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Вопросы вирусологии. 2021; 66: 29-39

Биологическая характеристика и пермиссивность к вирусам штамма диплоидных клеток почки летучей мыши нетопыря Натузиуса (Pipistrellus nathusii Keyserling & Blasius, 1839; (Chiroptera: Microchiroptera: Vespertilionidae)

Поволяева О. С., Юрков С. Г., Лаптева О. Г., Колбасова О. Л., Чадаева А. А., Кольцов А. Ю., Синдрякова И. П., Власов М. Е., Живодёров С. П., Луницин А. В.

https://doi.org/10.36233/0507-4088-12

Аннотация

Введение. Летучие мыши (Microchiroptera) являются эпидемиологически важным естественным резервуаром вирусов различных таксономических групп, включая возбудителей особо опасных болезней человека и животных. Учитывая актуальность арбовирусных инфекций, представляется целесообразным проведение исследований по изучению спектра чувствительности клеток из тканей летучих мышей, обитающих и мигрирующих на территории Российской Федерации, к вирусам векторных инфекций сельскохозяйственных животных.

Цель исследования - получение диплоидного штамма клеток почечной ткани летучей мыши (ПЛМ) вида нетопырь лесной, или нетопырь Натузиуса (Pipistrellus nathusii), изучение его биологических характеристик, а также оценка пермиссивности полученной клеточной культуры к вирусам блютанга, лихорадки долины Рифт (ЛДР), заразного узелкового дерматита (ЗУД) крупного рогатого скота (КРС), миксомы кроликов (Myxomatosis cuniculi), фибромы Шоупа, африканской чумы лошадей (АЧЛ) и африканской чумы свиней (АЧС).

Материал и методы. Донорами органов служили 2 особи клинически здоровых самцов летучей мыши Р. nathusii. Для получения диплоидного штамма культуры клеток почки этого вида и изучения свойств полученной клеточной культуры градации от 6-го и выше пассажных уровней использовали традиционные цитологические, вирусологические и молекулярные методы. Определяли пермиссивность данного штамма к вирусам блютанга, ЛДР, ЗУД, миксомы кроликов, фибромы Шоупа, АЧЛ и АЧС.

Результаты. Формирование конфлюэнтного монослоя наблюдали через 72 ч, при этом индекс пролиферации (ИП) был равен 2,7-3,3. Клеточный монослой сохранялся без смены среды в течение 45 сут (срок наблюдения). Показана стабильность кариотипа в условиях непрерывного субкультивирования на уровне 36-го пассажа. Культура клеток получила наименование «Штамм диплоидных клеток почки летучей мыши Pipistrellus nathusii (Diploid cell line Pipistrellus nathusii kidney)»; установлена её пермиссивность к вирусам блютанга, ЛДР, ЗУД и миксомы кроликов.

Обсуждение. Чувствительность полученного клеточного материала к вирусам блютанга и ЛДР согласуется с данными об идентификации реовируса и возбудителя ЛДР у египетских фруктовых летучих мышей (Rousettus aegyptiacus), а пермиссивность данного штамма к возбудителям ЗУД и миксомы кроликов - с результатами обнаружения поксвирусов у вида поздний кожан (Eptesicus fuscus).

Выводы. Получен и паспортизирован штамм диплоидных клеток ПЛМ P nathusii. Установленная пермиссивность к вирусам блютанга, ЛДР, ЗУД и миксомы кроликов позволяет использовать его для выделения и изучения этих патологических агентов. Репродукция возбудителей в клетках данного штамма из тканей ПЛМ вида P nathusii, обитающего и мигрирующего на территории европейской части Российской Федерации, указывает на его потенциальную роль в эпидемиологии значимых инфекций, особенно трансмиссивных.

Список литературы

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Problems of Virology. 2021; 66: 29-39

Biological characteristics and permissiveness to viruses of diploid kidney cells strain from the bat Nathusius’ pipistrelle (Pipistrellus nathusii Keyserling & Blasius, 1839; Chiroptera: Microchiroptera: Vespertilionidae)

Povolyaeva O. S., Yurkov S G., Lapteva O. G., Kolbasova O. L., Chadaeva A. A., Kol'tsov А. Yu., Sindryakova I. P., Vlasov M. E., Zhivoderov S. P., Lunitsin A. V.

https://doi.org/10.36233/0507-4088-12

Abstract

Introduction. Bats are an epidemiologically important natural reservoir of viruses of various taxonomic groups, including causative agents of especially dangerous infections of humans and animals. Considering the relevance of arbovirus infections, it seems advisable to study the spectrum of the sensitivity of cells derived from bats inhabiting and migrating on the territory of the Russian Federation to causative agents of vector-borne diseases of animals.

The study aimed to obtain a diploid strain of cells from renal tissue of bats Pipistrellus nathusii and to investigate its biological characteristics, as well as to assess its permissiveness for bluetongue (BTV); Rift Valley fever (RVFV); lumpy skin disease (LSDV); rabbit myxoma (Myxomatosis cuniculi); rabbit, or Shope fibroma (RFV); African horse sickness (AHSV) and African swine fever (ASFV) viruses.

Material and methods. There were 2 clinically healthy male individuals of P. nathusii who taken as donors of organs. To obtain diploid kidney cell culture strain and to study its properties, the level of the 6th passage was investigated by conventional cytological, virological, and molecular methods. The permissiveness of the obtained cell culture for BTV, RVFV, LSDV, Myxomatosis cuniculi, RFV, AHSV and ASFV was determined.

Results. The formation of a confluent monolayer was observed after 72 hours, while the proliferation index was 2.7-3.3. The cell monolayer had been maintained without changing the medium for 45 days (observation period). The stability of the karyotype had been demonstrated in continuous subculturing at the 36th passage. The cell culture named «Diploid cell line Pipistrellus nathusii kidney», and its permissiveness to BTV, RVFV, LSDV and Myxomatosis cuniculi had been demonstrated.

Discussion. The sensitivity of the strain to BTV and RVFV is consistent with the data on the identification of reovirus and RVFV in Egyptian fruit bats (Rousettus aegyptiacus), and its permissiveness for LSDV and rabbits myxoma virus is consistent with the results of detection of poxviruses in big brown bat (Eptesicus fuscus). Conclusion. A diploid kidney cell strain derived from P. nathusii was obtained and certified. Its permissiveness to BTV, RVFV, LSDV and rabbits myxoma viruses makes it possible to use this strain for isolation and studies of these viruses. Reproduction of the viruses in diploid kidney cells strain derived from P. nathusii living and migrating in the European part of the Russian Federation indicates their potential role in the epidemiology of significant infections, especially transmissible ones.

References

1. IUCN SSC Bat Specialist Group. Available at: https://www.iucn.org/commissions/ssc-groups/mammals/specialist-groups-a-e/bat (accessed January 18, 2021).

2. Baker M.L., Schountz T., Wang L.F. Antiviral immune responses of bats: a review. Zoonoses Public Health. 2013; 60(1): 104-16. https://doi.org/10.1111/j.1863-2378.2012.01528.x.

3. Calisher C.H., Childs J.E., Field H.E., Holmes K.V, Schountz T. Bats: Important reservoir hosts of emerging viruses. Clin. Microbiol. Rev. 2006; 19(3): 531-45. https://doi.org/10.1128/CMR.00017-06.

4. Wang L.F., Walker P.J., Poon L.L. Mass extinctions, biodiversity and mitochondrial function: are bats ‘special’ as reservoirs for emerging viruses? Curr. Opin. Virol. 2011; 1(6): 649-57. https://doi.org/10.1016/j.coviro.2011.10.013.

5. Drexler J.F., Corman V.M., Wegner T., Tateno A.F., Zerbinati R.M., Gloza-Rausch F., et al. Amplification of emerging viruses in a bat colony. Emerg. Infect. Dis. 2011; 17(3): 449-56. https://doi.org/10.3201/eid1703.100526.

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13. Kohl C., Lesnik R., Brinkmann A., Ebinger A., Radonic A., Nitsche A., et al. Isolation and characterization of three mammalian orthoreo-viruses from European bats. PLoS One. 2012; 7(8): e43106. https://doi.org/10.1371/journal.pone.0043106.

14. Chua K.B., Koh C.L., Hooi P.S., Wee K.F., Khong J.H., Chua B.H., et al. Isolation of Nipah virus from Malaysian Island flying-foxes. Microbes Infect. 2002; 4(2): 145-51. https://doi.org/10.1016/s1286-4579(01)01522-2.

15. Halpin K., Young P.L., Field H.E., Mackenzie J.S. Isolation of Hen-dra virus from pteropid bats: a natural reservoir of Hendra virus. J. Gen. Virol.2000; 81(Pt. 8): 1927-32. https://doi.org/10.1099/0022-1317-81-8-1927.

16. Albarino C.G., Foltzer M., Towne J.S., Rowe L.A., Campbell S., Jaramillo C.M., et al. Novel paramyxovirus associated with severe acute febrile disease, South Sudan and Uganda, 2012. Emerg. Infect. Dis. 2014; 20(2): 211-6. https://doi.org/10.3201/eid2002.131620.

17. Waruhiu C., Ommeh S., Obanda V., Agwanda B., Gakuya F., Ge X.Y, et al. Molecular detection of viruses in Kenyan bats and discovery of novel astroviruses, caliciviruses and rotaviruses. Virol. Sin. 2017; 32(2): 101-14. https://doi.org/10.1007/s12250-016-3930-2.

18. Zhang H., Todd S., Tachedjian M., Barr J.A., Luo M., Yu M., et al. A novel bat herpesvirus encodes homologues of major histocompatibility complex classes I and II, C-type lectin, and a unique family of immune-related genes. J. Virol. 2012; 86(15): 8014-30. https://doi.org/10.1128/jvi.00723-12.

19. Graves D.C., Ferrer J.F. In vitro transmission and propagation of the bovine leukemia virus in monolayer cell cultures. Cancer Res. 1976; 36(11 Pt. 1): 4152-9.

20. Sandekian V., Lim D., Prud’homme P., Lemay G. Transient high level mammalian reovirus replication in a bat epithelial cell line occurs without cytopathic effect. Virus Res. 2013; 173(2): 327-35. https://doi.org/10.1016/j.virusres.2013.01.010.

21. Slater T., Eckerle I., Chang K. Bat lung epithelial cells show greater host species-specific innate resistance than MDCK cells to human and avian influenza viruses. Virol. J. 2018; 15(1): 68. https://doi.org/10.1186/s12985-018-0979-6.

22. Jordan I., Horn D., Oehmke S., Leendertz F.H., Sandig V. Cell lines from the Egyptian fruit bat are permissive for modified vaccinia Ankara. Virus Res. 2009; 145(1): 54-62. https://doi.org/10.1016/).virusres.2009.06.007.

23. Crameri G., Todd S., Grimley S., McEachern J.A., Marsh G.A., Smith C., et al. Establishment, immortalisation and characterisation of pteropid bat cell lines. PLoS One. 2009; 4(12): e8266. https://doi. org/10.1371/journal.pone.0008266.

24. Banerjee A., Misra V., Schountz T., Baker M.L. Tools to study pathogen-host interactions in bats. Virus Res. 2018; 248: 5-12. https://doi.org/10.1016.

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