BACKGROUND: Coevolution between pathogens and their hosts decreases host morbidity and mortality. Bats host and can tolerate viruses which can be lethal to other vertebrate orders, including humans. Bat adaptations to infection include localized immune response, early pathogen sensing, high interferon expression without pathogen stimulation, and regulated inflammatory response. The immune reaction is costly, and bats suppress high-cost metabolism during torpor. In the temperate zone, bats hibernate in winter, utilizing a specific behavioural adaptation to survive detrimental environmental conditions and lack of energy resources. Hibernation torpor involves major physiological changes that pose an additional challenge to bat-pathogen coexistence. Here, we compared bat cellular reaction to viral challenge under conditions simulating hibernation, evaluating the changes between torpor and euthermia. RESULTS: We infected the olfactory nerve-derived cell culture of Myotis myotis with an endemic bat pathogen, European bat lyssavirus 1 (EBLV-1). After infection, the bat cells were cultivated at two different temperatures, 37 °C and 5 °C, to examine the cell response during conditions simulating euthermia and torpor, respectively. The mRNA isolated from the cells was sequenced and analysed for differential gene expression attributable to the temperature and/or infection treatment. In conditions simulating euthermia, infected bat cells produce an excess signalling by multitude of pathways involved in apoptosis and immune regulation influencing proliferation of regulatory cell types which can, in synergy with other produced cytokines, contribute to viral tolerance. We found no up- or down-regulated genes expressed in infected cells cultivated at conditions simulating torpor compared to non-infected cells cultivated under the same conditions. When studying the reaction of uninfected cells to the temperature treatment, bat cells show an increased production of heat shock proteins (HSPs) with chaperone activity, improving the bat's ability to repair molecular structures damaged due to the stress related to the temperature change. CONCLUSIONS: The lack of bat cell reaction to infection in conditions simulating hibernation may contribute to the virus tolerance or persistence in bats. Together with the cell damage repair mechanisms induced in response to hibernation, the immune regulation may promote bats' ability to act as reservoirs of zoonotic viruses such as lyssaviruses.

Department of Botany and Zoology Masaryk University Kotlářská 2 61137 Brno Czechia

Department of Ecology and Diseases of Zoo Animals Game Fish and Bees University of Veterinary Sciences Brno Palackého třída 1946 1 61242 Brno Czechia

Institut Pasteur Université Paris Cité Bioinformatics and Biostatistics Hub 28 rue du Docteur Roux 75724 Paris Cedex 15 France

Institut Pasteur Université Paris Cité Lyssavirus Epidemiology and Neuropathology Unit 28 rue du Docteur Roux 75724 Paris Cedex 15 France

Institute of Vertebrate Biology Czech Academy of Sciences Květná 8 60300 Brno Czechia

RECETOX Masaryk University Kotlářská 2 61137 Brno Czechia

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Bouma HR, Carey HV, Kroese FG. Hibernation: the immune system at rest? J Leukoc Biol. 2010;88(4):619–624. doi: 10.1189/jlb.0310174. PubMed DOI

Martínková N, Pikula J, Zukal J, Kovacova V, Bandouchova H, Bartonička T, et al. Hibernation temperature-dependent pseudogymnoascus destructans infection intensity in palearctic bats. Virulence. 2018;9:1734–1750. doi: 10.1080/21505594.2018.1548685. PubMed DOI PMC

Davis WH. Hibernation: ecology and physiological ecology. Biol Bats. 1970;1:265–300. doi: 10.1016/B978-0-12-758001-2.50013-7. DOI

Lyman CP. Thermoregulation and metabolism in bats. Biol Bats. 1970;1:301–330. doi: 10.1016/B978-0-12-758001-2.50014-9. DOI

Currie SE, Stawski C, Geiser F. Cold-hearted bats: uncoupling of heart rate and metabolism during torpor at sub-zero temperatures. J Exp Biol. 2018;221:jeb170894. PubMed

Field KA, Johnson JS, Lilley TM, Reeder SM, Rogers EJ, Behr MJ, et al. The white-nose syndrome transcriptome: activation of anti-fungal host responses in wing tissue of hibernating little brown Myotis. PLoS Pathog. 2015;11:e1005168. doi: 10.1371/journal.ppat.1005168. PubMed DOI PMC

Field KA, Sewall BJ, Prokkola JM, Turner GG, Gagnon MF, Lilley TM, et al. Effect of torpor on host transcriptomic responses to a fungal pathogen in hibernating bats. Mol Ecol. 2018;27:3727–3743. doi: 10.1111/mec.14827. PubMed DOI

Fritze M, Costantini D, Fickel J, Wehner D, Czirják GÁ, Voigt CC. Immune response of hibernating European bats to a fungal challenge. Biol Open. 2019;8:bio046078. doi: 10.1242/bio.046078. PubMed DOI PMC

Gerow CM, Rapin N, Voordouw MJ, Elliot M, Misra V, Subudhi S. Arousal from hibernation and reactivation of Eptesicus fuscus gammaherpesvirus (Ef HV) in big brown bats. Transbound Emerg Dis. 2019;66:1054–1062. doi: 10.1111/tbed.13102. PubMed DOI

Kurtz CC, Carey HV. Seasonal changes in the intestinal immune system of hibernating ground squirrels. Dev Comp Immunol. 2007;31:415–428. doi: 10.1016/j.dci.2006.07.003. PubMed DOI

Ganeshan K, Nikkanen J, Man K, Leong YA, Sogawa Y, Maschek JA, et al. Energetic trade-offs and hypometabolic states promote disease tolerance. Cell. 2019;177:399–413.e12. doi: 10.1016/j.cell.2019.01.050. PubMed DOI PMC

Irving AT, Zhang Q, Kong PS, Luko K, Rozario P, Wen M, et al. Interferon regulatory factors IRF1 and IRF7 directly regulate gene expression in bats in response to viral infection. Cell Rep. 2020;33:108345. doi: 10.1016/j.celrep.2020.108345. PubMed DOI PMC

Logan SM, Storey KB. Markers of tissue remodeling and inflammation in the white and brown adipose tissues of a model hibernator. Cell Signal. 2021;82:109975. doi: 10.1016/j.cellsig.2021.109975. PubMed DOI

Kurtz CC, Otis JP, Regan MD, Carey HV. How the gut and liver hibernate. Compart Biochem Physiol Part A Mol Integr Physiol. 2021;253:110875. doi: 10.1016/j.cbpa.2020.110875. PubMed DOI PMC

Banerjee A, Rapin N, Bollinger T, Misra V. Lack of inflammatory gene expression in bats: a unique role for a transcription repressor. Sci Rep. 2017;7:2232. doi: 10.1038/s41598-017-01513-w. PubMed DOI PMC

Clayton E, Munir M. Fundamental Characteristics of Bat Interferon Systems. Front Cell Infect Microbiol. 2020;10:527921. doi: 10.3389/fcimb.2020.527921. PubMed DOI PMC

Zhou P, Tachedjian M, Wynne JW, Boyd V, Cui J, Smith I, et al. Contraction of the type I IFN locus and unusual constitutive expression of IFN- PubMed DOI PMC

Kuzmin IV, Schwarz TM, Ilinykh PA, Jordan I, Ksiazek TG, Sachidanandam R, et al. Innate immune responses of bat and human cells to filoviruses: commonalities and distinctions. J Virol. 2017;91:e02471. doi: 10.1128/JVI.02471-16. PubMed DOI PMC

Xie J, Li Y, Shen X, Goh G, Zhu Y, Cui J, et al. Dampened STING-dependent interferon activation in bats. Cell Host Microbe. 2018;23:297–301. doi: 10.1016/j.chom.2018.01.006. PubMed DOI PMC

Ahn M, Anderson DE, Zhang Q, Tan CW, Lim BL, Luko K, et al. Dampened NLRP3-mediated inflammation in bats and implications for a special viral reservoir host. Nat Microbiol. 2019;4:789–799. doi: 10.1038/s41564-019-0371-3. PubMed DOI PMC

Serra-Cobo J, Amengual B, Abellán C, Bourhy H. European bat lyssavirus infection in Spanish bat populations. Emerg Infect Dis. 2002;8:413. doi: 10.3201/eid0804.010263. PubMed DOI PMC

Amengual B, Bourhy H, López-Roig M, Serra-Cobo J. Temporal dynamics of European bat Lyssavirus type 1 and survival of Myotis myotis bats in natural colonies. PLoS ONE. 2007;2:e566. doi: 10.1371/journal.pone.0000566. PubMed DOI PMC

Seidlova V, Zukal J, Brichta J, Anisimov N, Apoznański G, Bandouchova H, et al. Active surveillance for antibodies confirms circulation of lyssaviruses in Palearctic bats. BMC Vet Res. 2020;16:482. doi: 10.1186/s12917-020-02702-y. PubMed DOI PMC

Turmelle A, Jackson F, Green D, McCracken G, Rupprecht C. Host immunity to repeated rabies virus infection in big brown bats. J Gen Virol. 2010;91:2360. doi: 10.1099/vir.0.020073-0. PubMed DOI PMC

Davis AD, Rudd RJ, Bowen RA. Effects of aerosolized rabies virus exposure on bats and mice. J Infect Dis. 2007;195:1144–1150. doi: 10.1086/512616. PubMed DOI

Troupin C, Picard-Meyer E, Dellicour S, Casademont I, Kergoat L, Lepelletier A, et al. Host genetic variation does not determine spatio-temporal patterns of European bat 1 lyssavirus. Genome Biol Evol. 2017;9:3202–3213. doi: 10.1093/gbe/evx236. PubMed DOI PMC

Tjørnehøj K, Fooks A, Agerholm J, Rønsholt L. Natural and experimental infection of sheep with European bat lyssavirus type-1 of Danish bat origin. J Comp Pathol. 2006;134:190–201. doi: 10.1016/j.jcpa.2005.10.005. PubMed DOI

Dacheux L, Larrous F, Mailles A, Boisseleau D, Delmas O, Biron C, et al. European bat lyssavirus transmission among cats. Eur Emerg Infect Dis. 2009;15:280. doi: 10.3201/eid1502.080637. PubMed DOI PMC

Müller T, Cox J, Peter W, Schäfer R, Johnson N, McElhinney L, et al. Spill-over of European bat lyssavirus type 1 into a stone marten (Martes foina) in Germany. J Vet Med Ser B. 2004;51:49–54. doi: 10.1111/j.1439-0450.2003.00725.x. PubMed DOI

Regnault B, Evrard B, Plu I, Dacheux L, Troadec E, Cozette P, et al. First case of lethal encephalitis in Western Europe due to European bat lyssavirus type 1. Clin Infect Dis. 2022;74:461–466. doi: 10.1093/cid/ciab443. PubMed DOI

Hicks D, Nunez A, Healy D, Brookes S, Johnson N, Fooks A. Comparative pathological study of the murine brain after experimental infection with classical rabies virus and European bat lyssaviruses. J Comp Pathol. 2009;140:113–126. doi: 10.1016/j.jcpa.2008.09.001. PubMed DOI

Hicks D, Nunez A, Banyard A, Williams A, Ortiz-Pelaez A, Fooks A, et al. Differential chemokine responses in the murine brain following lyssavirus infection. J Comp Pathol. 2013;149:446–462. doi: 10.1016/j.jcpa.2013.04.001. PubMed DOI

Kuzmin IV, Botvinkin AD, Shaimardanov RT. Experimental lyssavirus infection in chiropters. Vopr Virusol. 1994;39(1):17–21. PubMed

Parize P, Travecedo Robledo IC, Cervantes-Gonzalez M, Kergoat L, Larrous F, Serra-Cobo J, et al. Circumstances of Human-Bat interactions and risk of lyssavirus transmission in metropolitan France. Zoonoses Public Health. 2020;67:774–784. doi: 10.1111/zph.12747. PubMed DOI

Robardet E, Borel C, Moinet M, Jouan D, Wasniewski M, Barrat J, et al. Longitudinal survey of two serotine bat (Eptesicus serotinus) maternity colonies exposed to EBLV-1 (European Bat Lyssavirus type 1): Assessment of survival and serological status variations using capture-recapture models. PLoS Negl Trop Dis. 2017;11:e0006048. doi: 10.1371/journal.pntd.0006048. PubMed DOI PMC

Colombi D, Serra-Cobo J, Métras R, Apolloni A, Poletto C, López-Roig M, et al. Mechanisms for lyssavirus persistence in non-synanthropic bats in Europe: insights from a modeling study. Sci Rep. 2019;9:1–11. doi: 10.1038/s41598-018-36485-y. PubMed DOI PMC

Freuling C, Vos A, Johnson N, Kaipf I, Denzinger A, Neubert L, et al. Experimental infection of serotine bats (Eptesicus serotinus) with European bat lyssavirus type 1a. J Gen Virol. 2009;90:2493–2502. doi: 10.1099/vir.0.011510-0. PubMed DOI

Eggerbauer E, Pfaff F, Finke S, Höper D, Beer M, Mettenleiter TC, et al. Comparative analysis of European bat lyssavirus 1 pathogenicity in the mouse model. PLoS Negl Trop Dis. 2017;11:e0005668. doi: 10.1371/journal.pntd.0005668. PubMed DOI PMC

Davy CM, Donaldson ME, Bandouchova H, Breit AM, Dorville NAS, Dzal YA, et al. Transcriptional host-pathogen responses of Pseudogymnoascus destructans and three species of bats with white-nose syndrome. Virulence. 2020;11:781–794. doi: 10.1080/21505594.2020.1768018. PubMed DOI PMC

He X, Korytář T, Zhu Y, Pikula J, Bandouchova H, Zukal J, et al. Establishment of Myotis myotis cell lines-model for investigation of host-pathogen interaction in a natural host for emerging viruses. PLoS ONE. 2014;9:e109795. doi: 10.1371/journal.pone.0109795. PubMed DOI PMC

Šimić I, Lojkić I, Krešić N, Cliquet F, Picard-Meyer E, Wasniewski M, et al. Molecular and serological survey of lyssaviruses in Croatian bat populations. BMC Vet Res. 2018;14:274. doi: 10.1186/s12917-018-1592-z. PubMed DOI PMC

Van Brussel K, Holmes EC. Zoonotic disease and virome diversity in bats. Curr Opin Virol. 2022;52:192–202. doi: 10.1016/j.coviro.2021.12.008. PubMed DOI PMC

Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T. Bats: Important Reservoir Hosts of Emerging Viruses. Clin Microbiol Rev. 2006;19:531–545. doi: 10.1128/CMR.00017-06. PubMed DOI PMC

Fuoco NL, Fernandes ER, dos Ramos Silva S, Luiz FG, Ribeiro OG, Katz ISS. Street rabies virus strains associated with insectivorous bats are less pathogenic than strains isolated from other reservoirs. Antivir Res. 2018;160:94–100. doi: 10.1016/j.antiviral.2018.10.023. PubMed DOI

Franka R, Johnson N, Müller T, Vos A, Neubert L, Freuling C, et al. Susceptibility of North American big brown bats (Eptesicus fuscus) to infection with European bat lyssavirus type 1. J Gen Virol. 2008;89:1998–2010. doi: 10.1099/vir.0.83688-0. PubMed DOI

Leopardi S, Priori P, Zecchin B, Poglayen G, Trevisiol K, Lelli D, et al. Active and passive surveillance for bat lyssaviruses in Italy revealed serological evidence for their circulation in three bat species. Epidemiol Infect 2019;147. PubMed PMC

Harazim M, Piálek L, Pikula J, Seidlovái V, Zukal J, Bachorec E, et al. Associating physiological functions with genomic variability in hibernating bats. Evol Ecol. 2021;35:291–308. doi: 10.1007/s10682-020-10096-4. DOI

Chionh YT, Cui J, Koh J, Mendenhall IH, Ng JH, Low D, et al. High basal heat-shock protein expression in bats confers resistance to cellular heat/oxidative stress. Cell Stress Chaperones. 2019;24:835–849. doi: 10.1007/s12192-019-01013-y. PubMed DOI PMC

Faherty SL, Villanueva-Cañas JL, Blanco MB, Albà MM, Yoder AD. Transcriptomics in the wild: Hibernation physiology in free-ranging dwarf lemurs. Mol Ecol. 2018;27:709–722. doi: 10.1111/mec.14483. PubMed DOI

Chang YF, Imam JS, Wilkinson MF. The nonsense-mediated decay RNA surveillance pathway. Annu Rev Biochem. 2007;76:51–74. doi: 10.1146/annurev.biochem.76.050106.093909. PubMed DOI

Lei M, Dong D, Mu S, Pan YH, Zhang S. Comparison of brain transcriptome of the greater horseshoe bats (Rhinolophus ferrumequinum) in active and torpid episodes. PLoS ONE. 2014;9:e107746. doi: 10.1371/journal.pone.0107746. PubMed DOI PMC

Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, Henson PM. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF- PubMed DOI PMC

Schuster N, Krieglstein K. Mechanisms of TGF- PubMed DOI

Wang ZW, Sarmento L, Wang Y, Li Xq, Dhingra V, Tseggai T, et al. Attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system. J Virol. 2005;79:12554–12565. doi: 10.1128/JVI.79.19.12554-12565.2005. PubMed DOI PMC

Yang BH, Wang K, Wan S, Liang Y, Yuan X, Dong Y, et al. TCF1 and LEF1 control Treg competitive survival and Tfr development to prevent autoimmune diseases. Cell Rep. 2019;27:3629–3645. doi: 10.1016/j.celrep.2019.05.061. PubMed DOI PMC

Martínez Gómez JM, Periasamy P, Dutertre CA, Irving AT, Ng JHJ, Crameri G, et al. Phenotypic and functional characterization of the major lymphocyte populations in the fruit-eating bat Pteropus alecto. Sci Rep. 2016;6:37796. doi: 10.1038/srep37796. PubMed DOI PMC

Feldman EL, Sullivan KA, Kim B, Russell JW. Insulin-like growth factors regulate neuronal differentiation and survival. Neurobiol Dis. 1997;4:201–214. doi: 10.1006/nbdi.1997.0156. PubMed DOI

Peirce MJ, Brook M, Morrice N, Snelgrove R, Begum S, Lanfrancotti A, et al. Themis2/ICB1 is a signaling scaffold that selectively regulates macrophage Toll-like receptor signaling and cytokine production. PLoS ONE. 2010;5:e11465. doi: 10.1371/journal.pone.0011465. PubMed DOI PMC

Guito JC, Prescott JB, Arnold CE, Amman BR, Schuh AJ, Spengler JR, et al. Asymptomatic infection of Marburg virus reservoir bats is explained by a strategy of immunoprotective disease tolerance. Curr Biol. 2021;31:257–270. doi: 10.1016/j.cub.2020.10.015. PubMed DOI

Begeman L, Suu-Ire R, Banyard AC, Drosten C, Eggerbauer E, Freuling CM, et al. Experimental Lagos bat virus infection in straw-colored fruit bats: A suitable model for bat rabies in a natural reservoir species. PLoS Negl Trop Dis. 2020;14:e0008898. doi: 10.1371/journal.pntd.0008898. PubMed DOI PMC

Zhang G, Cowled C, Shi Z, Huang Z, Bishop-Lilly KA, Fang X, et al. Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science. 2013;339:456–460. doi: 10.1126/science.1230835. PubMed DOI PMC

Schad J, Voigt CC. Adaptive evolution of virus-sensing toll-like receptor 8 in bats. Immunogenetics. 2016;68:783–795. doi: 10.1007/s00251-016-0940-z. PubMed DOI PMC

Harazim M, Horáček I, Jakešová L, Luermann K, Moravec JC, Morgan S, et al. Natural selection in bats with historical exposure to white-nose syndrome. BMC Zool. 2018;3:8. doi: 10.1186/s40850-018-0035-4. DOI

Delmas O, Holmes EC, Talbi C, Larrous F, Dacheux L, Bouchier C, et al. Genomic diversity and evolution of the lyssaviruses. PLoS ONE. 2008;3:e2057. doi: 10.1371/journal.pone.0002057. PubMed DOI PMC

Mareuil F, Doppelt-Azeroual O, Ménager H. A public Galaxy platform at Pasteur used as an execution engine for web services. F1000Research. 2017;6.

Dacheux L, Dommergues L, Chouanibou Y, Doméon L, Schuler C, Bonas S, et al. Co-circulation and characterization of novel African arboviruses (genus Ephemerovirus) in cattle, Mayotte island, Indian Ocean, 2017. Transbound Emerg Dis. 2019;66:2601–2604. doi: 10.1111/tbed.13323. PubMed DOI PMC

Cokelaer T, Desvillechabrol D, Legendre R, Cardon M. ‘Sequana’: a set of Snakemake NGS pipelines. J Open Source Softw. 2017;2:352. doi: 10.21105/joss.00352. DOI

Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011;17:10–12. doi: 10.14806/ej.17.1.200. DOI

Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. doi: 10.1093/bioinformatics/bts635. PubMed DOI PMC

Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–930. doi: 10.1093/bioinformatics/btt656. PubMed DOI

Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. doi: 10.1186/s13059-014-0550-8. PubMed DOI PMC

Fabregat A, Sidiropoulos K, Viteri G, Forner O, Marin-Garcia P, Arnau V, et al. Reactome pathway analysis: a high-performance in-memory approach. BMC Bioinform. 2017;18:142. doi: 10.1186/s12859-017-1559-2. PubMed DOI PMC

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:1–12. doi: 10.1186/gb-2002-3-7-research0034. PubMed DOI PMC

Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45–e45. doi: 10.1093/nar/29.9.e45. PubMed DOI PMC

Higher antibody titres against Pseudogymnoascus destructans are associated with less white-nose syndrome skin lesions in Palearctic bats

Phylogeographic Aspects of Bat Lyssaviruses in Europe: A Review