Bats harbor a myriad of viruses and some of these viruses may have spilled over to other species including humans. Spillover events are rare and several factors must align to create the “perfect storm” that would ultimately lead to a spillover. One of these factors is the increased shedding of virus by bats. Several studies have indicated that bats have unique defense mechanisms that allow them to be persistently or latently infected with viruses. Factors leading to an increase in the viral load of persistently infected bats would facilitate shedding of virus. This article reviews the unique nature of bat immune defenses that regulate virus replication and the various molecular mechanisms that play a role in altering the balanced bat–virus relationship.

Luman is a human basic leucine zipper transcription factor that, like the herpes simplex virus transcription factor VP16, requires the host cell factor, HCF, for activity. Although both HCF and Luman have been implicated in cell growth, their biological roles have not been clearly defined. Luman conforms to a type II membrane-associated glycoprotein with its carboxyl terminus embedded in cellular membranes and its amino terminus, which contains all its identified functional domains, in the cytoplasm. Here we show that Luman is processed by regulated intramembrane proteolysis (RIP). The site 1 protease (S1P), a Golgi apparatusresident enzyme responsible for catalyzing the first step in the RIP pathway of the sterol regulatory element binding proteins (SREBPs) and ATF6, may also be involved in the processing of Luman. Thus, processing of Luman was highly stimulated by brefeldin A, a compound that causes the reflux of Golgi apparatus enzymes to the endoplasmic reticulum (ER). In addition, coexpression of Luman with S1P containing a KDEL ER retrieval signal resulted in virtually quantitative cleavage of Luman in the absence of any treatment. Finally, Luman contains a sequence, RQLR, immediately downstream from the transmembrane domain which bears similarity to the consensus S1P cleavage site identified by others. Substitution of arginine residues within this motif abolished S1P cleavage, providing robust evidence that S1P is involved in Luman processing. We observed that following S1P cleavage, the majority of the cleaved Luman was retained in cytoplasmic membranes, indicating that an additional step or enzymes yet to be identified are involved in complete cleavage and release to yield the product which ultimately enters the nuclei of cells.Luman (also known as LZIP and CREB3) is a basic leucine zipper transcription factor of the CREB/ATF gene family. It possesses a potent N-terminal acidic activation domain and a basic-leucine zipper motif (bZIP) (15,(23)(24)(25)(26). The primary structure of Luman appears to be strongly conserved, and Luman homologues in mice (LZIP [8]), cattle (our unpublished results), and fruit flies (dCREB-A/BBF-2 [1, 37]) have been identified. We and others (15, 24) originally identified Luman when screening for cellular ligands of the human host cell factor (HCF, also known as C1 factor), a protein required by the herpes simplex virus (HSV) transactivator VP16. Luman interacts with HCF through the tetrapeptide DHTY (15,24,25), which is homologous to the EHAY HCF binding sequence of VP16. This motif, as (D/E)HXY, is conserved in the VP16 homologues of other alphaherpesviruses as well as in the homologues of Luman in mice, cattle, and fruit flies. Luman can bind and activate genes containing cyclic AMP response elements (CREs), although its natural target has not been identified. Similarly, although Luman has been implicated in the regulation of cell growth (18), its biological role in this process has not been clearly defined. Luman mRNA is present in a wide range of adult and fetal tissues (...

In recent years viruses similar to those that appear to cause no overt disease in bats have spilled-over to humans and other species causing serious disease. Since pathology in such diseases is often attributed to an over-active inflammatory response, we tested the hypothesis that bat cells respond to stimulation of their receptors for viral ligands with a strong antiviral response, but unlike in human cells, the inflammatory response is not overtly activated. We compared the response of human and bat cells to poly(I:C), a viral double-stranded RNA surrogate. We measured transcripts for several inflammatory, interferon and interferon stimulated genes using quantitative real-time PCR and observed that human and bat cells both, when stimulated with poly(I:C), contained higher levels of transcripts for interferon beta than unstimulated cells. In contrast, only human cells expressed robust amount of RNA for TNFα, a cell signaling protein involved in systemic inflammation. We examined the bat TNFα promoter and found a potential repressor (c-Rel) binding motif. We demonstrated that c-Rel binds to the putative c-Rel motif in the promoter and knocking down c-Rel transcripts significantly increased basal levels of TNFα transcripts. Our results suggest bats may have a unique mechanism to suppress inflammatory pathology.

Host cell factor (HCF) was initially discovered as a cellular co-factor required for the activation of herpes simplex virus immediate early gene expression by the virion associated transactivator VP16. HCF also participates in a variety of cellular processes, although the mechanism of its action is not known. VP16 binds to HCF through a 4-amino acid motif (EHAY), which closely resembles the HCF binding domain of two cellular basic leucine-zipper proteins, Luman and Zhangfei. Luman is a powerful transcription factor that, in transient expression assays, activates promoters containing cAMP or unfolded protein response elements (UPRE). In contrast, Zhangfei neither binds consensus recognition elements for basic leucinezipper proteins nor does it activate promoters containing them. Here we show that Zhangfei suppresses the ability of Luman to activate transcription. HCF appeared to be required for efficient suppression. A mutant of Zhangfei, which was unable to bind HCF, was impaired in its ability to suppress Luman. Zhangfei did not suppress ATF6, a transcription factor closely related to Luman but that does not bind HCF, unless the HCF binding motif of Luman was grafted onto it. Zhangfei inhibited the HCF-dependent activation of a UPRE-containing promoter by a Gal4-Luman fusion protein but was unable to inhibit the HCF-independent activation by Gal4-Luman of a promoter that contained Gal4 binding motifs. Binding of HCF by Zhangfei was required for the co-localization of Luman and Zhangfei to nuclear domains, suggesting that HCF might target the proteins to a common location.

Bats are important reservoir hosts for emerging viruses, including coronaviruses that cause diseases in people. Although there have been several studies on the pathogenesis of coronaviruses in humans and surrogate animals, there is little information on the interactions of these viruses with their natural bat hosts. We detected a coronavirus in the intestines of 53/174 hibernating little brown bats (Myotis lucifugus), as well as in the lungs of some of these individuals. Interestingly, the presence of the virus was not accompanied by overt inflammation. Viral RNA amplified from little brown bats in this study appeared to be from two distinct clades. The sequences in clade 1 were very similar to the archived sequence derived from little brown bats and the sequences from clade 2 were more closely related to the archived sequence from big brown bats. This suggests that two closely related coronaviruses may circulate in little brown bats. Sequence variation among coronavirus detected from individual bats suggested that infection occurred prior to hibernation, and that the virus persisted for up to 4 months of hibernation in the laboratory. Based on the sequence of its genome, the coronavirus was placed in the Alphacoronavirus genus, along with some human coronaviruses, bat viruses and the porcine epidemic diarrhoea virus. The detection and identification of an apparently persistent coronavirus in a local bat species creates opportunities to understand the dynamics of coronavirus circulation in bat populations.

Insectivorous bats are speculated to be ancestral hosts of Middle-East respiratory syndrome (MERS) coronavirus (CoV). MERS-CoV causes disease in humans with thirty-five percent fatality, and has evolved proteins that counteract human antiviral responses. Since bats experimentally infected with MERS-CoV do not develop signs of disease, we tested the hypothesis that MERS-CoV would replicate less efficiently in bat cells than in human cells because of its inability to subvert antiviral responses in bat cells. We infected human and bat (Eptesicus fuscus) cells with MERS-CoV and observed that the virus grew to higher titers in human cells. MERS-CoV also effectively suppressed the antiviral interferon beta (IFNβ) response in human cells, unlike in bat cells. To determine if IRF3, a critical mediator of the interferon response, also regulated the response in bats, we examined the response of IRF3 to poly(I:C), a synthetic analogue of viral double-stranded RNA. We observed that bat IRF3 responded to poly(I:C) by nuclear translocation and post-translational modifications, hallmarks of IRF3 activation. Suppression of IRF3 by small-interfering RNA (siRNA) demonstrated that IRF3 was critical for poly(I:C) and MERS-CoV induced induction of IFNβ in bat cells. Our study demonstrates that innate antiviral signaling in E. fuscus bat cells is resistant to MERS-CoV-mediated subversion.

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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