**2. Innate immune adaptation of bats as preventing to develop severe infections**

The innate immune system is primary or first line of the defense against invading pathogens. The pattern recognition receptors (PRRs), including toll-like receptors (TLRs), Nod-like receptors (NLRs), absent in melanoma-2 (AIM2)-like receptors (ALRs), retinoic acid-inducible gene-1 (RIG-1)-like receptors (RLRs, RIG-1 and melanoma differentiation-associated protein 5 or MDA5), C-type lectin receptors (CLRs), and cyclic GMP (guanosine monophosphate)–AMP (adenosine monophosphate) synthase (cGAS) and stimulator of interferon genes (STING) signaling pathways play a crucial role in the host defense and the generation of pro-inflammatory immune response (cytokine, chemokines, reactive oxygen and nitrogen species (ROS and RNS), and type 1 interferon (IFN) production) [29–34]. TLR4 is a crucial PRR to recognize Gram-negative bacterial lipopolysaccharide (LPS) as a potent microbial or pathogen-associated molecular pattern (MAMP or PAMP) to induce a potent pro-inflammatory immune response to clear the infection. However, its overactivation may cause severe inflammation. Pallas's mastiff bats (*Molossus molossus*) upon exposure to the *Escherichia coli* (*E. coli*)-derived LPS do not develop leucocytosis and hyperthermia or fever independent of their sex (**Figure 1**) [35]. However, they show weight loss upon exposure to the LPS. This study indicates the presence of defective TLR4 signaling responsible for the NF-κB-dependent pyrogenic cytokines (IL-1 and IL-6) (**Figure 1**). This defect may also prevent the further activation of cytosolic NLRP3-dependent inflammasomes responsible for generating IL-1β and IL-18. Bat (little brown bat or *Myotis lucifugus*) mitochondria produce lesser ROS (a potent inducer of NLRP3 activation) [36].

#### **Figure 1.**

*Schematic representation of immune response in bats preventing development of severe infection and inflammation. The gram-negative bacteria or its PAMP (LPS) recognition in bats do not stimulate proinflammatory cytokine production through NF-*κ*B activation and increase in body temperature. The increase in autophagy further increases cellular longevity, acts as an antiviral mechanism to clear or control the infection, decreases or suppresses inflammation. The PYHIN domain containing AIM2 and IFI16 inflammasomes are absent and hence, do not take part in cytosolic DNA recognition as DAMP to inflammasome activation-based maturation of IL-1*β *and IL-18. This further decreases the incidence of inflammation and associated tissue damage. The cGAS-STING-based signaling mechanism recognizing cytosolic dsDNA as DAMP also does not work in bats due to the presence of serine at 358 AA position in STING that is unable to activate IRF3 and type 1 IFN production. Hence, this further prevents inflammatory events in response to the self-DNA. Only the cytosolic RNAs activate different PRRs (RIG-1, MDA5, and TLR3) that via IRF3 and IRF7 activation synthesize type 1 and 3 IFNs, which exert antiviral action, but damp pro-inflammatory action of NRLP3 and NLRP1 inflammasomes. Mx1 is an IFN-inducible antiviral protein with a GTPase activity. APOBEC3 also directly acts as an antiviral host factor without inducing inflammation. Hence, only protective antiviral immune response works in bats to control their number without inducing severe inflammation.*

#### *Learning from Bats to Escape from Potent or Severe Viral Infections DOI: http://dx.doi.org/10.5772/intechopen.98916*

The reduced mitochondrial ROS (mtROS) production in Seba's short-tailed bats involves a mild depolarization of the inner mitochondrial membrane that decreases the membrane potential to a level sufficient to produce ATP molecules but insufficient to synthesize mtROS (**Figure 1**) [37]. This mechanism decreases with age in mice but remains intact in these bats. For example, in 2.5 years old mice this mechanism of mild mitochondrial depolarization disappears in different organs (lungs, liver, spleen, skeletal muscles, heart, brain, and kidneys). Hence, mtROS-mediated DNA and protein damage is seen in mice or other mammals but not in bats.

The immune challenge among bats does not alter their oxidative stress irrespective of their pre-migration and migration seasons [38]. However, bats have higher baseline leukocytes but lower neutrophil numbers during their migratory seasons as compared to their pre-migratory season. Their plasma haptoglobin (a humoral innate immune component) levels also remain same during both seasons [38]. However, plasma haptoglobin level of migratory bats increases upon an immune (LPS) challenge that remains unchanged in non-migratory or pre-migratory bats under the same immunogenic stimulation. Of note, bats do not upregulate genes associated with chronic inflammation with the advancement of age that is seen in other mammals, including humans [39]. Hence, this protects them from age related inflammatory diseases and predisposes them towards healthy aging and longevity along with tolerance to infections, including Ebola, Nipah, and many more. Also, the bat microbiota (Firmicutes and Proteobacteria are dominant bacteria) differs from other terrestrial mammals (strict anaerobic phylum Bacteroidetes in mice and humans), and remains intact throughout their life that further protects them from age-associated inflammation and inflammatory diseases [40, 41]. On the other hand in mice and humans gut microbiota changes with time and aging that predispose them to age-associated inflammatory diseases associated with gut bacteria dysbiosis [42–44].

A study has shown the TLR3, TLR7, and TLR9 expression at mRNA levels in different organs of Leschenault's Rousette bats (*Rousettus leschenaulti*) [45]. Another study has shown the expression of full length mRNA transcripts of TLR1-TLR10 in the Australian flying fox or *P. alecto* [46]. This bat species also expresses the pseudogene for TLR13. However, their functional protein level expression in different bat species needs further investigation. The evolutionary studies have shown that the bats evolved under the influence of positive selection for TLR7, TLR8, and TLR9 that is highest for TLR9 and lowest for TLR7 [47]. The TLR3 in bats has evolved under a negative selection process. This study indicates the adaptation of host-pathogen interaction in bats, particularly in bat TLR9. The bat TLR8 has an extensive sequence variation within them that separates them from other mammals, including humans [48]. Bat TLRs are evolving slowly under purifying selection in response to the functional constraints with a divergence process that is overall congruent with the species tree [49]. The bat TLRs show unique mutations in their ligand-binding domains even involving their non-conservative amino acid (AA) change and/or targets of positive selection. These changes can modify the binding of the corresponding TLR ligands. Hence, bat TLRs may vary in recognizing the same ligand recognized by other mammalian or human TLRs.

Flying fox bats (*P. alecto*) have other cytosolic dsRNA recognizing receptors called RLRs, including RIG-1, MDA5, and laboratory of genetics and physiology 2 (LGP2), like humans that upon recognizing cytosolic dsRNA induce the type 1 IFN production [50]. LGP2 synergy with MDA5 to generate antiviral immune response during RLR-dependent dsRNA recognition [51]. LGP2 interacts with the IFN-inducible, dsRNA binding protein PACT (a cofactor of DICER in the processing of microRNAs) through its regulatory C-terminal domain that inhibits RIG-1-dependent signaling but promotes MDA5-dependent antiviral

immune response [52]. TLR3, RLRs (RIG-1), and MDA5 serve as potent antiviral immune response inducers in bats to protect them from severe infection caused by Encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV) (**Figure 1**) [53]. The functionally conserved RLR adaptor called mitochondrial antiviral signaling (MAVS) protein has been demonstrated in the Chinese rufous horseshoe bat (*Rhinolophus sinicus*) and straw-colored fruit bat (*Eidolon helvum*) that upon RLR (RIG-1 and MDA5)-based activation transmits signals to produce type 1 IFNs (IFN-β) and interferon stimulated gene (ISG) called IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) that further enhances IFN gene program (IFN-β, IRF7, and OAS1 or 2′-5'oligadenylate synthase 1), which activates ISGs, immune homeostasis, and cell's internal antiviral immune response (**Figure 1**) [54–56].

The activation of MAVS involves the RIG-1 and MDA5 dimer formation [57]. Also, the IFIT1 generated exerts an anti-inflammatory action via suppressing TLR-dependent NF-κB-mediated pro-inflammatory cytokines (TNF-α, IL-1β) and chemokines (CCL3) through activating Sin3A-histone deacetylase 2 (HDAC2) transcriptional regulatory complex containing SAP25 that has an inhibitory action (**Figure 1**) [56]. Hence, these PRRs protect bats from developing severe viral infections through increased type 1 IFN production but low tissue damaging pro-inflammatory immune response. It should be interesting to observe that viruses harboring bats as their primary reservoirs may have evolved strategies to escape this innate immune mechanism to recognize cytosolic dsRNA viruses or bats have developed other mechanisms to escape from exaggerated pro-inflammatory innate immune response upon recognizing cytosolic dsRNA viruses. The MERS-CoV replicates efficiently in Jamaican fruit bats (*Artibeus jamaicensis*) without causing a productive infection with clinical signs of the disease [58]. The interferon regulatory factor (IRF3) transcription factor activation plays a crucial role in generating the potent antiviral immune response in the bat (*Eptesicus fuscus*) against MERS-CoV (**Figure 1**) [59]. In comparison to humans or other mammals, MERS-CoV fails to subvert the IRF3 activation and dependent type 1 IFN response generation in *E. fuscus*. The IRF3 in bats differs from humans due to the presence of serine185 (S185) that provides an enhanced antiviral protection (**Figure 1**) [60]. The S185 insertion in the human IRF3 increases its antiviral action. Hence, the positive selection of S185 in the bat IRF3 increases its antiviral action. Also, the bats persistently infected with MERS-CoV have increased type 1 IFN levels than non-infected ones and its disruption increases the virus replication [61].

The bat cells repeatedly select for the mutant MERS-CoV called delta open reading frame (ΔORF5) MERS-CoV and are resistant to superinfection by wild type (WT) MERS-CoV due to deficiency of MERS-CoV binding receptor dipeptidyl peptidase 4 (DPP4) and increased type 1 IFN levels [61]. Additionally, the Australian black flying foxes in response the cytosolic TLRs and RLRs recognizing viral PAMPs (dsRNA) also activate IRF7, which also induces type 1 IFNs mediated antiviral immune response (**Figure 1**) [62]. The deficiency or the defective activation of IRF7 in bats enhances viral replication and the development of the productive infection. Of note, virus (bat paramyxovirus, Tioman virus) infection to bats also induces protective type III IFN production that further provides protection from the development of productive infection (**Figure 1**) [63]. Egyptian rousette bats (*Rousettus aegyptiacus*) are the naturally harbor Marburg virus (MARV) and do not develop clinical symptoms of the disease as compared to humans due to generation of IFN-based immune response by DCs and suppressing pro-inflammatory immune response [64, 65]. This is because these bats secrete IFN-ω, which have antiviral action against RNA viruses (**Figure 1**). Also, the 13% of genes induced by IFN-ω in bats are not found in the interferome and other ISG databases, indicating their uniqueness to bats [64].

#### *Learning from Bats to Escape from Potent or Severe Viral Infections DOI: http://dx.doi.org/10.5772/intechopen.98916*

Bat immune cells exert protective type 1 (IFN-α, β, and ω) type II (IFN-γ) IFNs against Filoviruses (Marburg and Ebola viruses) but human immune cells fail to do so (**Figure 1**) [66]. Myxovirus resistance 1 (Mx1, a GTPase) is another antiviral protein induced in response to the IFNs is evolutionary conserved in vertebrates and can restrict a wide range of viruses in host cells (**Figure 1**) [67]. In bats these Mx1 proteins protect against Ebola and Influenza viruses through reducing the polymerase activity of these viruses along with other circulating viruses [68]. However, bat Mx1 does not inhibit Thogoto virus (enveloped negative sense ssRNA virus of *Orthomyxoviridae* family) as it does not infect them. On the other hand, mice Mx1 in hematopoietic cells inhibits Thogoto virus infection [67]. Hence, Mx1 is another IFN-induced antiviral protein in bats to protect against severe viral infections (**Figure 1**). Also, the production of type 1 IFN inhibits the NLRP1 and NLRP3 inflammasome-induced IL-1β and IL-18 production and induces IL-10 synthesis via STAT1 transcription factor (**Figure 1**) [69]. The IL-10 further activates STAT3 to reduce the IL-1β and IL-1α levels. IFNs also inhibit inflammasome-mediated Caspase 11 (CASP11) to inhibit the pro-inflammatory IL-1β and IL-18 release via activating immunity-related GTPases M clade 2 (Irgm2) and Gate16 (an ATG8 family member), which inhibit CASP11 maturation or activation [70]. Hence, IFN levels control exaggerated inflammation through different mechanisms.

The cGAS-STING signaling-mediated type 1 IFN production against DNA viruses is lost in bats due to the loss of serine AA at 358 (S358) position of the STING (**Figure 1**) [71, 72]. The S358 AA of the STING from other non-bat mammals is conserved and its phosphorylation is crucial for STING-dependent IRF3 activation and type 1 IFN release. For example, in human STING the S3666 and S358 phosphorylation is crucial for IRF3 binding and activation, but not for TBK1 [73]. Also, the TLR9-dependent cytosolic DNA recognition in bats is not as functional as in other mammals, including humans as result to adapt its high metabolic rate that increases the body temperature over 41°C during migratory flight that can induce DNA damage and its migration to the cytosol (**Figure 1**) [49]. Along with, defective cGAS-STING and TLR9 signaling for cytosolic DNA recognition, absent in melanoma 2 (AIM2) and gamma-interferon-inducible protein Ifi-16 (IFI16 or p204 in mouse) or interferon-inducible myeloid differentiation transcriptional activator are the PYRIN and HIN domain containing (PYHIN) proteins also recognizing cytosolic DNA are absent the genome of most bats, including *P. alecto* and *M. davidii* [74–76]. Both, AIM2 and IFI16 are involved in the cytosolic DNA recognition-induced inflammasome activation, and the maturation and release of pro-inflammatory cytokines (IL-1β and IL-18) (**Figure 1**) [75]. Only, a bat called *Pteronotus parnellii* has a truncated AIM2. Hence, the removal of cytosolic DNA sensors or PRRs adds to escape from the inflammatory immune response generated due to DNA damage observed high metabolic rate-induced rise in temperature during long migratory flights and helps in the coexistence of host and pathogens. Also, the killer immunoglobulin-like receptors (KIRs) encoded by genes in the leukocyte receptor complex (LRC), and killer cell lectin-like receptors (KLRs, also called Ly49 receptors), encoded within the natural killer gene complex (NKC) are required for potent antiviral function of NK cells. However, *P. alecto* lacks both KLRs and KIRs and *M. davidii* has only one Ly49 pseudogene [76].

The pteropodidae or cave nectar bat (*Eonycteris spelae*) monocytes, macrophages and granulocytes resemble human counterparts depending on the immune parameters that are divergent between mice and humans [77]. However, mast cells, eosinophils, basophils, platelets or thrombocytes have not been identified and characterized in different bat species [54]. Further studies are required in this direction. Also, the genome-wide comparison of immune-related genes have indicated their much closer phylogenetic relationship with humans than rodents. Also, bats express largest and most diverse array of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) genes (encode antiviral DNA cytosine deaminases), which are potent antiviral proteins and act as antiviral restriction factors for viruses, including hepadnaviruses (hepatitis DNA virus), and parvoviruses [78, 79]. The potent antiviral immune response of APOBEC3 involves its cytosine deaminase activity that deaminates cytosine residues in the nascent retroviral DNA to block retrovirus replication via hypermutation (**Figure 1**) [80]. This hyper-mutated retroviral DNA, then gets degraded or becomes non-functional [81]. In other mammals, including humans and laboratory mice the expression and action of APOBEC3 might threaten the integrity of the host genome triggering the incidence of cancer [82]. For example, a common APOBEC3 overexpression in humans is associated with the incidence of breast cancer in humans and the overexpression of APOBEC1 (A1) in mice is associated with hepatocellular carcinoma [83–85]. However, bats are more resistant to developing cancer despite expressing APOBEC3 as they express a higher quantity of ABC transporter called ABCB1 than humans and efficiently removes cytotoxic agents (doxorubicin) and damaged DNA [86]. Hence, in bats APOBEC exerts its only antiviral action and remains sans to increase susceptibility to cancer. However, further studies are warranted. Of note, even minor levels of IFNs are able to induce APOBEC3 family of proteins (A3A, A3G, and A3F) expression and their antiviral action [87].

Lower NLRP3 inflammasome activation in the cytosol prevents exaggerated inflammatory immune response in immune cells bats due to lower ROS production (crucial for NLRP3 activation) and apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and secretion of interleukin-1β (**Figure 1**) [88]. Also, bats produce less TNF-α due to the interaction of c-Rel (a member NF-κB family) with the promoter sequence of TNF-α [89]. The antiviral innate immune response in bat macrophages in response to the virus-derived PAMPs is also accompanied by sustained production of an increased amount of anti-inflammatory cytokine (IL-10) (**Figure 1**) [90]. These unique anti-inflammatory mechanisms in bats, including greater mouse-eared bats, *Myotis myotis* may have evolved due to their high metabolic rate (but produce low ROS that regulates NLRP3 inflammasome activation) and long distance flights [90]. For example, this bat species along with other long-distance traveling bats exhibit a delayed aging process as indicated by the absence of shortened telomerase and due to strategies to check induction of severe inflammation, but the induction of potent anti-inflammatory mechanisms [91, 92]. Also, the expression of high basal levels of heat shock proteins (HSP70 and HSP90) in bats protects them from increased metabolic stress that further contributes to their longevity and healthy aging [93]. Hence, these processes may contribute to longevity and healthy aging among bats.

Autophagy is an essential cellular process through which cells maintain homeostasis, including immune homeostasis [94–96]. Autophagy involves the breakdown of cellular components and the sequestration of the portion of cytoplasm into the double or multi-membraned vesicle called autophagosomes, which then fuse with cellular suicide or waste bags or lysosomes (contain hydrolases in their lumen and their membranes have permeases) to form autophagolysosomes or autolysosomes [96–98]. Autolysosomes are the junk crashers of the cell, in which luminal materials, including internal membrane, are degraded and exported out of the cell through permeases to recycle in the cytosol [96]. Hence, autophagy is the renewal process for cytosolic components through which cytoplasmic macromolecules mobilize to generate energy-rich compounds to meet cellular energy requirements during conditions with decreased internal and external energy resources. The impaired autophagy predisposes the host towards premature aging and inflammatory and degenerative diseases. Hence, autophagy helps the host to escape from

#### *Learning from Bats to Escape from Potent or Severe Viral Infections DOI: http://dx.doi.org/10.5772/intechopen.98916*

premature aging and different diseases (cancer, neurodegeneration, and other chronic inflammatory conditions) through cellular self-digestion [99].

Autophagy also plays a crucial role in immune response to infections and inflammation that works downstream to different PRRs (TLRs, NLRs, RLRs, and cGAS-STING signaling) discussed earlier (**Figure 1**) [100–102]. The increased autophagy in Australian black fly foxes also dampens the severity of the lyssavirus infection through suppressing the virus replication and increases the tolerance to the prolonged infection with lesser cell death than humans (**Figure 1**) [103]. Autophagy increases with the increases in the viral load in bats. The pharmacological activation of the autophagy decreases the virus replication that shows its antiviral action. Another virus called Nelson Bay Orthoreovirus (NBV that in humans causes severe respiratory tract infection) isolated from the Australian fruit bat increases autophagy in host cells depending on the viral replication without causing severe infection [104]. Hence, increased autophagy along with increasing longevity and suppressing aging mechanisms among bats also increases their antiviral immune response to protect them from severe productive infection.
