White-nose syndrome is associated with increased replication of a naturally persisting coronaviruses in bats

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White-nose syndrome is associated with increased replication of a naturally persisting coronaviruses in bats

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White-nose syndrome is associated with increased replication of a naturally persisting coronaviruses in bats
Series Title:
Scientific Reportsvolume
Davy, Christina M.
Donaldson, Michael E.
Sobudhi, Sonu
Rapin, Noreen
Warnecke, Lisa
Turner, James M.
Bollinger, Trent K.
Kyle, Christopher J.
Dorville, Nicole. A. S,-Y.
Kunkel, Emma L.
Norquay, Kaleigh J. O.
Dzal, Yvonne A.
Willis, Craig K. R.
Misra, Vikram
Springer Nature
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Subjects / Keywords:
Fungal Pathogenesis ( local )
Molecular Ecology ( local )
Virus-Host Interactions ( local )
serial ( sobekcm )


Spillover of viruses from bats to other animals may be associated with increased contact between them, as well as increased shedding of viruses by bats. Here, we tested the prediction that little brown bats (Myotis lucifugus) co-infected with the M. lucifugus coronavirus (Myl-CoV) and with Pseudogymnoascus destructans (Pd), the fungus that causes bat white-nose syndrome (WNS), exhibit different disease severity, viral shedding and molecular responses than bats infected with only Myl-CoV or only P. destructans. We took advantage of the natural persistence of Myl-CoV in bats that were experimentally inoculated with P. destructans in a previous study. Here, we show that the intestines of virus-infected bats that were also infected with fungus contained on average 60-fold more viral RNA than bats with virus alone. Increased viral RNA in the intestines correlated with the severity of fungus-related pathology. Additionally, the intestines of bats infected with fungus exhibited different expression of mitogen-activated protein kinase pathway and cytokine related transcripts, irrespective of viral presence. Levels of coronavirus antibodies were also higher in fungal-infected bats. Our results suggest that the systemic effects of WNS may down-regulate anti-viral responses in bats persistently infected with M. lucifugus coronavirus and increase the potential of virus shedding.
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Scientific Reportsvolume, Vol. 8 (2018-10-19).

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1 White-nose syndrome is associated with increased replication of a naturally persisting coronaviruses in batsChristinaþ  M.þ  DavywáxዅŠƒ‡Žþ  bäþ  Donaldsonwᑐ—þ  ubudhiþ n þ n yᑔ‡‡þ  Rapi nyá Lisaþ  Warneck ezá{árƒ‡•þ  äþ  T urnerzá|ᔇ–þ  äþ  Bollinger}ኔ‹•–‘’Š‡”þ  räþ  yle~á ‹…‘Ž‡þ  äþ  äæäþ  Dorvi llezábƒþ  äþ  unk elz჎‡‹‰Šþ  räþ  äþ  orquayzᘑ‡þ  äþ  Dzalzá ”ƒ‹‰þ  äþ  äþ  Wi lli sz & Vikramþ  Mi sraþ n þ n y’‹ŽŽ‘˜‡”‘ˆ˜‹”—•‡•ˆ”‘„ƒ–•–‘‘–Š‡”ƒ‹ƒŽ•ƒ›„‡ƒ••‘…‹ƒ–‡†™‹–Š‹…”‡ƒ•‡†…‘–ƒ…–„‡–™‡‡–Š‡á ƒ•™‡ŽŽƒ•‹…”‡ƒ•‡†•Š‡††‹‰‘ˆ˜‹”—•‡•„›„ƒ–•ä‡”‡á™‡–‡•–‡†–Š‡’”‡†‹…–‹‘–Šƒ–Ž‹––Ž‡„”‘™„ƒ–• (Myotis lucifugus) co-infected with the M. lucifugus coronavirus (Myl-CoV) and with Pseudogymnoascus destructans (Pd fᖊ‡ˆ—‰—•–Šƒ–…ƒ—•‡•„ƒ–™Š‹–‡æ‘•‡•›†”‘‡fᇚŠ‹„‹–†‹¡‡”‡–†‹•‡ƒ•‡ •‡˜‡”‹–›á˜‹”ƒŽ•Š‡††‹‰ƒ†‘Ž‡…—Žƒ””‡•’‘•‡•–Šƒ„ƒ–•‹ˆ‡…–‡†™‹–Š‘Ž› Myl-CoV or only P. destructans . We took advantage of the natural persistence of Myl摋„ƒ–•–Šƒ–™‡”‡‡š’‡”‹‡–ƒŽŽ› inoculated with P. destructans ‹ƒ’”‡˜‹‘—••–—†›ä‡”‡á™‡•Š‘™–Šƒ––Š‡‹–‡•–‹‡•‘ˆ˜‹”—•æ‹ˆ‡…–‡† „ƒ–•–Šƒ–™‡”‡ƒŽ•‘‹ˆ‡…–‡†™‹–Šˆ—‰—•…‘–ƒ‹‡†‘ƒ˜‡”ƒ‰‡|v我Ž†‘”‡˜‹”ƒŽ–Šƒ„ƒ–• ™‹–Š˜‹”—•ƒŽ‘‡äf…”‡ƒ•‡†˜‹”ƒŽ‹–Š‡‹–‡•–‹‡•…‘””‡Žƒ–‡†™‹–Š–Š‡•‡˜‡”‹–›‘ˆˆ—‰—•æ”‡Žƒ–‡† ’ƒ–Š‘Ž‘‰›ä††‹–‹‘ƒŽŽ›á–Š‡‹–‡•–‹‡•‘ˆ„ƒ–•‹ˆ‡…–‡†™‹–Šˆ—‰—•‡šŠ‹„‹–‡††‹¡‡”‡–‡š’”‡••‹‘ ‘ˆ‹–‘‰‡æƒ…–‹˜ƒ–‡†’”‘–‡‹‹ƒ•‡’ƒ–Š™ƒ›ƒ†…›–‘‹‡”‡Žƒ–‡†–”ƒ•…”‹’–•á‹””‡•’‡…–‹˜‡‘ˆ˜‹”ƒŽ ’”‡•‡…‡ä‡˜‡Ž•‘ˆ…‘”‘ƒ˜‹”—•ƒ–‹„‘†‹‡•™‡”‡ƒŽ•‘Š‹‰Š‡”‹ˆ—‰ƒŽæ‹ˆ‡…–‡†„ƒ–•ä—””‡•—Ž–••—‰‰‡•– –Šƒ––Š‡•›•–‡‹…‡¡‡…–•‘ˆƒ›†‘™æ”‡‰—Žƒ–‡ƒ–‹æ˜‹”ƒŽ”‡•’‘•‡•‹„ƒ–•’‡”•‹•–‡–Ž›‹ˆ‡…–‡† with M. lucifugus coronavirus and increase the potential of virus shedding. Bats are hosts for many viruses and are thought to be the source of some viruses that have spilled over to humans and other mammals, causing fatal disease. ese include coronaviruses causing severe acute respiratory syndrome (SARS1), Middle East respiratory syndrome (MERS2 – 5), porcine epidemic diarrhoea (PED6) and swine acute diarrhoea syndrome (SADS7); paramyxoviruses such as Hendra8 and Nipah9; and loviruses like Marburg10 and Ebola11. Four families of viruses that are pathogenic for other mammalian species (Coronaviridae12, Paramyxoviridae13, Rhabdoviridae14 and Filoviridae15) may also have originated in bats. ese viruses oen cause serious disease in their secondary hosts, but most do not appear to cause clinical signs or pathology in bats16– 18, suggesting that uniquely benign relationships have co-evolved between the viruses and their primary bat hosts19, 20. While relatively little is known about the dynamics of viral infections in bats, these viruses may be maintained in bat populations as a result of either persistently infected individuals, reinfection aer waning immunity, or spatial transmission dynamics21,22.wEnvironmental and Life Sciences Graduate Program, Trent University, Peterborough, ON, Canada. xOntario Ministry of Natural Resources and Forestry, Wildlife Research and Monitoring Section, Trent University, Peterborough, ON, Canada. yDepartment of Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. zDepartment of Biology, University of Winnipeg, Manitoba, Canada. {Present address: Department of Animal Ecology and Conservation, University Hamburg, Hamburg, Hamburg, Germany. |Present address: Institute for Land Water and Society, Charles Sturt University, Albury, New South Wales, Australia. }Canadian Wildlife Health Cooperative and Department of Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. ~Forensic Science Department, Trent University, Peterborough, ON, Canada. Christina M. Davy, Michael E. Donaldson, Sonu Subudhi and Noreen Rapin contributed equally. Correspondence and requests for materials should be addressed to V.M. (email: vikram.misra@usask.ca ) Received: 16 July 2018 Accepted: 9 October 2018 Published: xx xx xxxxb


2 e rare spill-over of bat viruses to other animals may require a “perfect storm” of conditions that include increased contact between bats or fomites and other mammals, possibly due to human impacts on habitat quality23, and the ability of the virus to infect, replicate, and transmit in the secondary host. e rate of viral shedding and the amount of detectable virus associated with bat colonies uctuates, with periodic increases oen linked to parturition, waning maternal immunity, nutritional stress or increased energy consumption17, 24– 29. Increased shedding of virus by a colony of bats may reect an increase in the proportion and number of susceptible individuals, or an increase in the replication of persistent or latent virus normally suppressed by the host. For herpesviruses, reactivation from latency is linked to perturbations caused by a variety of physiological, immunological and psychological stressors30. e mechanisms that trigger the reactivation of latent or persistently infecting viruses are not clearly understood, but the increased shedding of viruses is correlated with some incidents of spill-over of bat viruses to other animals31. e Canadian prairies are home to three species of bats, including the little brown bat (Myotis lucifugus ), big brown bat ( Eptesicus fuscus), and northern long-eared bat (Myotis septentrionalis ). All three species hibernate from October to May, sometimes in shared hibernacula. We recently demonstrated that ~30% of hibernating M. lucifugus sampled over two years from hibernacula in Manitoba were infected with a coronavirus (Myl -CoV), which persisted at low levels in the intestine32. A closely related coronavirus also infects E. fuscus33. Whereas bats appear to be relatively resistant to viral infections, a cold-adapted fungus that was recently introduced to North America has caused widespread mortality in some species of bats in eastern United States and Canada34– 37. e fungus (Pseudogymnoascus destructans) causes white-nose syndrome (WNS) in hibernating bats, which is characterized by the growth of white fungal mycelia on the face and exposed skin of the wings and tail membranes. e visual and microscopic eects of P. destructans on the skin of the wings are associated with increased expression of several genes devoted to innate immunity and inammation in wing tissue38, 39. Profound systemic eects include dehydration, hypovolemia, metabolic acidosis, and fat depletion, which can lead to death40–42. Other systemic eects of bat WNS include an accumulation of neutrophils in the lungs, which is accompanied by an increase in the expression of several cytokine genes43 suggesting that even the most severely aicted hibernating bats are capable of at least some systemic immune response to fungal infection. Previous studies on other species have demonstrated that a fungus and a virus could interact during co-infection and aect each other44, 45. Similar interactive impacts of co-infection with P. destructans and viruses on bat immune responses are not known. We used M. lucifugus experimentally-infected with P. destructans and/ or naturally infected with Myl -CoV as a model to understand how co-infections inuence bat-virus interactions. is system allows us to avoid confounding factors of direct pathogen-pathogen interactions, because the fungus aects the skin, while the coronavirus infections occur internally, almost exclusively in the ileum and lungs32. We hypothesized that co-infection would alter the molecular response of bats to a persistent viral infection, and that viral shedding would change as a result of the increased or disrupted host immune response. To test this prediction, we examined tissues collected from M. lucifugus at the termination of an earlier study that quantied the eects and pathogenesis of P. destructans in hibernating bats experimentally infected with the fungus37, some of which were naturally infected with MylCoV32. is combination of uninfected, virus-infected, fungus-infected and co-infected M. lucifugus allowed us to test our hypothesis that host responses to co-infection are synergistic and not simply additive.ResultsQuantitation of MylCoV and M. lucifugus RNA through reverse transcription quantitative PCR (RT-qPCR) and dual-RNA-sequencing indicated that co-infected bats had signicantly higher levels of Myl -CoV RNA than bats infected with virus alone. e amount of MylCoV RNA correlated with the severity of WNS pathology in co-infected bats. is phenomenon was associated with specic molecular responses to co-infection, even in the intestines of bats where only one of the two pathogens was directly interacting with the host tissue. e levels of antibodies against Myl -CoV nucleocapsid (N) protein were also higher in co-infected bats. Each key result is discussed in detail below.Bats co-infected with the fungus P. destructans and the virus Myl-CoV contained higher levels of Mylæ‘äþ Myl -CoV genomic RNA was detected in bats infected only with MylCoV (virus-infected; 7/18), co-infected bats (European P. destructans (3/13), or with North American P. destructans (7/16)37). ere was no dierence in the frequency of Myl -CoV detected among these treatments (p-valueþ t þ t 0.801). We pooled bats infected with the two P. destructans isolates for all further analyses and tested whether co-infection with P. destructans and MylCoV correlated with an increase in viral replication. Our RT-qPCR data showed that the co-infected bats contained 60-fold more MylCoV RNA on average than the virus-infected bats (Mann Whitney test; p-valueþ t þ t 0.014; Fig. 1). Relative quantities of Myl -CoV RNA detected in the ileum of the virus-infected bats were low and showed low variation (Standard Deviation of 1/ CTþ t þ t 0.005), compared to the relative quantities of Myl -CoV RNA in the co-infected bats (Standard Deviation of 1/CTþ t þ t 0.108; Fig. 1). e severity of WNS fungal pathology varied in co-infected bats, and we therefore tested whether relative quantities of viral RNA in the ileum correlated with the severity of WNS symptoms. Levels of WNS severity were scored based on fungal hyphae on the wings, secondary bacteria in wing lesions, oedema, necrosis and inammation in wing lesions, and levels of neutrophils in lung, spleen and liver interstitium. Severity scores for wing tissue, secondary bacteria in lesions, and neutrophils in the lung interstitium positively correlated with relative amounts of coronavirus RNA in hibernating bats (Table 1).ƒ–”‡•’‘•‡•–‘…‘拐ˆ‡…–‹‘‡š…‡‡†–Š‡•—‘ˆ”‡•’‘•‡•–‘˜‹”—•‘”ˆ—‰ƒŽ‹ˆ‡…–‹‘ƒŽ‘‡äþ To determine the extent to which Myl -CoV and P. destructans infection interact to inuence gene expression in bat intestines, we performed a transcriptomic analysis on bat intestines comparing gene expression among the


3 uninfected, virus-infected, fungus-infected, and co-infected treatments (Fig. 2(A)). RNA sequencing resulted in ~700 million paired-end reads passing lters, 65% of which aligned to the M. lucifugus genome (TableS1). Pairwise dierential gene expression varied widely among the four treatments with relatively low overlap in dierentially expressed transcripts (Fig. 2B,C), Supplementary Fig.1). Similar transcript expression occurred between the uninfected and virus-infected bats, and between the fungus-infected and co-infected bats. e fungus-infected bats exhibited a much stronger response, dierentially expressing 324 transcripts compared to the uninfected bats (TableS3). ese transcripts were enriched for only two gene ontology (GO) terms (cell-cell junction and plasma membrane part; TableS8). e co-infected bats dierentially expressed 634 transcripts relative to the uninfected bats (TableS4). ese transcripts showed signicant enrichment for 16 GO terms (TableS8). e co-infected and fungus-infected bats shared 108 similar dierentially expressed transcripts and overlapped in one enriched GO term relative to the uninfected bats (plasma membrane part; TableS8).b¡‡…–‘ˆ‹ˆ‡…–‹‘™‹–Š–Š‡ˆ—‰—• P. destructans ‘–Š‡‡š’”‡••‹‘‘ˆ‰‡‡•Ž‹‡†–‘‹ƒ–‡ responses in the intestines of bats infected with the virus Myl-CoV.þ When we directly compared responses of bats among the four treatments, response of the virus-infected bats diered strongly from the responses of fungus-infected or co-infected bats (virus-infected vs. fungus-infected: 461 dierentially expressed transcripts and 9 signicantly enriched GO terms; virus-infected vs. co-infected: 473 transcripts and 43 enriched GO terms; TablesS5, S6, S7; Supplementary Fig.1). ese dierences in gene expression patterns included genes that clustered in two processes relevant to host-pathogen interactions – the mitogen-activated protein kinase (MAPK) pathways and cytokine and innate immune responses. Table 2 lists genes from the two processes that were signicantly either up or down-regulated when virus-infected bats were compared to co-infected bats. For the MAPK pathway-related transcripts, genes such as RSU1 and RERG were up-regulated while those, such as STYK1, RRAD, MAP3K and SRC were down-regulated. For cytokine-related genes several transcripts were suppressed. When we compared the expression of the same genes for bats with WNS (combining fungus-infected and co-infected bats) and all bats without WNS (combining uninfected and virus-infected bats), we found similar dierences (last two columns of Table 2 ). is suggested that supercial infection with fungus, P. destructans , was the driving factor for altered gene expression in the bat intestines. Virus-infected Co-Infected 0.0 0.1 0.2 0.3 0.4 0.5 Bat group 1/ CT(Myl-CoV -GAPDH )L evel of Coronavirus RN A *p<0.05 *Mann Whitney test 60X Vi rus-infe ct ed bats Co -infec ted bats Av erage C T (Myl-C oV GAPDH) Av erage 1/ C T (Myl-C oV GAPDH) Standar d Deviation (1/ C T) 16.96 10.96 0.005 0.131 0.059 0.108 F old Change 2(CTMock-inf ec te d-CTP. destruct an s-infec te d)= 64 Figure 1.þ Eect of white-nose syndrome on level of Myotis lucifugus coronavirus (Myl -CoV) RNA in hibernating little brown bats (M. lucifugus). Relative transcript levels for the coronavirus RNA polymerase gene for each bat are depicted as reciprocal of Cycle threshold (Ct) normalized separately (Ct) for levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcripts in each sample. e horizontal bar represents the mean while the vertical bar indicates standard deviation from the mean. Signicance (p value) is as calculated with an independent Mann-Whitney test. Virus-infected bats had lower 1/Ct values for coronavirus RNA than co-infected bats. e average fold-dierences between virus-infected and co-infected bats were calculated from the dierence between the average Ct values. Correlate Level of coronavirus RNA Pearson CorrelationaSignicance N Virus-infected/Co-infected 0.610 0.009 17 Average hyphae score 0.630 0.016 14 Average bacterial score 0.680 0.007 14 Lung interstitial neutrophils 0.618 0.043 11Table 1.þ Correlation between level of Myotis lucifugus coronavirus RNA and disease severity of white-nose syndrome (WNS) in co-infected M. lucifugus, based on three measures of WNS severity and pathology. aPearson’s coecients were calculated for the Ct levels for cytokine transcripts for bats in each treatment class and lung interstitial neutrophil scores and mean bacterial and hyphae scores for 5 wing sections for each bat.


4 To conrm the results of the RNA-seq analysis, we selected 4 genes from Table 2 , namely IRF1, RERG, SRC and IL22RA1, to be veried by RT-qPCR. We also included interleukin 10 (IL10) due to its biological relevance to immune regulation and because we had previously observed an increase in its expression related to fungal infection43. As we wanted to conrm whether WNS was driving gene expression in the intestines of bats, we per formed a two-group analysis for the RT-qPCR data. We combined all the bats without WNS into a single group (uninfectedþ t þ t virus-infected) and all the bats with WNS into the other group (fungus-infectedþ t þ t co-infected; Fig. 3(A)). Expression of Ras-like estrogen regulated growth inhibitor (RERG) increased while expression of Interleukin 22 receptor subunit alpha 1 (IL22 RA1) genes decreased in bats with WNS, irrespective of viral infection (Fig. 3C,E ). Expression of the immune modulatory cytokine IL10 tended to be higher in bats with WNS than in bats without WNS, but the dierence was not statistically signicant (p-valueþ t þ t 0.07) (Fig. 3(F)).White-nose syndrome is associated with increased coronavirus antibody levels in the co-infected bats.þ In 2017, we performed a similar study, experimentally exposing 63þ t M. lucifugus to P. destructans as described in Warnecke et al .37. We performed IgG ELISA on blood plasma to detect Myl -CoV (coronavirus) N protein antibodies and found that 21/63 were positive for antibodies against the coronavirus. Of those 21 bats, 7 had detectable coronavirus RNA in their intestines suggesting an active infection, and 3 out of the 7 had been experimentally infected with P. destructans during the course of the study. We compared the ELISA optical density (O.D.) values of these virus-infected bats to co-infected bats (Fig. 4(A)) and found that the presence of P. destructans was associated with increased levels of coronavirus antibodies (Mann Whitney test, p valueþ t þ t 0.03; Fig. 4(B)).DiscussionOur ndings suggest that systemic responses of bats to WNS results in increased coronavirus replication and consequently, increased viral shedding, which may lead to subsequent infection of susceptible animals. Coronavirus infection may in turn increase the severity of WNS pathology. is is the rst study to examine the systemic eects of co-infection on either bat coronavirus or WNS, and our results raise important questions in regard to zoonotic spillover events. Although events of successful viral spillover to distantly related species are thought to be extremely rare, in recent years several coronaviruses have spilled over, including SARS-CoV1, MERS-CoV2–5, Uninfected vs Virus-infected Uninfected vs fungus-infected Virus-infected vs Fungus-infecte d Co-infected vs Fungus-infecte d Co-infected vs Virus-infecte d Uninfected vs Co-infecte dContro l No MylCoV or P .destructans n = 5 Naturally inf ec te d with MylCoV onl y n = 4 Experimentally inf ec te d with P. destructans only n = 3 Coin fe ct ed with both pathogens n = 4 30 0 0 56 52 316 10 0 0 160 56 210 AC BUpregulated Downr egulated Bat Groups Uninfec te d Vi rus-infe ct ed F ungus-infe ct ed Co -infe ct edW ith fungusW ithout fungus log2Fo ld Change -log10(padj) log2Fo ld Change -log10(padj) log2Fo ld Change -log10(padj) log2Fo ld Change -log10(padj) log2Fo ld Change -log10(padj) log2Fo ld Change -log10(padj) Figure 2.þ Co-infection of little brown bats (Myotis lucifugus ) with M. lucifugus coronavirus (MylCoV) and Pseudogymnoascus destructans results in non-additive patterns of gene expression compared to sole infection with the virus or fungus. (A) Experimental design, showing the four treatments of little brown bat (Myotis lucifugus ) established by experimental inoculation with Pseudogymnoascus destructans and by qPCR detection of persistent Myl -CoV infections: uninfected, virus-infected, fungus-infected and co-infected. (B) Dierential gene expression identied by DESeq2 among virus-infected, fungus-infected and Co-infected bats as compared to the change each exhibited relative to uninfected bats. (C) Dierential gene expression among the four treatments, detected by DESEQ2 and visualized in volcano plots. e log of the adjusted p-value is plotted as a function of the log ratio of dierential expression. Colored data points represent dierent groups of genes based on fold change and false discovery rate (FDR) cuto; red (2 fold change, FDR 0.05), dark grey (2 fold change, FDRþ t þ t 0.05), light grey (2 fold change, FDRþ t þ t 0.05), black (2 fold change, FDRþ t þ t 0.05).


5 PEDV-CoV6 and SADS-CoV7. ese viruses are thought to have originated in bats. In addition, circumstantial evidence suggests that most alpha and beta coronaviruses that parasitize other mammals may have originated in bats as well46. If so, then understanding host-pathogen interactions between bats and coronaviruses could inform our ability to predict or manage the risk of spillover. In this study, we showed that a coronavirus exhibits low activity in its natural host, M. lucifugus, but that co-infection with a fungus increases the quantity of viral RNA Ensembl Gene Name Ensembl Description Virus-infected vs. Coinfected All bats “without fungus” vs. all bats “with fungus”aLog2 Fold ChangebPadj Log2 Fold ChangebPadj MAPK pathway-related transcripts STYK1 serine/threonine/tyrosine kinase 1 1.268 0.025 1.516 0.0001 RSU1 Ras suppressor protein 1 1.102 0.004 RRAD RRAD, Ras related glycolysis inhibitor and calcium channel regulator 1.297 0.028 1.3 0.025 RERG RAS like estrogen regulated growth inhibitor 1.562 0.018 1.416 0.005 MAP3K11 mitogen-activated protein kinase 11 1.14 0.040 1.13 0.0002 SRC SRC proto-oncogene, non-receptor tyrosine kinase 1.539 0.013 1.297 0.0037 Cytokine-related transcripts IRF1 Interferon regulatory factor 1 1.551 0.001 1.444 0.0001 IFI6 Interferon alpha inducible protein 6 1.798 0.014 1.352 0.039 IL22RA1 Interleukin 22 receptor subunit alpha 1 1.411 0.015 1.314 0.002 SOCS6 Suppressor of cytokine signaling 6 1.278 0.008 1.534 0.0001Table 2.þ RNA-sequencing identied dierential expression of transcripts related to the MAPK pathway and to cytokine-related processes, comparing gene expression in the ileum of little brown bats (Myotis lucifugus ) infected only with the M. lucifugus coronavirus (MylCoV; virus-infected) or co-infected with MylCoV and Pseudogymnoascus destructans. e last 2 columns show the same comparisons made aer grouping bats that were not exposed to the fungus, and bats that were exposed to the fungus and exhibiting symptoms of WNS (irrespective of their viral infection status). a(Uninfectedþ t þ t virus-infected) vs. (fungus-infectedþ t þ t co-infected) bPositive log2 fold-change values indicate higher expression in the second listed treatments relative to the rst. Without fungus With fungu s 0.00 0.02 0.04 0.06 0.08 0.10Bat Group1/ CTSR C *p = 0.71 *Mann Whitne yT est (D) Without fungus With fungu s 0.00 0.05 0.10 0.15 0.20 0.25Bat Group1/ C T IRF1 *p = 0.61*Mann Whitne yT est(B) Without fungus With fungu s 0.00 0.05 0.10 0.15 0.20Bat Group1/ CTIL22RA1 *p = 0.0072 *Mann Whitne yT est (E) Without fungus With fungu s 0.00 0.05 0.10 0.15 0.20 0.25Bat Group1/ CTRERG *p < 0.0001*Mann Whitney Te st (C) Without fungus With fungus 0.00 0.05 0.10 0.15Bat Group1/ CTIL10 *p = 0.07 *Mann Whitne yT est (F) Uninfected Vi rus-infecte d Fungus-infected C o-infec te dW ith fungusW ithout fungus (A) Figure 3.þ Eect of white-nose syndrome (WNS) on the levels of immune genes IRF1, RERG, SRC, IL22RA1 and IL10 expressed in the ileum of little brown bats (Myotis lucifugus ). (A) Summary of the four treatments, with a red arrow indicating the two groups (“with fungus” and “without fungus”) that were compared. (B–F) e relative transcript levels of each gene for bats with and without WNS, depicted as reciprocal of Cycle threshold (Ct) normalized separately (Ct) for levels of transcripts for GAPDH in each sample. Statistical signicance was calculated based on the independent Mann Whitney test. e dierence in the two groups was signicant for RERG and IL22RA1 genes.


6 in the intestines. We have no reason to expect zoonotic transmission of the coronavirus i.e. Myl -CoV, but similar co-infection mechanisms may operate in tropical bat species harbouring potentially zoonotic viruses. Our results suggest that secondary skin infection with the fungus, P. destructans , substantially increases the level of viral RNA in the intestine of hibernating bats. We showed that infection of the skin with P . destructans can cause profound changes in gene expression in the intestines, despite a lack of direct contact between intestinal tissue and the fungus. Infection with P. destructans causes modulation of a number of immune responses, including down-regulation of interleukin and cell proliferation genes which may compromise bats’ ability to suppress viral activity (Fig. 5 ). Taken together, our results have implications for epidemiological studies of P. destructans , the WNS fungus and for research into viral spillovers, which should consider the potential implications of co-infections that increase viral shedding. Complex strategies allow viruses to remain endemic in populations. ese include a continuously replenished source of susceptible hosts for viruses that cause short-term acute infections with long-lasting immunity (e.g. measles virus), antigenic dri of virus (e.g. inuenza virus) or waning immunity (e.g. respiratory syncytial virus) that allows reinfection, and long-lasting latent (e.g. herpesviruses) or persistent infections (e.g. pestiviruses) with sustained or periodic shedding. It is not yet clear how bat viruses are maintained in their natural host populations, or how they avoid extinction as host populations become immune and less susceptible. Persistent infections can be established in cultured cells with viruses that may have originated in bats, including Ebola virus47 and SARS-CoV48– 50, but whether these viruses persist in their primary hosts is not known. Studies of persistence of bat viruses in infected bats have produced equivocal results. e lack of direct evidence supporting specic models of persistence or transmission dynamics represents a major knowledge gap in bat-virus ecology21. We maintained M. lucifugus in controlled laboratory hibernation chambers for four months during these experiments, and we detected the coronavirus i.e. Myl -CoV, at the end of hibernation. ese data imply that the coronavirus can persist in its host for at least the duration of hibernation, particularly as nucleotide variability among the detected coronavirus isolates showed that spread of coronavirus among bats within a chamber was unlikely32. In an extensive study of New World alphacoronaviruses, no target viruses were detected in the rectal swabs of individual bats sampled over time46, suggesting that persistence and intensity of shedding varies among species or viruses. e authors concluded that the targeted coronaviruses do not persist in their hosts but are maintained in populations by the introduction of new susceptible individuals. However, their results could also reect viral persistence in individual animals, with low baseline levels of virus replication and undetectable shedding interspersed with periods of increased replication and shedding that did not occur during the sampling period. Periodic or seasonal increases in virus shedding associated with parturition, lactation, nutritional deprivation or environmental stress21, 29 suggest persistent or latent viruses may be activated by hormonal or other systemic cues. Direct evidence linking a specic trigger to increased shedding has not yet been found. However, viral replication in rodent and bat cells persistently infected with Ebola virus increased greatly following modulation of the Ras/MAPK pathway with lipopolysaccharides or phorbol esters, and with the resulting suppression of the cells’ interferon response47– 49. In experimental systems, the immune modulatory cytokine IL10 also inuences viral persistence and replication51– 53, although more study is required to clarify the eects of circulating cytokines on the replication of persistently infecting viruses. Nevertheless, these results suggest that circumstances which induce anti-inammatory cytokines or suppress anti-viral innate responses, may provide a trigger for increased shedding of persistently infecting virus. We discovered that bats with WNS (fungus-infected and co-infected) had signicantly lower intestinal levels of transcripts for IL22RA1 and other interferon-related genes as compared to uninfected bats, and we observed the same trend in IL10 (although it was not signicant; p valueþ t þ t 0.07). IL22RA1 is the receptor present on host cells, including intestinal cells, which help in initiating cellular signalling in response to IL22 produced by V irus-infected Co-infected 0.0 0.5 1.0 1.5 2.0 2.5 Bats with Vi ru sA ntibodies and RNAIgG ELIS A O.D at 405n m Effect of coinfection on coronaviru st iters *p = 0.03*Mann Whitney Te st(B) Uninfected Virus-infected F ungus-infected C o-infe ct edW ith fungusW ithout fungus (A) Figure 4.þ Little brown bats (Myotis lucifugus) coinfected with M. lucifugus coronavirus (Myl -CoV) and Pseudogymnoascus destructans produce more antibodies against Myl -CoV than bats infected only with MylCoV. (A) Diagram summarizes the four treatments; the red arrow shows the two groups between which antibody levels were compared. (B) Antibody levels against the Myl -CoV N protein detected by antibody capture ELISA expressed as optical density (O.D.) values at 405þ t nm. Co-infected bats had signicantly higher antibody levels than bats infected only with Myl -CoV (independent Mann Whitney test; p valueþ t þ t 0.03).


7 T-cells54. IL-22 leads to an increase in anti-microbial peptide production, cellular protection against damage and increases cellular proliferation55. erefore, reduced IL-22 signalling in the intestines of bats with WNS, might suppress the bat defences that control the coronavirus infection. Additionally, previous studies have shown that the anti-inammatory gene, IL10, is expressed more in the lungs of bats with WNS than in bats without it43. We saw a similar trend with the levels of IL10 in the intestines which might play a role in suppressing the immune response against the coronavirus. Another altered cytokine gene which was of interest was the suppressor of cytokine signalling-6 (SOCS6) gene. Fungal-infected bats showed lower levels of SOCS6 transcripts, lack of which has been implicated in mild growth retardation in mice56. Overall, our results suggest that WNS triggers changes in gene expression in the ileum (Fig. 5 ). ese may inuence expression of interferon-stimulated-genes (ISGs), thereby leading to increased viral replication at the site of viral persistence. Interferon-related transcripts were more highly expressed in the ileum of virus-infected bats that did not have WNS, suggesting that the bat’s response to WNS causes down-regulation of interferon activity. Interferons may control coronavirus replication, as seen in cases of SARS-CoV57 and MERS-CoV58. erefore, a decrease in interferon activity might cause an increase in coronavirus (Myl -CoV) replication. In addition to interferon-related genes, we also found that RERG, which is related to growth inhibition, was upregulated in the fungus-infected bats when compared to virus-infected bats. Upregulation of RERG could aect the rate of cell proliferation in the intestines59. Finally, this cascade of responses is associated with increased severity of WNS symptoms. Bats with WNS experience a range of systemic disturbances including dehydration, hypovolemia, metabolic acidosis and fat depletion40, 41, neutrophil inltration of the lung interstitium, and increased expression of transcripts related to anti-microbial and proand anti-inammatory cytokines43. Taken together, this evidence suggests that hibernating bats respond systemically to supercial fungal infection, and this hypothesis is further supported by our observations of altered gene expression in the ileum of fungus-infected bats. Based on our results, we propose a model for how secondary infections may increase the replication and subsequent shedding of persistently infecting virus (Fig. 5 ). e establishment of WNS (or other secondary infec tion) impacts the tissue with which that pathogen interacts (in the case of P. destructans , the skin). Direct inter actions between the host and the secondary pathogen are limited to the aected tissue, but the systemic response to the disease triggers a cascade of immune responses, including increased release of cytokines or neutrophils. Aected cells such as intra-alveolar macrophages in the lungs or cells lining the intestine, may produce proor anti-inammatory molecules and inuence cells that harbour viral genomes. is cascade of host responses disrupts the equilibrium between the persistently infecting virus and the cell’s innate immune response, leading to a dramatic increase in the expression of coronavirus (MylCoV) replication. Vi rus-infec te d Co -infe ct ed F ungal infe ct ion (P. destructans ) Fu ngal infe ct ion presen tIntestinesFu ngal infe ct ion absen t Higher le vels of Vi rus in Fu ngus infe ct ed bats L ess IL22 signalling Reduced ISG expression (Hernandez, 2015)Wi ngs Suppression of MAPK inase related genes Lowe ring of innate immune r esponse leading to increased viral load My l-C oV (V irus) Higher IL10 lev els Figure 5.þ Hypothesized model of pathways involved in increased coronavirus shedding and white-nose syndrome (WNS) severity in little brown bats (Myotis lucifugus ) co-infected with M. lucifugus coronavirus (MylCoV) and Pseudogymnoascus destructans. Diagram summarizes the changes observed by comparing co-infected bats with virus-infected bats. Bats with persistent Myl -CoV infection exhibit relatively low viral shedding. When bats are also infected with P. destructans (shown in yellow arrow) and develop WNS, the level of coronavirus increases. ere is a change in the level of some immune genes, such as IL22, RERG and possibly IL10, which may have an eect on immune response and cell proliferation. e increase in coronavirus levels in co-infected bats is possibly due to the bats’ systemic response to WNS reducing innate anti-viral responses.


8 Our assays were unfortunately limited to analysing viral and cytokine transcripts rather than protein, because reagents for detecting bat viral and host proteins are not yet available. We were not able to perform serial dilutions of the plasma to precisely quantify anti-viral titre due to the limitation in the amount of plasma obtained from each bat. e sample size was also small for this assay, because only 7 of the sampled bats had detectable levels of coronavirus in their intestines and were positive for viral antibodies. Despite these limitations, we demonstrated higher antibodies against the coronavirus in the plasma of co-infected bats when compared with virus-infected bats. is increased antibody level in co-infected bats might reect an adaptive immune response to increased coronavirus replication in the intestines. Our proposed hypothesis for the mechanism driving increased viral replication following pathogenic co-infection was worth testing, but our results are also consistent with an alternative hypothesis. Increased viral replication or viral load may aect the severity and population-level impacts of WNS. Bat mortality following the arrival of WNS varies widely from site to site, with populations decreasing from 30% to 99%35. Variation in the microclimate, and other ecological factors may drive some of this variation60, but our data suggest that cryptic viral infections may also play a role in determining survival rates for bats hibernating in sites colonized by P. destructans . We recommend that future studies on population-wide impact of WNS incorporate viral sampling to help better understand the role of co-infections on bat populations in the wild.Materials and Methodsƒ’Ž‡ƒ…“—‹•‹–‹‘äþ Fiy-four male M. lucifugus were collected from a WNS-free cave in Manitoba, Canada in November 2010. Details of the experimental design as well as protocols for collecting and transporting bats, infection with P. destructans , maintenance of bats in hibernation and sample collection have been described previously37, 43. Briey, bats in groups of 18 were either sham-inoculated or inoculated with North American or European isolates of P. destructans . Bats were housed at 7þ t °C and 97% relative humidity with ad libitum water. All bats were equipped with data loggers to monitor skin temperatures. Bats were euthanized during the experiment when humanely required or at the termination of the experiment 120 days aer inoculation. Immediately following euthanasia samples from segments of wing as well as various tissues were preserved in RNAlater (Qiagen, 76016) or in formalin. Samples in RNAlater were kept at 20þ t °C until they were processed. North American and European isolates of P. destructans caused similar disease outcomes37, so we did not dier entiate between the strains in subsequent analysis. e procedures for care, handling and euthanasia of bats were approved by the University Committee on Animal Care and Supply of the University of Saskatchewan (Protocol #20100120). Bats were collected under the province of Manitoba Wildlife Scientic Permit WB11145. In 2017, a further 129þ t M. lucifugus were collected from a WNS-free cave in Manitoba, Canada in January under the Manitoba Sustainable Development Wildlife Scientic Permit No. SAR16009. Bats were euthanized during the experiment when humanely required or at the termination of the experiment 70 days aer infection and a similar experiment was performed at the University of Winnipeg as described above (Protocol #AE08399).‹•–‘Ž‘‰‹…ƒŽ…Žƒ••‹¤…ƒ–‹‘äþ During necropsy, we collected representative samples for histopathology from all major organ systems. In addition, representative samples were taken from all areas of the wing and rolled on dental wax before placing in 10% neutral buered formalin. Tissues were processed routinely for histology. Five m sections were cut and stained with periodic acid-Schi stain to highlight fungal hyphae. Liver and other tissues were processed routinely and stained with hematoxylin and eosin. Wings were scored on a scale of 0 to 5 with 5 being very severe with 50% of wing covered in fungal hyphae. We used a bacterial score from 0 to 5, with 5 indicating wide-spread and abundant bacteria being present in many areas within the dermis and underlying connective tissues. Average scores from 5 sections of wing were used for analysis. Interstitial lung neutrophil assessment was similarly evaluated on a scale of 0 to 5, with 5 being very severe. Average scores from the 5 sections were used for analysis.bš–”ƒ…–‹‘äþ Tissues preserved in RNAlater were homogenized in 2þ t ml sealed vials with a 5þ t mm stainless steel bead, 0.1þ t g of 0.1þ t mm zirconia/silica beads and 350þ t l Buer RLT Plus (with -mercaptoethanol, RNeasy Plus Mini Kit, Qiagen, 74136) using a Retsch MM400 Oscillating Mill at 30þ t Hz for 4þ t min. Total RNA was extracted fol lowing the manufacturers protocol. RNA integrity was assessed using RNA 6000 Nano Kit (Agilent, 5067-1511) with the Agilent 2100 Bioanalyzer.…›–Š‡•‹•äþ cDNA was synthesized from 1þ t g of RNA (or less if concentrations were too low) per reaction using QuantiTect Reverse Transcription Kit (Qiagen 205313). cDNA samples were stored at 80þ t °C until they were used for PCR.‘Ž›‡”ƒ•‡Šƒ‹‡ƒ…–‹‘fäþ Tissue samples were identied by their submission numbers with no reference to treatment class prior to analysis with PCR, so that evaluation of the results could not be inadvertently biased by knowledge of the treatment. We used semi-nested PCR to detect MylCoV. Primers were designed from the partial sequence of Rocky Mountain bat coronavirus replicase (accession number EF544563) (TableS9). e primary reaction used primers MyCVF1 and MyCVR1 to yield a 441þ t bp product. e secondary or nested reaction used primers MyCVF2 and MyCV R1 to give a 273þ t bp product. PCR were performed in a MJ Research PTC-200 thermal cycler using TopTaq DNA Polymerase (Qiagen, 200205). Each reaction (50þ t l) contained 2þ t l cDNA (or 1þ t l primary reaction), 200þ t nM of each primer, 200þ t M of each dNTP (Invitrogen, 10297018), TopTaq PCR buer and 0.25þ t l TopTaq. e thermal prole for the primary reaction was: 94þ t °C for 3þ t min (denaturation), followed by 30 cycles of 94þ t °C for 30þ t sec, 45þ t °C for 30þ t sec (annealing), 72þ t °C for 1þ t min and nally 72þ t °C for 10þ t min. e thermal prole used for the secondary reaction was 94þ t °C for 3þ t min (denaturation), then 30 cycles of 94þ t °C for 30þ t sec, 55þ t °C for 30þ t sec (annealing), 72þ t °C for 1þ t min and nally 72þ t °C for 10þ t min. PCR products were ana lyzed on ethidium bromide stained 1.0% agarose gels (Invitrogen 15510-027 in 0.5X TBE). PCR products were


9 puried using MinElute PCR Purication Kit (Qiagen, 28006) and veried by sequencing (Macrogen, Korea). If more than one DNA band was present, the appropriate size band was cut out and puried using QIAquick Gel Extraction Kit (Qiagen, 28706) before sequencing.‡˜‡”•‡æ”ƒ•…”‹’–‹‘—ƒ–‹–ƒ–‹˜‡æ“fäþ e Stratagene MX3005P qPCR System was used in conjunction with QuantiFast SYBR Green PCR Kit (Qiagen 204056). We quantied coronavirus with RNA primers MyCVF2 and MyCV R1 (TableS9). For initial experiments data were normalized to two transcripts – glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin43. As there were no dierences in results, all subsequent experiments used only GAPDH as a normalizer using primers GAPDH US and GAPDH DS (designed for use in humans but also amplify M. lucifugus transcripts – TableS9). As well, a no-template (negative) control was included with every set of primers. Each 25þ t l reaction contained: 1þ t M of each primer set, 12.5þ t l SYBR Green Master Mix and 8.5þ t l of diluted cDNA. To verify the RNAseq data, cDNA from ileum samples in which coronavirus RNA had been detected via RT-qPCR were analysed using the following primers, IL22RA1, IRF1, RERG and SRC (for sequence of primers see TableS9). Primers were designed by aligning primers described for quantitating human cytokines (PrimerBank) with annotated transcripts of M. lucifugus genes: c-jun (Accession number: XM_006096110.1), cyclin D1 (XM_006098046.1), IL10 (XM_006094865.1) and TNF alpha (XM_006104644.1). e interferon beta primers were designed using the annotated transcript for the E. fuscus gene (XM_008145044.1), which also amplify transcripts from M. lucifugus. Primer eciencies were determined from cycle threshold (Ct) values of puried PCR products serially diluted and re-amplied. Primers amplied targets with an eciency of about 100% and in all cases the identities of the PCR products were conrmed by their specic dissociation temperature, specic sizes on agarose gels and by sequencing. We observed primer-dimers in some reactions in addition to the PCR product. e dimers dissociated at 77þ t °C, while the specic coronavirus polymerase product dissociated at 83þ t °C. To avoid false positives due to primer-dimers, the thermocycler was programmed to read at 80þ t °C (in the cycle aer the primer-dimer had dissociated, and before dissociation of the target product). e thermal prole used was 95þ t °C for 5þ t min followed by 40 cycles of 95þ t °C for 10þ t sec, 60þ t °C for 30þ t sec (readings taken at 80þ t °C), and a nal cycle of dissociation of product 95þ t °C for 1þ t min, 55þ t °C for 30þ t sec and 95þ t °C for 30þ t sec (readings taken at every degree between 55þ t °C and 95þ t °C). Only results from reactions that yielded unambiguous results were used for analysis.敇“ƒŽ›•‹•äþ To explore the mechanisms driving high virus load in bats with WNS, we performed RNA-seq analysis which could potentially screen all targets in the bat intestinal cells. We targeted the ileum transcriptome because this is the tissue in which Myl -CoV is present in detectable concentrations32. Extraction of RNA from ileum tissue, which includes the ileum and potential gut contents have been described in previous sections. Bats were screened for Myl -CoV using RT-qPCR, and bats were assigned post hoc to treatment groups representing four infection histories (Fig. 1(A)): 1) Uninfected (bats were not infected with virus or fungus; nþ t þ t 5), 2) Virus-infected (bats were naïve to the fungus but had a persistent Myl -CoV infection; nþ t þ t 4), 3) Fungus-infected (bats were experimentally infected with P. destructans and no virus was detected; nþ t þ t 3), or 4) Co-infected (bats with persistent Myl -CoV infections that were also experimentally infected with P. destructans ; nþ t þ t 4). All samples had adequate RNA quality for sequencing (i.e. RIN value 7).‹•‘Žƒ–‹‘äþ Tissues were homogenized in 2þ t ml sealed vials with a 5þ t mm steel bead, 0.1þ t g of 0.1þ t mm zir conium silica beads, 350þ t L of RLT buer (with -mercaptoethanol) (RNeasy Plus Kit, Qiagen) using a Retsch MM400 tissue homogenizer at 30þ t Hz twice for 2þ t minutes each. Total RNA from tissues was extracted using the procedure provided with the RNeasy Plus Kit.…Ž‹„”ƒ”›’”‡’ƒ”ƒ–‹‘ƒ†æ•‡“—‡…‹‰äþ Total RNA was sent to The Centre for Applied Genomics at e Hospital for Sick Children (Toronto, Canada). RNA quality was assessed using a Bioanalyzer (Agilent Technologies). We retained all samples with a DV200 (percentage of RNA fragments greater than 200 nt) greater than 85% (TableS1), discarding one Co-infected sample with a DV200þ t þ t 42%. Poly(A) mRNA was enriched using oligo dT-beads, and cDNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs). Barcoded libraries were pooled in equimolar quantities, and the sixteen libraries were sequenced on three lanes of a HiSeq. 2500 System (Illumina Inc.), which generated 126þ t bp paired-end reads.敇“—‡…‹‰”‡ƒ†ƒŽ‹‰‡–ƒ†ƒƒŽ›•‹•äþ We used FastQC v0.11.561 to assess sequence quality and Trimmomatic v0.3662 to remove the adapter sequences and low-quality bases from reads with the following settings: Illumina clop:2:30:10, leading:3, tailing:3, slidingwindow:4:15, minlength:36. We used TopHat v2.1.163 to align the trimmed paired-end reads from each library, separately, to the Ensembl M. lucifugus genome sequence (Myoluc2.064) in strand-specic mode (fr-rststrand) with mate-inner-dist values specic for the insert size of each library. We used featureCounts65 to count reads mapped to the Myoluc2.0 genome annotation in strand-specic mode (reversely stranded), counting paired-end reads as fragments, counting only those fragments where both reads aligned successfully, counting multi-mapping fragments, and excluding chimeric fragments. We assessed the variability within and between the treatments using the R package SARTools v.1.3.066. e featureCount-estimated gene counts were transformed by a variance stabilizing method (VST) using SARTools. We identied dierentially expressed genes between each of the treatments using DESeq2þ t v.1.12.3, run in SARTools. Custom SARTools-based DESeq settings included: cooksCutoþ t þ t TRUE (perform outliers detection), independentFilteringþ t þ t TRUE, alphaþ t þ t 0.05 (threshold of statistical signicance), pAdjustMethodþ t þ t BH (benjamini hochberg p -value adjustment method), and locfuncþ t þ t median (estimate size factors). Dierentially expressed genes were identied as having a fold-change 2 and false discovery rate (FDR)-corrected p-valuesþ t þ t 0.0567. We


10 produced volcano plots representing the dierential expression comparisons by plotting the log of the adjusted p value as a function of the log ratio of dierential expression. We used the Ensembl gene IDs identied by DESeq2 as input for the web-based g:Proler68 to test for gene ontology (GO) term enrichment among the dierentially expressed genes, using a FDR signicance threshold 0.05. ese GO-terms and their corresponding p -values were used in REViGO69 to visualize signicant enrichment of biological processes.f‰n…ƒ’–—”‡bfƒ‰ƒ‹•– Myl 摒”‘–‡‹äþ Purified, glutathione-s-transferase (GST)-tagged Myl -CoV N protein expressed in infected E. coli BL21 cells was used as positive antigen, and GST-tagged protein expressed in uninfected BL21 cells was used as negative antigen. 96-well Costar high-binding round-bottom assay plates were coated with 0.05þ t g/well of either antigen diluted in 0.1þ t M phosphate buered saline (pH 7.4) in a total volume of 100þ t l. Plates were covered and incubated overnight at 4þ t °C and washed three times with 300þ t l of PBS-Tween 20 (0.1%) immediately prior to use. Serum samples were diluted to 1:100 in PBS-Tween 20 (0.2%) supplemented with 5% fetal bovine serum (Gibco, ermosher). 100þ t l of each sample was added in parallel to a positive and negative antigen plate and incubated at 37þ t °C for one hour and washed as above. A peroxidase-labelled goat anti-bat IgG secondary antibody (0.05þ t g in 100þ t l per well, Bethyl labs) was added, incubated for one hour at 37þ t °C and washed as above. Peroxidase substrate (2,2’-azino-bis (3-ethylbenzthiazoline-6sulfonic acid)) was added to each well and colour development was quantied 30þ t minutes later by measuring the optical density at 405þ t nm using an ELISA microplate reader. e ELISA cut-o value (0.39) was calculated as the [(mean bat plasma O.D. values for bats that were PCR-negative for Myl -CoV in the ileum)þ t þ t (3x standard deviations of those O.D values)].–ƒ–‹•–‹…ƒŽƒƒŽ›•‹•äþ Data from RT-qPCR and histopathological scores were analysed with SPSS Statistics version 23. e relative levels of a transcript for each bat were calculated as RT-qPCR Cycle threshold (Ct) nor malized separately ( Ct) to the “house-keeping” gene GAPDH. A Ct reduction of one (1) indicates an approximately two-fold higher concentration of RNA. e signicance of dierences of mean values of Ct between co-infected bats and virus-infected bats were determined using an independent-samples Mann-Whitney U test. We calculated Pearson’s coecients to test the correlation between Ct levels for coronavirus polymerase cDNA for bats in each treatment class, and average scores for fungal hyphae, secondary bacteria, oedema, necrosis and inammation in wing lesions, as well as bacteremia and levels of neutrophils in lung, spleen and liver interstitium.b–Š‹…ƒŽ–ƒ–‡‡–äþ Bat studies were carried out in strict compliance with Canadian Council on Animal Care guidelines and the procedure for care, handling, and euthanasia of bats were approved by the University Committee on Animal Care and Supply of the University of Saskatchewan (protocol s#20100120).ƒ–ƒ……‡••‹„‹Ž‹–›All RNA-seq fastq les have been submitted to the NCBI Sequence Read Archive database (accession number SRX3752319SRX3752333). ese data les will be released to the public upon acceptance of this manuscript for publication.Referencesþt 1.þt Ge, X. Y. et al . Isolation and characterization of a bat SAS-lie coronavirus that uses the ACE2 receptor. 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