Transcriptomic responses of bat cells to European bat lyssavirus 1 infection under conditions simulating euthermia and hibernation

Abstract

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.

Keywords: Antiviral state; Chiroptera; EBLV-1; Heat shock proteins (HSPs); Hibernation; In vitro infection model; Innate immunity; Lyssaviruses; Myotis myotis; Transcriptome.

Fig. 1

Experimental design of cultivation of bat cells at temperatures simulating euthermia and torpor. Green—untreated cell culture, magenta—cell culture infected with EBLV-1

Fig. 2

EBLV-1 infection in the experimental treatments of the Myotis myotis olfactory nerve cells MmNOl. A Measured viral load in 500 ng of RNA, virus titer in supernatant and proportion of reads mapped to EBLV-1 reference. Note that virus RNA and virus production is present after 48 h cultivation at 5 ${}^{\circ }$C, demonstrating that the cells at 5 ${}^{\circ }$C were infected. B Fluorescent images of the cell culture (blue) infected with the EBLV-1 virus (green). I – infected, NI – not infected, t – cultivation temperature

Fig. 3

A Principal components of the gene expression profiles obtained from the sequencing data. B Sample to sample dissimilarity of the sequencing data inferred from complete-linkage clustering based on Euclidean distances. I – infected, NI – not infected, t – cultivation temperature

Fig. 4

Differential expression between the infection-treated and control cells cultivated A at 37 ${}^{\circ }$C and B at 5 ${}^{\circ }$C. Horizontal lines – $\text{FDR}=0.05$, vertical lines – ${log}_2(FC)>1$