(6):1387–92.Сугобаева БП, Дадабаева ЖС, Сайдулдин ТС. Выявление рабических антител с помощью РСК. В кн.: Диагностика, лечение и профилактика инфекционных болезней животных Казахстана. Алма-Ата; 1989. С. 45–53.Sugobaeva BP, Dadabaeva ZhS, Sajduldin TS. Detection of rabid antibodies using CFT. In: Diagnostics, Treatment and Prevention of Infectious Animal Diseases in Kazakhstan. Alma-Ata; 1989. P. 45–53 (In Russ.)Сухарьков АЮ, Назаров НА, Метлин АЕ. Диагностика бешенства животных методом иммуноферментного анализа, сравнение прямого и непрямого сэндвич-варианта. Ветеринария Кубани. 2011;(6):12–4.Sukharkov AYu, Nazarov NA, Metlin AE. Diagnostics of animal rabies by enzyme immunoassay. Comparison of direct and indirect sandwich ELISA options. Veterinaria Kubani = Kuban Veterinary Medicine. 2011;(6):12–4 (In Russ.)Жилин ЕС, Кошеметов ЖК, Татыбаева АТ, Матвеева ВМ, Алиева АБ, Мыктыбаева ГБ. Способ получения антиидиотипических антител к антигенам вируса бешенства. Патент Республики Казахстан № 27436; 2013.Zhilin ES, Koshemetov ZhK, Tatybaeva AT, Matveeva VM, Alieva AB, Myktybaeva GB. Method for producing antiidiotypic antibodies to antigens of the rabies virus. Patent of the Republic of Kazakhstan No. 27436; 2013(In Russ.)Shankar BP. Advances in diagnosis of rabies. Veterinary World. 2009;2(2):74–8.Shankar BP. Advances in diagnosis of rabies. Veterinary World. 2009;2(2):74–8.Цетлин ЕМ, Волкова ВА. Отработка оптимальной схемы учета результатов при применении иммуноферментной тест-системы для определения антигенной активности культуральной антирабической вакцины. Вопросы вирусологии. 1996;(1):21–4.Cetlin EM, Volkova VA. Development of the optimal scheme for recording the results when using the enzyme immunoassay test system to determine the antigenic activity of the cultural rabies vaccine. Voprosy virusologii = Problems of Virology. 1996;(1):21–4 (In Russ.)Маркова ЕВ, Матвеева ИН, Попова ВМ. Инновационный подход к количественной оценке гликопротеина в вакцинах против бешенства. Таврический вестник аграрной науки. 2017;(2):17–28.Markova EV, Matveeva IN, Popova VM. An innovative approach to quantitative evaluation of glycoprotein in vaccines against rabies. Tavricheskiy vestnik agrarnoy nauki = Tavrichesky Bulletin of Agrarian Science. 2017;(2):17–28 (In Russ.)Salvi NC, Deopurkar RL, Waghmare AB, Khadilkar MV, Kalolikar MY, Gade SK, Mohite LS. Validation of indirect ELISA for quantitative testing of rabies antibodies during production of antirabies serum using equines. Proc Vaccinol. 2010;2(1):3–11. https://doi.org/10.1016/j.provac.2010.03.001Salvi NC, Deopurkar RL, Waghmare AB, Khadilkar MV, Kalolikar MY, Gade SK, Mohite LS. Validation of indirect ELISA for quantitative testing of rabies antibodies during production of antirabies serum using equines. Proc Vaccinol. 2010;2(1):3–11. https://doi.org/10.1016/j.provac.2010.03.001Jallet C, Tordo N. In Vitro ELISA test to evaluate rabies vaccine potency. J Vis Exp. 2020;159:e59641. https://doi.org/10.3791/59641Jallet C, Tordo N. In Vitro ELISA test to evaluate rabies vaccine potency. J Vis Exp. 2020;159:e59641. https://doi.org/10.3791/59641Korimbocus J, Dehay N, Tordo N, Cano F, Morgeaux S. Development and validation of a quantitative competitive ELISA for potency testing of equine anti rabies sera with other potential use. Vaccine. 2016;34(28):3310–6. https://doi.org/10.1016/j.vaccine.2016.04.086Korimbocus J, Dehay N, Tordo N, Cano F, Morgeaux S. Development and validation of a quantitative competitive ELISA for potency testing of equine anti rabies sera with other potential use. Vaccine. 2016;34(28):3310–6. https://doi.org/10.1016/j.vaccine.2016.04.086Dykman LA, Khlebtsov NG. Immunological properties of gold nanoparticles. Chem Sci. 2017;8(3):1719–35. https://doi.org/10.1039/C6SC03631GDykman LA, Khlebtsov NG. Immunological properties of gold nanoparticles. Chem Sci. 2017;8(3):1719–35. https://doi.org/10.1039/C6SC03631GAsgary V, Shoari A, Baghbani-Arani F, Shandiz SAS, Khosravy MS, Janani A, et al. Green synthesis and evaluation of silver nanoparticles as adjuvant in rabies veterinary vaccine. Int J Nanomedicine. 2016;(11):3597–605. https://doi.org/10.2147/IJN.S109098Asgary V, Shoari A, Baghbani-Arani F, Shandiz SAS, Khosravy MS, Janani A, et al. Green synthesis and evaluation of silver nanoparticles as adjuvant in rabies veterinary vaccine. Int J Nanomedicine. 2016;(11):3597–605. https://doi.org/10.2147/IJN.S109098Kasempimolporn S, Saengseesom W, Huadsakul S, Boonchang S, Sitprija V. Evaluation of a rapid immunochromatographic test strip for detection of Rabies virus in dog saliva samples. J Vet Diagn Invest. 2011;23(6):1197–201. https://doi.org/10.1177/1040638711425576Kasempimolporn S, Saengseesom W, Huadsakul S, Boonchang S, Sitprija V. Evaluation of a rapid immunochromatographic test strip for detection of Rabies virus in dog saliva samples. J Vet Diagn Invest. 2011;23(6):1197–201. https://doi.org/10.1177/1040638711425576Wang H, Feng N, Yang S, Wang C, Wang T, Gao Y, et al. A rapid immunochromatographic test strip for detecting rabies virus antibody. J Virol Methods. 2010;170(1–2):80–5. https://doi.org/10.1016/j.jviromet.2010.09.002Wang H, Feng N, Yang S, Wang C, Wang T, Gao Y, et al. A rapid immunochromatographic test strip for detecting rabies virus antibody. J Virol Methods. 2010;170(1–2):80–5. https://doi.org/10.1016/j.jviromet.2010.09.002Shiota S, Mannen K, Matsumoto T, Yamada K, Yasui T, Takayama K, et al. Development and evaluation of a rapid neutralizing antibody test for rabies. J Virol Methods. 2009;161(1):58–62. https://doi.org/10.1016/j.jviromet.2009.05.018Shiota S, Mannen K, Matsumoto T, Yamada K, Yasui T, Takayama K, et al. Development and evaluation of a rapid neutralizing antibody test for rabies. J Virol Methods. 2009;161(1):58–62. https://doi.org/10.1016/j.jviromet.2009.05.018David D, Yakobson B, Rotenberg D, Dveres N, Davidson I, Stram Y. Rabies virus detection by RT-PCR in decomposed naturally infected brains. Vet Microbiol. 2002;87(2):111–8. https://doi.org/10.1016/S0378-1135(02)00041-XDavid D, Yakobson B, Rotenberg D, Dveres N, Davidson I, Stram Y. Rabies virus detection by RT-PCR in decomposed naturally infected brains. Vet Microbiol. 2002;87(2):111–8. https://doi.org/10.1016/S0378-1135(02)00041-XДедков ВГ, Девяткин АА, Полещук ЕМ, Сафонова МВ, Маркелов МЛ, Шипулин ГА. Разработка и апробация набора реагентов для определения РНК классического вируса бешенства методом ОТ-ПЦР в реальном времени. Вопросы вирусологии. 2016;61(5):235–40. https://doi.org/10.18821/0507-4088-2016-61-4-235-240Dedkov VG, Deviatkin AA, Poleshchuk EM, Safonova MV, Markelov ML, Shipulin GA. Development and evaluation of the RT-PCR kit for the rabies virus diagnosis. Voprosy virusologii = Problems of Virology. 2016;61(5):235–40 (In Russ.) https://doi.org/10.18821/0507-4088-2016-61-4-235-240Nagaraj T, Vasanth JP, Desai A, Kamat A, Madhusudana SN, Ravi V. Ante mortem diagnosis of human rabies using saliva samples: comparison of real time and conventional RT-PCR techniques. J Clin Virol. 2006;36(1):17–23. https://doi.org/10.1016/j.jcv.2006.01.009Nagaraj T, Vasanth JP, Desai A, Kamat A, Madhusudana SN, Ravi V. Ante mortem diagnosis of human rabies using saliva samples: comparison of real time and conventional RT-PCR techniques. J Clin Virol. 2006;36(1):17–23. https://doi.org/10.1016/j.jcv.2006.01.009Bourhy H, Reynes J, Dunham E, Dacheux L, Larrous F, Huong VTQ, et al. The origin and phylogeography of dog rabies virus. J Gen Virol. 2008;89(11):2673–81. https://doi.org/10.1099/vir.0.2008/003913-0Bourhy H, Reynes J, Dunham E, Dacheux L, Larrous F, Huong VTQ, et al. The origin and phylogeography of dog rabies virus. J Gen Virol. 2008;89(11):2673–81. https://doi.org/10.1099/vir.0.2008/003913-0Moreira BLC, Pereira LA, Gimenez APL, Inagaki JMF, Raboni SM. Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines. J Virol Methods. 2019;270:46–51. https://doi.org/10.1016/j.jviromet.2019.04.025Moreira BLC, Pereira LA, Gimenez APL, Inagaki JMF, Raboni SM. Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines. J Virol Methods. 2019;270:46–51. https://doi.org/10.1016/j.jviromet.2019.04.025Moreira BLC, Gimenez APL, Inagaki JMF, Raboni SM. Inactivated rabies vaccines: Standardization of an in vitro assay for residual viable virus detection. PLoS Negl Trop Dis. 2020;14(3):e0008142. https://doi.org/10.1371/journal.pntd.0008142Moreira BLC, Gimenez APL, Inagaki JMF, Raboni SM. Inactivated rabies vaccines: Standardization of an in vitro assay for residual viable virus detection. PLoS Negl Trop Dis. 2020;14(3):e0008142. https://doi.org/10.1371/journal.pntd.0008142Sekar T, Premkumar AA, Mohan GC, Sekar B, Sundaran B, Sivakumar S. Quantification of rabies virus by Real Time PCR in comparison with mouse inoculation test (MIT) and fluorescent antibody test (FAT). Madridge J Vaccines. 2019;3(1):80–5. https://doi.org/10.18689/mjv-1000118Sekar T, Premkumar AA, Mohan GC, Sekar B, Sundaran B, Sivakumar S. Quantification of rabies virus by Real Time PCR in comparison with mouse inoculation test (MIT) and fluorescent antibody test (FAT). Madridge J Vaccines. 2019;3(1):80–5. https://doi.org/10.18689/mjv-1000118Игнатьев ГМ, Оксанич АС, Антонова ЛП, Самарцева ТГ, Мосолова СВ, Мефед КМ и др. Молекулярно-генетическое исследование стабильности и подтверждение подлинности штамма Внуково-32, применяемого для производства вакцины антирабической культуральной концентрированной очищенной инактивированной сухой. БИОпрепараты. Профилактика, диагностика, лечение. 2020;20(2):107–15. https://doi.org/10.30895/2221-996X-2020-20-2-107-115Ignatyev GM, Oksanich AS, Antonova LP, Samartseva TG, Mosolova SV, Mefed KM, et al. Molecular genetic testing of stability and identification of Vnukovo-32 strain used for production of the cultural concentrated purified inactivated dry rabies vaccine. BIOpreparaty. Profilaktika, diagnostika, lechenie = BIOpreparations. Prevention, Diagnosis, Treatment. 2020;20(2):107–15 (In Russ.) https://doi.org/10.30895/2221-996X-2020-20-2-107-115Тучков ИВ, Краснов ЯМ, Горяев АА, Матвеева ЖВ, Степанов АВ, Майоров НВ, Никифоров АК. Нуклеотидная последовательность и филогенетический анализ G гликопротеина российского фиксированного штамма «Москва 3253» вируса бешенства. Проблемы особо опасных инфекций. 2013;4:73–5. https://doi.org/10.21055/0370-1069-2013-4-73-75Tuchkov IV, Krasnov YM, Goryaev AA, Matveeva ZV, Stepanov AV, Mayorov NV, Nikiforov AK. Nucleotide sequence and phylogenetic analysis of G glycoprotein of the rabies virus strain “Moscow 3253” from Russia. Problemy osobo opasnykh infektsii = Problems of Particularly Dangerous Infections. 2013;4:73–5 (In Russ.) https://doi.org/10.21055/0370-1069-2013-4-73-75Лосич МА, Зайкова ОН, Непоклонова ИВ, Гребенникова ТВ, Верховский ОА, Одноворов АИ, Алипер ТИ. Молекулярно-биологическая характеристика вакцинного штамма ERA-CB 20M вируса бешенства. Вопросы вирусологии. 2018;63(5):224–32. https://doi.org/10.18821/0507-4088-2018-63-5-224-232Losich MA, Zajkova ON, Nepoklonova IV, Grebennikova TV, Verhovskij OA, Odnovorov AI, Aliper TI. Molecular and biological characteristics of vaccinary ERA-CB 20M of rabies virus. Voprosy virusologii = Problems of Virology. 2018;63(5):224–32 (In Russ.) https://doi.org/10.18821/0507-4088-2018-63-5-224-232Tao X, Han N, Guo, Z, Tang Q, Rayner S, Liang G. Molecular characterization of China human rabies vaccine strains. Virol Sin. 2013;28(2):116–23. https://doi.org/10.1007/s12250-013-3314-9Tao X, Han N, Guo, Z, Tang Q, Rayner S, Liang G. Molecular characterization of China human rabies vaccine strains. Virol Sin. 2013;28(2):116–23. https://doi.org/10.1007/s12250-013-3314-9Zhu S, Wang C, Zhang P, Li H, Luo S, Guo C. Sequencing and molecular characterization of CTNCEC25, a China fixed rabies virus vaccine strain CTN-1 adapted to primary chicken embryo cells. Virol J. 2014;11:176. https://doi.org/10.1186/1743-422X-11-176Zhu S, Wang C, Zhang P, Li H, Luo S, Guo C. Sequencing and molecular characterization of CTNCEC25, a China fixed rabies virus vaccine strain CTN-1 adapted to primary chicken embryo cells. Virol J. 2014;11:176. https://doi.org/10.1186/1743-422X-11-176Зайкова ОН, Гребенникова ТВ, Гулюкин АМ, Шабейкин АА, Полякова ИВ, Метлин АЕ. Молекулярно-генетическая характеристика полевых изолятов вируса бешенства, выявленных на территории Владимирской, Московской, Тверской, Нижегородской и Рязанской областей. Вопросы вирусологии. 2017;62(3):101–8. https://doi.org/10.18821/0507-4088-2017-62-3-101-108Zaykova ON, Grebennikova TV, Gulyukin AM, Shabeykin AA, Polyakova IV, Metlin AE. Molecular-genetic characterization of field isolates of rabies virus identified in the territory of Vladimir, Moscow, Tver, Nizhny Novgorod and Ryazan regions. Voprosy virusologii = Problems of Virology. 2017;62(3):101–8 (In Russ.) https://doi.org/10.18821/0507-4088-2017-62-3-101-108

The authors declare that there are no conflicts of interest present.