Abstract As the only flying mammal, bats harbor a number of emerging and re-emerging viruses, many of which cause severe diseases in humans and other mammals yet result in no clinical symptoms in bats. As the master regulator of the interferon (IFN)-dependent immune response, IFN regulatory factor 7 (IRF7) plays a central role in innate antiviral immunity. To explore the role of bat IRF7 in the regulation of the IFN response, we performed sequence and functional analysis of IRF7 from the pteropid bat, Pteropus alecto. Our results demonstrate that bat IRF7 retains the ability to bind to MyD88 and activate the IFN response despite unique changes in the MyD88 binding domain. We also demonstrate that bat IRF7 has a unique expression pattern across both immune and non-immune related tissues and is inducible by double-strand RNA. The broad tissue distribution of IRF7 may provide bats with an enhanced ability to rapidly activate the IFN response in a wider range of tissues compared to other mammals. The importance of IRF7 in antiviral activity against the bat reovirus, Pulau virus was confirmed by siRNA knockdown of IRF7 in bat cells resulting in enhanced viral replication. Our results highlight the importance of IRF7 in innate antiviral immunity in bats.

Citation: Zhou P, Cowled C, Mansell A, Monaghan P, Green D, Wu L, et al. (2014) IRF7 in the Australian Black Flying Fox, Pteropus alecto: Evidence for a Unique Expression Pattern and Functional Conservation. PLoS ONE 9(8): e103875. https://doi.org/10.1371/journal.pone.0103875 Editor: Sebastian D. Fugmann, Chang Gung University, Taiwan Received: May 6, 2014; Accepted: July 2, 2014; Published: August 6, 2014 Copyright: © 2014 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The P. alecto IRF7 sequence has been submitted to GenBank under accession number KJ534586. Funding: This work was supported by funding from the Australian Research Council Future Fellowship (FT110100234) to M. L. B. and a CSIRO OCE Science Leaders award to L. -F. W. and support from the Victorian Government’s Operational Infrastructure Support Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: M. L. B. confirms that they are a PLOS One Editorial board member. This does not alter the authors’ adherence to PLOS One Editorial policies and criteria.

Introduction Bats have been implicated in the spillover of many deadly viruses including rabies, henipaviruses (Hendra and Nipah), ebola virus, and the coronaviruses (CoV): severe acute respiratory syndrome (SARS-CoV) and the recently emerged Middle Eastern respiratory syndrome virus (MERS-CoV), all of which impose a significant threat to human health [1], [2], [3], [4], [5], [6]. As natural hosts, bats rarely show clinical signs of disease during infection [7]. How bats co-exist with viruses and the role of the bat innate immune system in controlling viral replication remain poorly understood [8]. Identifying the mechanisms responsible for controlling viral replication in bats has profound implications for the development of therapeutic strategies targeting viral infections in humans and other species. One of the most important early anti-viral defenses in mammals is the IFN system, which not only provides pivotal protection immediately following infection but also shapes the adaptive immune response [9]. Of the three IFN families discovered, type I (including α and β) and type III (λ) IFNs respond directly to viral infection. Due to the importance of IFNs in controlling viral replication, the regulation of the IFN response has been extensively studied in humans and other mammals. Key to the regulation of IFN production and signaling is the IFN regulatory factor (IRF) transcription factor family. The IRF family consists of nine members which share functional and structural characteristics. However, only IRF1, IRF3, IRF5 and IRF7 have been implicated as positive regulators of type I IFN transcription, and only IRF3 and IRF7 are designated as antiviral IRFs [10],[11]. Since their first discovery within the biological context of Epstein-Barr virus latency, IRF7 was identified as the master regulator of the type I IFN-dependent immune response, and perhaps that of type III IFN as well [12],[13],[14]. IRF7 is expressed only at low levels in most cells but is constitutively expressed in certain immune cells such as plasmacytoid dendritic cells (pDC) which specialize in IFN production. Correspondingly, the tissue distribution of human IRF7 is restricted to immune tissues which contain large numbers of specialized immune cells including spleen, thymus and peripheral blood lymphocytes whereas non-immune tissues including the intestine and colon express almost undetectable levels of IRF7 [12]. Although IRF7 is expressed at low levels in most cell types, it is induced strongly by type I IFN mediated signaling in all cells [15]. Interestingly, multiple fish species (Japanese flounder, crucian carp, mandarin fish, snakehead fish and Atlantic salmon) have been demonstrated to express IRF7 constitutively in all tissues including both immune and non-immune tissues [16],[17],[18],[19],[20]. Viral sensing either by Toll like receptors (TLRs) or retinoic acid-inducible gene 1 (RIG-I)-like receptors can result in the activation of IRF7 and subsequent induction of IFNs [21],[22]. All TLRs with the exception of TLR3 activate IRF7 through the adaptor protein, MyD88 (myeloid differentiation primary response gene 88) through the MyD88-dependent pathway. MyD88 forms a complex with the kinases IRAK-4 (interleukin 1 receptor associated kinase 4), IRAK-1 and TRAF-6 (TNF receptor associated factor). This complex binds directly to IRF7 leading to ubiquitination by TRAF-6 and phosphorylation by IRAK1 or IKK-1 (IκB kinase-1) and translocation from the cytosol to the nucleus where IRF7 binds to promoter elements inducing IFN production [21],[23]. TLR3 and TLR4 activate IRF7 through the MyD88-independent pathway through the adaptor molecule TRIF (TIR-domain-containing adapter-inducing IFN-β) which forms a complex with TBK1 (TANK binding kinase 1), IKK-ε (inhibitor of nuclear factor-κb kinase e) and IRF7. In this case the phosphorylated IRF7 forms a homodimer or heterodimer with IRF3 and translocates to the nucleus where it binds to the IFN promoter via its DNA-binding domain to induce type I or type III IFN [24]. In IRF7 knockout mice, viral induced IFN production through the TLR3 (MyD88-independent) pathway is greatly impaired. As a result, mice become more susceptible to viral infection [13]. Although IRF3 has been reported to preferentially activate IFN-β over IFN-α genes, IRF7 is believed to efficiently activate both IFN-α and IFN-β [25]. The human IFN-β promoter region contains four positive regulatory domains (PRDs 1 to IV) that serve as binding sites for IRFs. In the human IFN-α promoter region there may be two or three PRD modules depending on the IFN-α subtype [26]. Due to the importance of IRF7 in the innate immune response, it is an active target for viruses to evade the host immune response [27]. A role for IRF7 in immunosurveillance has also been identified in breast cancer [28]. Using the Australian black flying fox (P. alecto) as a model species we have begun to explore the role of the IFN system in the control of viral replication in bats. We have demonstrated that TLRs, RIG-I-like receptors, and some IFN stimulated genes (PKR, Mx1 and OAS1) appear to be conserved in sequence compared to other mammals [29],[30],[31]. However, bats appear to have relatively higher expression of type III IFN and wider distribution of type III IFN receptors consistent with a role for type III IFNs in antiviral immunity [32],[33]. Bat genome analysis has also provided evidence for positive selection of genes within the IFN pathway, including TLR7, c-Rel, TBK-1, IFN-γ, ISG15 and RIG-I [34]. These changes may have occurred in response to the co-evolution of bats with viruses and may have consequences for the clearance of viral infections and the ability of bats to coexist with viruses. Due to the central role of IRF7 in the regulation of the IFN response, we performed sequence and functional analysis of P. alecto IRF7. Our results provide the first description of IRF7 in any species of bat and evidence for conserved IRF7 functional activity despite variation at the sequence level in the bat IRF7 gene.

Materials and Methods Cells lines All animal experiments were approved by the Australian Animal Health Laboratory (AAHL) animal ethics committee (protocol number 1389). Immortalized and cloned P. alecto kidney (PaKiT03) and lung (PaLuT02) cells established previously [35] were cultured in DMEM/F12-Hams (Sigma), supplemented with 10% foetal calf serum (FCS, Hyclone), 100 units/ml penicillin, 100 µg/ml streptomycin and 50 µg/ml gentamycin (Sigma). Human embryonic kidney HEK293T cells were cultured in DMEM supplemented with 10% FCS (Hyclone), 15 mM L-glutamine, 100 µg/ml penicillin, NEAA/Na-py/fungizone. All cells were maintained in a humidified atmosphere of 5% CO 2 at 37°C. Viruses Sendai virus (SeV, Cantell strain) was prepared in chicken embryos as described previously [29]. Pulau virus (PulV) was prepared and titered as described previously [33]. For infection of cells, virus was incubated with cells for one hour at 37°C, then replaced with normal cell culture medium for the indicated time. Genome and sequence analysis Full-length IRF7, IRF3 and MyD88 open reading frames (ORFs) were identified in the P. alecto genome (NCBI ID P. alecto ASM32557v1) [34] using BLASTX. For comparative purposes, sequences were obtained from the current genome assemblies from the Ensembl database for the following species: ENSP00000380697, Homo sapiens (human); ENSMUSP00000095565, Mus musculus (mouse); ENSECAP00000007698, Equus caballus (horse); ENSSSCP00000013664, Sus scrofa (pig); ENSBTAP00000056564, Bos taurus (cow). The microbat Myotis davidii IRF7 amino acid sequence was deduced from IRF7 ORF annotated from the published M. davidii genome (NCBI ID M. davidii ASM32734v1) [34]. The P. alecto IRF7 sequence has been submitted to GenBank under accession number KJ534586. Sequence alignment was performed using ClustalX and visualized using GeneDoc (http://www.nrbsc.org/gfx/genedoc/index.html). Alignment files were visualized using EMBOSS Plotcon to determine the conservation of IRF7 proteins among different species. Genomic intron-exon maps of the genes were drawn using Fancy Gene v1.4 by comparing individual IRF7 ORFs of P. alecto, horse and human (http://host13.bioinfo3.ifom-ieocampus.it/fancygene/). Phylogenetic trees were constructed using the neighbour joining method and MEGA4.1 program with 1000 bootstrap replicates [36]. Plasmid constructs Primers listed in Table 1 were designed based on the P. alecto genomic sequences and used in RT-PCR to amplify IRF7, IRF3 and MyD88 from RNA extracted from freshly isolated bat splenocytes. To construct expression plasmids, PCR products corresponding to full-length IRF3 and IRF7 were ligated directly to Vivid Colors pcDNA 6.2/EmGFP TOPO vector (Life Technologies) with an N-terminal GFP tag. The MyD88 ORF was ligated to the pFLAG-CMV2 expression vector (Sigma) using restriction enzymes NotI and SalI with an N-terminal FLAG tag for detection. To generate a truncated bat IRF7 (tIRF7) that lacked the MyD88 binding region at amino acids (aa) 234–298, overlapping PCR was performed [37]. The resulting tIRF7 PCR product was ligated to the pcDNA 6.2/EmGFP TOPO vector. The human IRF7 (hu-IRF7) and hu-MyD88 plasmids have been described previously [38]. Hu-IRF7 is in the pEGFP-N1 vector and hu-MyD88 is in the pEF-Bos vector with an N-terminal FLAG tag. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. https://doi.org/10.1371/journal.pone.0103875.t001 Mouse IFN-α4, IFN- α6 and human IFN-β promoter plasmids, abbreviated as Mu_IFN- α4P, -α6P and Hu_IFN- βP respectively, have been described previously [21]. The bat IFN-β promoter plasmid was constructed using sequence 1000 bp upstream from the start codon of IFN-β ORF from the P. alecto genome. Promoter prediction was performed using the online transcriptional start site prediction tool, Matinspector in the Genomatix software suite (http://www.genomatix.de/cgi-bin//matinspector_prof). Regions containing putative IRF3 or IRF7 binding sites were identified from −221 to −70 bp from the ATG of the bat IFN-β gene by comparison with human IFN promoters and cloned into the pGL4.1 expression vector (Promega). A transfection control pRL-Tk plasmid containing Renilla luciferase was obtained from Promega. Details of primers used during plasmid construction can be found in Table 1. Luciferase promoter assay HEK293T cells were transfected using Fugene 6 (Promega) according to the manufacturer’s instructions. Approximately 2×105 cells per well in a 24-well plate were co-transfected with 100 ng of the relative IFN promoter plasmids, 50 ng of pRL-TK (Promega) served as an internal control. Where indicated, expression plasmids for bat IRF7, human IRF7 or bat MyD88 were included in the transfection mix. Cells were harvested 30 h post-transfection and lysed using passive lysis buffer provided in the following kit. Luciferase activities were determined using the dual-luciferase assay system (Promega) using a Thermo Fluoroskan ascent FL machine. For promoter experiments in PaKiT03 cells, similar transfections were performed using lipofectamine 2000 (Life Technologies). IRF7 knockdown A smartpool consisting of four small interfering RNAs (siRNAs) targeting bat IRF7 (siIRF7) was designed based on the full-length IRF7 ORF sequence using Dharmacon custom services (Thermo). Information of the four siIRF7 can be found in Table 1. Transfection of siIRF7 was performed in PaKiT03 cells using the Neon transfection system (Life Technologies) according to the manufacturer’s instructions. Briefly, 20 nM of each of the four siIRF7 was used for every 105 cells. Cells were harvested into RLT lysis buffer (RNeasy kit, Qiagen) 48 h post-transfection and stored at −80°C prior to RNA extraction. Quantitative reverse transcription PCR (qRT-PCR) RNA was extracted from cell lysate from the siRNA knockdown experiment using a Qiagen RNeasy kit and converted to cDNA using the Quantitect reverse transcription kit for real-time PCR (Qiagen). All experiments were performed according to manufacturer’s protocols. Preparation of cDNA from 12 P. alecto tissues including brain, kidney, liver, lung, lymph nodes, spleen, heart, small intestine, wing, salivary gland, thymus and testis from three individual bats and from polyI:C stimulated PaLuT02 cells has been described previously [33]. For each sample, 1 µg RNA was applied to reverse transcription using the Quantitech reverse transcription kit (Qiagen). IRF7 qRT-PCR primers were designed using Primer Express 3.0 (Applied Biosystems) with default parameter settings and are listed in Table 1. Primers for IFN-β and 18s rRNA have been described previously [33]. Reactions were carried out using EXPRESS SYBR GreenER qPCR Supermix Universal (Life technologies) in an Applied Biosystems 7500 Fast Real-Time qRT-PCR instrument. For each reaction from cDNA, 2 µl of 1∶5 diluted cDNA were used with a final concentration of 200 nmol of each primer. The cycling profile for cDNA samples consisted of an initial denaturation at 94°C for 2 minutes followed by 40 cycles of 94°C for 15 seconds, 60°C for 1 minute, followed by melt curve analysis. Expression levels of target genes were calculated using the standard curve method after normalisation to the housekeeping gene 18s rRNA. Indirect immunofluorescence assay (IFA) confocal microscopy PaKiT03 cells were seeded onto glass coverslips in 24-well plates at 105 cells per well one day before transfection. They were transfected with 200 ng each of GFP-IRF7 and FLAG-tagged MyD88 (human or bat) using Lipofectamine 2000 (Life Technologies). At 16 h post-transfection, cells were fixed in 4% paraformaldehyde in PBSA at room temperature for 40 minutes. After removal of fixative, cells were washed three times with PBSA, followed by treatment with 0.1% Triton X-100 for 10 minutes and blocked with 0.5% BSA in PBSA for 30 minutes. Mouse anti-human MyD88 antibody (Santa Cruz, CAT. sc11356) was diluted 1∶1000 in 0.5% BSA and applied to cells and incubated at 37°C for 1 h. Cells were then incubated with Alexa 488 conjugated goat anti-mouse secondary antibody at 37°C for 1 h and washed three times in PBSA. Nuclei were labelled with DAPI and coverslips were mounted on glass slides for analysis. All slides were examined under a Leica confocal microscope (Leica, Germany). Immunoprecipitation analysis The immunoprecipitation method for analysis of IRF7 and MyD88 has been described previously [38]. In brief, 2×106 HEK293T cells were seeded 24 h prior to co-transfection with 1.25 µg of both FLAG-MyD88 and GFP-IRF7 (human or bat) using Fugene 6 (Promega). Cells were then lysed 24 h later with lysis buffer (50 mM Tris (pH7.4), 1.0% Triton X-100, 150 mM NaCl, 1 mM EDTA, 2 mM Na 3 VO 4 , 10 mM NaF, 1 mM PMSF and protease cocktail inhibitor mixture; Promega). Cell lysates were cleared by centrifugation (9000 g, 10 min, 4°C), and then pre-cleared with protein G-Sepharose beads for 30 min at 4°C and FLAG-MyD88 immune complexes were immune-precipitated from supernatant using anti-FLAG M2-agarose (Sigma) conjugated beads for 2 h at 4°C. Beads were washed with 3 X lysis buffer and eluted by boiling beads in 5 volumes of SDS PAGE sample buffer. Lastly, SDS PAGE and western blot analysis were performed using the samples obtained. For the blotting, anti-human MyD88 rabbit antibody and anti-human IRF7 goat antibody (both from Santa Cruz) were used at 1∶1000 dilution during primary antibody incubation and AP-conjugated goat anti-rabbit or rabbit anti-goat secondary antibody (both from Life technologies) were used at 1∶2000 dilution.

Discussion IRF7 is a master regulator of IFN expression in mammals and is therefore central to the innate antiviral immune response. In humans, IRF7 acts predominately in pDCs via activation of TLR7/9 and the MyD88 dependent signaling pathway [49]. Regulation of the IFN response may play an important role in the ability of bats to coexist with viruses in the absence of clinical signs of disease. This report describes the analysis of IRF7 from our model bat species, the Australian black flying fox, P. alecto, an important reservoir for viruses including Hendra virus, which has resulted in the deaths of numerous horses and humans since its discovery in 1994 [4],[46]. Our results support the conservation of functional activity of IRF7 in P. alecto but provide evidence of a wider tissue distribution which has implications for broader activation of the IFN response in bats. Our results provide the first functional characterization of IRF7 in any species of bat and contribute to our understanding of the function and evolution of IRF7 in mammals. Bat IRF7 was identified from the bat genome and bat transcriptome data [39],[40], together with RT-PCR results from spleen cDNA resulting in the identification of a single full length variant of IRF7. In humans and mice, IRF7 expression is very low in most tissues and cells with the exception of pDCs and cells that have been activated by IFN [12],[15]. In contrast, the transcription of P. alecto IRF7 was detected not only in immune-related tissues but comparable expression was observed in many other organs as well. Although there is a lack of data on the tissue distribution of IRF7 in mammals besides human and mouse, there have been several studies on fish IRF7. Interestingly, at least five species of fish including crucian carp, mandarin fish, snakehead fish, Atlantic salmon and Japanese flounder express IRF7 constitutively in a wide variety of tissue types although different IRF7 transcripts were expressed in each species. These tissues were neither primarily immune-related nor serve as portals for microbial infection, where the immune response is easily initiated. Since these fish IRF7s can also be induced by dsRNA, they were hypothesised to play an important role in fish immunity [16],[17],[18],[19],[20]. Although further analysis of the cell types responsible for constitutive IRF7 expression in bats is required, a constitutively expressed IRF7 in a broad range of cells and organs may result in faster and stronger IFN production upon viral infection [49]. This observation is similar to the pattern of type III IFN receptor expression which has a wide distribution in bats but only limited distribution in other mammals [50]. Thus, bats may maintain the potential to rapidly activate the innate immune response in a broader subset of tissues and cells than other mammals. Induction of IRF7 by treatment or transfection of our bat kidney cell line with the dsRNA ligand, polyI:C resulted in a peak in the induction of IRF7 at 9 h post-treatment, which is 3 h later than the peak in bat type I and type III IFNs but similar to that of ISGs Mx1, OAS1 and PKR described previously in bat cells [29],[33]. This result is consistent with the induction of IRF7 through type I IFN feedback similar to other species. In humans, IRF7 is generated through multiple pathways following IFN induction. Following the production of IFN and binding to the IFN-αR, a complex consisting of activated STAT1, STAT2 and IRF9, called the IFN stimulated gene factor 3 (ISGF3) is formed, which in turn binds to the ISRE on the IRF7 promoter and induces IRF7 transcription. The human IRF7 promoter region contains an NF-κB binding site and a single functional ISRE approximately 1.3-kb upstream from the ATG start site, both of which are important in the induction of IRF7 [49],[51]. Analysis of the putative bat IRF7 promoter region resulted in the identification of two ISREs and one NF-κB binding site indicating that multiple mechanisms for IRF7 activation may also exist in bats. However, two ISRE sites were also identified in the IRF7 promoters of other species examined (mouse, horse, cow, dog, cat and rat). Thus, whether the broad distribution of constitutively expressed IRF7 is the result of the presence of a more efficient IRF7 promoter region driven by transcription factors other than IRFs, or simply due to enriched immune-related cells in all tissues will require further study. Sequence differences in the MyD88 binding domain of bat and human IRF7 led to the hypothesis that there may be functional differences in the activation of bat IRF7 and the regulation of the IFN response that may contribute to the ability of bats to resist the clinical outcomes of viral infection. Our results demonstrate that these sequences differences do not appear to affect IRF7 function either in IFN transactivation activity or activation by MyD88. Bat IRF7 was capable of activating both IFN-α and IFN-β promoters and the levels of transactivation were equivalent to or higher than that of human IRF7. Similarly, bat MyD88 and bat IRF7 maintained binding capability similar to their human counterparts. Deletion of the MyD88-binding region of bat IRF7 impaired its ability to activate IFN, demonstrating functional conservation of the MyD88 binding domain with that of human IRF7. Collectively, these data demonstrate that bat IRF7 is capable of inducing IFN and MyD88 binding in a similar manner to human IRF7. Although the MyD88 binding domain of bat IRF7 has low sequence conservation with the equivalent region in human IRF7, experimental data demonstrate a fully functionally IRF7 exists in pteropid bats. Our results describing the experimental knockdown of IRF7 using siRNA is to our knowledge the first description of the use of siRNAs in bat cells. The successful knockdown of IRF7 is consistent with the presence of an RNA-silencing mechanism in bats similar to that in other mammals. IRF7 knockdown resulted in impaired IFN-β induction in SeV infected cells and enhanced PulV replication. Although further work will be required to determine whether IRF7 is the master regulator of the bat IFN response, these results confirm that IRF7 plays an important role in anti-viral defense and the early innate immune response in bats [13]. Analysis of the bat IFN-β promoter also revealed one residue difference in the PRDI module, known to be associated with activation by IRF3 or IRF7 [43]. A similar change in the human IFN-αP impairs its inducibility by IRFs [26]. Although bat IFN-β is strongly inducible in bat cells following either stimulation or viral infection, further work will be necessary to determine whether this mutation affects the induction of IFN-β under conditions other than those described here [29]. The presence of this mutation may also indicate that transcription factors other than IRF3 and IRF7 are involved in the regulation of IFNs in bats. Therefore, future work focusing on IFN promoters (including IFN-α and IFN-λ) will be necessary to explore whether the bat IRF-IFN induction pathway is as critical to IFN induction as it is in other species. In humans, there are around 1400 transcription factors that have been recognized [52]. Whether these factors play similar roles in bats or whether they perform different functions resulting in differences in the expression of downstream genes remains to be determined. Understanding the mechanisms responsible for the regulation of the IFN response will assist in discovering how bats successfully coexist with viruses. In conclusion, our data clearly demonstrate that bat IRF7 exhibits a constitutive expression pattern across a broad range of immune and non-immune related organs, intact IFN transactivation function, and binds to and is activated by bat MyD88. These findings not only provide the first information on the MyD88-IRF7 dependent IFN production pathway in bats, but also explore the functionality of bat IRF3 and identify critical point mutations in the bat IFN-β promoter. Ultimately, we hope these advances may help uncover the mechanisms underlying the ability of bats to co-exist with deadly viruses, and that this will in turn lead to the development of potential therapeutic strategies targeting viral infections in other mammals.

Supporting Information Figure S1. Phylogenetic analysis based on amino acid alignment of IRF7 from representative vertebrate species. Branch support is indicated as the percentage of 1000 bootstrap replicates. Sequences are from the Ensembl database with the exception of P. alecto IRF7. P. alecto IRF7 is highlighted in bold. https://doi.org/10.1371/journal.pone.0103875.s001 (TIF) Figure S2. Alignment of the IRF7 DNA binding motif of P. alecto with other mammals. Residues important for IRF7 binding to the IFN promoter (described in Genin et al, 2009) are highlighted in grey. Sequences from species other than P. alecto and M. davidii were obtained from the Ensembl database. Bat sequences from P. alecto, P. vampyrus and M. davidii genome are colored red. https://doi.org/10.1371/journal.pone.0103875.s002 (TIF) Figure S3. Alignment of MyD88 (in bold) from P. alecto with sequences from other mammals. Residues which are identical to human MyD88 are shown as dashes while gaps are indicated by dots. The death domains which are responsible for activation of IRF7 proteins are boxed. Sequences from species other than P. alecto were obtained from the Ensembl database. https://doi.org/10.1371/journal.pone.0103875.s003 (TIF) Figure S4. Alignment of the P. alecto IFN-β promoter region with the corresponding region from human, horse, mouse, dog, pig and microbat. If more than one IFN-β sequence was present, the gene identified as IFN-β1 was used in the analysis. Residues which are identical to the human sequence are shown as dashes while gaps are indicated by dots. Two modules named positive regulatory domain (PRD) III and I, responsible for binding to IRF3 and IRF7 in humans (Maniatis et al, 1998) are boxed. The TATA binding motif has also been boxed. The residue in the P. alecto IFN-β promoter which may have disabled PRDI has also been highlighted. https://doi.org/10.1371/journal.pone.0103875.s004 (TIF) Figure S5. Bat IRF3 can’t be activated by bat MyD88 (A) but can induce IFN-βP by itself (B). HEK293T cells were transiently co-transfected with bat MyD88 expression plasmid and mouse IFN-α4 or Bat IFN-βP promoter plasmids along with bat IRF3. After 30 h, cells were analysed for promoter activity by reporter gene luciferase assay. Data are mean values of two independent experiments and error bars represent standard errors. https://doi.org/10.1371/journal.pone.0103875.s005 (TIF)

Acknowledgments We thank Drs Prasad Paradkar and Glenn Marsh for critical review of the manuscript. Contributions Conceived and designed the experiments: PZ MLB AM PM. Performed the experiments: PZ PM DG LW. Analyzed the data: PZ PM DG LW. Contributed reagents/materials/analysis tools: AM CC ZS LFW. Contributed to the writing of the manuscript: PZ MLB.

Author Contributions Conceived and designed the experiments: PZ MLB AM PM. Performed the experiments: PZ PM DG LW. Analyzed the data: PZ PM DG LW. Contributed reagents/materials/analysis tools: AM CC ZS LFW. Contributed to the writing of the manuscript: PZ MLB.