**3. Adaptive immune response in bats to make them resistant severe viral infections**

We do not have greater immunological data for adaptive immunity in bats as compared to humans due to lack of experimental reagents specific for bats and corresponding appropriate animal models. The genes [MHC-I and II, TCR (TCR-α and –β) and co-receptors, including CD3, CD4, CD8, and CD28 along with B cellspecific markers (CD22, CD19, CD20, CD27, and Igs)] involved in adaptive immunity in other species are conserved in bats [21–23]. The transcripts of both pro- and anti-inflammatory cytokines (IL-2, IL-4, IL-5, IL-6, IL-12a, IL-12b, IL-17a, IL-23, IL-10, TGF *β*, TNF, IFN *γ*, IL-1 *β*, CCL2, CCL5, and CXCL10) are also present [23]. The alpha1 (α1) domain of the H chain of MHC-I of *P. alecto* have three sequential AAs (Met, Asp, and Leu), which are absent in other mammals, including humans [105]. These 3 extra AAs in bat MHC-I help to form an extra salt-bridge chain between the H chain and the N-terminal of aspartic acid (Asp) of the antigenic peptide that promotes peptide presentation to the MHC I with high affinity during antigen presentation process. This study indicates the induction of stronger MHC-1-dependent T cells (CD8+ cytotoxic T cells) immune response against viruses that helps them to survive otherwise lethal viral infections as seen in other mammals.

*P. alecto* has a predominant population of CD8<sup>+</sup> T cells in their spleen and CD4+ T cells are predominantly present in blood, lymph nodes (LNs), and bone marrow [106]. Forty percent of these splenic T cells constitutively express IL-17, IL-22, and TGF-β mRNA, indicating the polarization of these T cells towards, Th17 and regulatory T cells (Tregs) [106]. Recent identification and development of batspecific cross-reactive Abs and establishment of captive experimental bat colonies have advanced the field. Immunoglobulins or Abs, including IgG, IgA, IgM, and IgE have been detected in bats (*P. alecto*) [107, 108]. However, IgA in secretion is lesser than expected but that is compensated by increased presence of IgG in the mucosal surfaces [108]. IgM is the second most abundant Ab in the serum after IgG in *P. alecto*. Of note, bats have a bigger repertoire of germline genes encoding Ig variable (V), diversity (D), and joining (J) segments than humans, indicating a provision of a larger number of antigen (Ag) specificities in their naïve B cell receptor (BCR) repertoire [54]. For example, little brown bats (*Myotis lucifugus*) rely more on the germline encoded repertoire to fight against infections than somatic hypermutation (SHM) [109]. On the other hand, SHM in humans increase the affinities

of Abs for diverse antigens [110]. Thus, human Ab response generates more diverse Abs in humans than bats.

The maternal Abs transferred to Egyptian Rousette bats against the Marburg virus last for their first five months after birth and Abs last for approximately 1 year in these bats infected naturally [111]. However, the reinfection of bats with the same virus induces anamnestic immune or Ab response within 5 days of the post viral infection clearing the virus systemically as well as from major organs (salivary glands, intestine, urinary bladder, and the reproductive tract). Hence, reinfection with the virus to bats in the natural environment is not sufficient to induce the productive infection. Another study indicates that the maternally-derived Abs (MDAs) in seasonally breeding bats (African fruit bats) do not last long for other viruses, including Lagos bat lyssavirus (LBV, a member of genus lyssavirus and gamily *rhadoviridae*) [112]. Also, the Abs developed in captive bats decay more slowly than these MDAs, indicating the fast decay of these MDAs. However, Abs produced in captive bats decay faster than seasonally breeding bats living in their natural environment, indicating the Ab may persist for life in natural environment harboring bats.

The Abs-mediated virus neutralization is not a universal mechanism for protection against Ebola, Marburg, and Sosuga (a recently discovered pathogenic Paramyxovirus in Uganda) viruses in the Egyptian Rousette bats [113, 114]. Similarly, maternal Abs to the Henipavirus become undetectable between 4 and 12 months after birth [115]. The seasonal horizontal transmission of the virus makes seronegative bats seropositive for Abs and seasons of late pregnancy/lactation in bats may increase the risk of zoonotic diseases. Further studies have shown that in the straw colored fruit bats (*Eidolon helvum*) fruit bats maternal Abs provide protection against Lagos bat lyssavirus and African Henipavirus for 6 months and acquired immunity in developed adult bats against them lasts for 12 years (Lagos bat virus) and 4 years (Henipavirus) [116]. However, the disturbed pregnancy and lactation (seasonal birth pulse) impacts the maternal Ab-based immunity on persisting virus that depends on the transmission characteristics (prolonged infection period or within host latency). It is interesting to note that despite the diminished Abs level the Egyptian Rousette bats exert a protective immune response against severe Marburg infection that may be due to the anamnestic response generating Abs and type 1 IFNs [117].

Abs specific to the glycoprotein GP2 to another *Filoviridae* family member called Lloviu virus (LLOV) have been detected in insectivorous Schreiber's Bent-winged bats in the caves of Northern Spain [118]. A study has shown that the reinfection with the particular virus is essential to explain the shortness (hours to days) of acute infections and development of immunity lasting for another 1–2 years [119]. Hence, recurring latent infections are warranted for immunoprotection in bats to severe viral infections. The migrating status of the bats or other migratory animals//birds also determine the reactivation or suppression of the latent infection depending on the immune status [120, 121]. For example, the relapse at either the start or end of migration may increase the prevalence across the year and may maintain pathogens with low transmissibility and short infectious periods in the migratory population [120]. For example, relapse at the beginning of the migration may reduce the prevalence of highly virulent or infectious viruses by amplifying death of infected hosts during migration, especially for highly transmissible viruses and those transmitted during migration or breeding season. The long-distance migratory Nathusius' pipistrelles (*Pipistrellus nathusii*) show difference in the immune status, for example, during migration they have increased number of lymphocytes with decreased neutrophils as compared to the non or pre-migratory period [38]. The oxidative stress is higher during migration period without any association between blood oxidative status and immunological impact. Of note, the

immune challenge does not induce any changes in the oxidative stress irrespective of the migratory or pre-migratory season.
