Revising the paradigm: Are bats really pathogen reservoirs or do they possess an efficient immune system?

Summary While bats are often referred to as reservoirs of viral pathogens, a meta-analysis of the literature reveals many cases in which there is not enough evidence to claim so. In many cases, bats are able to confront viruses, recover, and remain immune by developing a potent titer of antibodies, often without becoming a reservoir. In other cases, bats might have carried an ancestral virus that at some time point might have mutated into a human pathogen. Moreover, bats exhibit a balanced immune response against viruses that have evolved over millions of years. Using genomic tools, it is now possible to obtain a deeper understanding of that unique immune system and its variability across the order Chiroptera. We conclude, that with the exception of a few viruses, bats pose little zoonotic danger to humans and that they operate a highly efficient anti-inflammatory response that we should strive to understand.


INTRODUCTION
Bats (Chiroptera) comprise the only order of mammals with the ability for powered flight, and with nearly 60 million years of physiological adaptions for this ability (Lei and Dong, 2016). With over 1,400 species, bats account for more than 20% of all mammalian species, second only to rodents, and can be found everywhere on earth except the poles (Calisher et al., 2006). Bats play an important role in insect control, reseeding deforested areas, and pollinating a variety of plants (Boyles et al., 2011;Zaho, 2020). Despite these useful roles, bats are mostly perceived as posing a threat to public health, as major transmitters of pathogenic and potentially zoonotic viruses (Dobson, 2005;Leroy et al., 2005;Li et al., 2005;Calisher et al., 2006). COVID-19 is only one recent example of media reports (Zhou et al., 2020) connecting bats to human disease and targeting them as reservoir animals, despite a lack of evidence (Andersen et al., 2020). Although the coronavirus isolated from bats in Wuhan (China) was found to be 96% genetically identical to the beta coronavirus that started the current pandemic, this degree of similarity accounts for a temporal distance of several to many years between the two, when taking the mutation rate of the virus into account (Boni et al., 2020;Ruiz-Aravena et al., 2022). Notably, the receptor-binding domain (RBD) of the bat virus cannot bind to human cells, indicating that it is not the direct source of the pandemic (Andersen et al., 2020;Chan et al., 2020;Ruiz-Aravena et al., 2022). Although there is some evidence that the potential ancestral COVID-19 virus had originated in bats (Shereen et al., 2020), to date, two years after the pandemic first struck, we still do not know the direct source of the human pathogenic COVID-19 variant (Ruiz-Aravena et al., 2022;Frutos et al., 2022). The bats' widespread image as a danger to public health will, however, be difficult to rehabilitate (Zaho, 2020;MacFarlane and Rocha, 2020). In this review, we scrutinized the literature in order to assess the evidence and determine whether bats are or are not reservoir animals for more than a hundred pathogenic viruses, as is often claimed (Calisher et al., 2006;Epstein and Newman, 2011;Hayman, 2016;Wang and Anderson, 2019). Our findings suggest that in many cases the confidence regarding the bats' role as reservoir animals is not sufficiently supported. Although we do not claim that bats are never the origin of human pathogens, we suggest that their role has been consistently exaggerated and often without the necessary scientific basis.

ARE BATS VIRAL RESERVOIR ANIMALS?
A reservoir animal is defined as an epidemiologically connected population in which the pathogen can be permanently maintained and from which infection is transmitted to the target population (Haydon et al., 2002). A slightly broader interpretation of this term is discussed by Ashford (Ashford, 2003).  (Tandler, 1996) 53 Nipah virus Orthoparamyxovirinae, Henipavirus yes no yes (Lvov et al., 1979;Kelkar et al., 1981)  iScience Review Moreover, many of the reported isolations are unconvincing: (1) Several viruses were only isolated from a single individual bat (Charlier et al., 2002); (2) In some cases isolation was performed from a homogenate of internal tissues from which transmission is unlikely (e.g., the liver and spleen) and not from oral swabs or saliva glands, urine, feces, or even blood or sera. (Mortlock et al., 2015;Hayman, 2016); (3) Several of the local viruses were also isolated from other animals in the region, including non-bat-specific ectoparasites (Ramírez-Martínez et al., 2021); and (4) Some isolations were taken from sick or dead individuals (Osborne et al., 2003;Kuzmin et al., 2010), which would probably not have transmitted the disease-sick bats have been shown to remain in the roost and refrain from social interactions (Moreno et al., 2021). Seroprevalence by itself doesn't reflect the ability or even the potential for being a reservoir or creating spillover events (Barrantes Murillo et al., 2022).
Another common type of research attempts to intentionally infect bats with human pathogens, such as Nipah virus (Middleton et al., 2007), various Coronaviruses Munster et al., 2016), and Ebola (Zaire) virus (Swanepoel et al., 1996), among many others. Such research has revealed that the viruses can replicate and circulate in bats until they eventually die out. These experiments are insufficient to consider bats as reservoirs. If anything, they indicate that humans are reservoirs of potential bat-pathogens. Moreover, in some cases, such as Ebola, infection experiments have demonstrated that the virus can also infect other animals (e.g., mice) (Swanepoel et al., 1996). Intentional infection of the grey-headed fruit bat (Pteropus poliocephalus) with Nipah virus (Middleton et al., 2007) nicely demonstrated how bats contend with the virus up to full recovery (zero viruses isolated 21 days post-infection from urine or any other bat tissue), resulting in immunity (virus-neutralizing antibodies detected 15 days in all the tested bats). This response is probably owing to the bat's unique immune system (discussed below).
To date, the evidence regarding the isolation of actual harmful pathogen viruses in bats is limited, with only a few well-known cases, including the Marburg virus that was isolated from Rousettus aegyptiacus fruit bats in Uganda (Towner et al., 2009), and Hendra virus (HeV) that was isolated from Australian fruit bats. In the case of the Marburg virus, some knowledge gaps regarding the full host range and circulation remain (Leendertz et al., 2016). In the case of Hendra, humans are infected by horses, which are supposedly infected by bats. Direct infection from bats is rare at most, as indicated by a serological survey of 128 people with prolonged and close contact with Pteropid bats, and in whom no evidence of infection with HeV (Selvey et al., 1996) was detected. This is important when considering the general public's fear of bats (Ló pez- Baucells et al., 2018;MacFarlane and Rocha, 2020;Lu et al., 2021). Moreover, as pointed out by (Scott, 2001) virus isolation alone is not sufficient for considering an animal a reservoir, as evidence of transmission is also required. The mere detection of a virus in bats does not imply that spillover will occur, and many additional biological, ecological, and anthropogenic conditions must be in place for such an event to occur . Some human pathogenic viruses are also known to infect and affect bats, including most lyssavirus species (Banyard et al., 2011), Tacaribe arenavirus (Cogswell-Hawkinson et al., 2012), and the Zwiesel bat banyangvirus (Kohl et al., 2020), among others that are known to harm bats.
As these examples show, bats are often perceived as reservoirs of viral diseases solely owing to being serologically positive, which merely means that the bats have survived the disease and developed an immune response to it Li et al., 2005;Swanepoel et al., 2007). In other cases, a virus closely related to the human pathogen but not pathogenic to humans may be present in bats, which is not sufficient to make bats its reservoir. Whereas bats might have been the ancestral origin of such a human virus, an intermediate host in which the viral mutations occurred, and where the virus reached significant prevalence, is probably needed for zoonotic spillover of the virus to humans to occur. According to (Olival et al., 2017) not only bats but also primates and rodents have a higher proportion of observed zoonotic viruses compared to other groups of mammals. Species in other orders (e.g. Cingulata, Pilosa, Didelphimorphia, Eulipotyphla) also share a majority of their observed viruses with humans, but the data is limited in these less diverse and poorly studied orders.
Unraveling the unique bat immune system Interestingly, it seems that bats can contend with deadly viruses better than humans and most other mammals can. After over a century of focusing on the viruses that bats carry, there is increasing interest in understanding the uniquely potent bat immune system. Here, we summarize the findings to date, focusing on the ability of the bat immune system to fend off viral diseases.
Most early research focused on isolating the basic immune components of the innate and acquired bat immune system and comparing them with what was already known in mice and humans. Some of the main findings are as follows: Cells resembling follicular dendritic cells (FDCs) were described in Pteropus giganteus (Sarkar and Chakravarty, 1991) and macrophages, B cells, and T cells were identified in the spleen and lymph nodes of the Indian fruit bat. The complement cascade was found in Tadarida brasiliensis bats (Allen et al., 2009). A variety of immune cells, including lymphocytes, neutrophils, eosinophils, basophils, and macrophages, was also identified by morphological means in histological sections from the Brazilian free-tailed bat (T. brasiliensis) (Turmelle et al., 2010). The pattern recognition factor of toll-like receptors (TLR) was described in two species of fruit bat, Pteropus alecto and R. leschenaultia Cowled et al., 2011), and found to be highly conserved between bats and other mammals. Several bat cytokine genes have now been characterized, including cDNAs corresponding to interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12p40, and tumor necrosis factor (TNF) from R. leschenaultii (Iha et al., 2009), which both play an important role in the antiviral immune state. Bats have demonstrated a highly diverse antibody repertoire, exceeding that of most species and on a par only with humans and mice (Baker et al., 2010;Bratsch et al., 2011). Another study examined the interferon (IFN) signaling pathway following IFN production, to determine the importance of IFNs in inducing an ''antiviral state'' in bat cells through the simultaneous suppression of type I IFN and induction of type III IFN post virus infection (Virtue et al., 2011). The IFN systems in bats were later found to be highly diverse and much more complex than expected. A thorough review summarizing these innate and acquired immunological findings was published by (Baker et al., 2013b), showing that although bats appear to share most features of their immune system with other mammals, there are qualitative and quantitative differences in their immune responses.
Several of the early publications already provided initial evidence of one of the main characteristics of the bat immune system-a delayed immune response, on which we will elaborate later in discussion. McMurray and Thomas (McMurray and Thomas, 1979) and Paul (Paul and Chakravarty, 1986) found that T-cell proliferation as part of the immune response peaked at 120 h post-infection in comparison to 48 h in mice. Moreover, Chakraborty (Chakraborty, 1983) found that cell-mediated immunity in bats is slower than in other mammals. Prolonging the immune response was later found to be a beneficial antiviral strategy in bats (Hayman, 2019).

Resistance versus tolerance in the bat immune response
A deeper understanding of the bat immune system was obtained using comparative genomics. Zhang et al.  sequenced the genomes of two distantly related bat species (P. alecto and Myotis davidii) and revealed genetic evidence of a uniquely evolved immune system. Although some immune genes have been lost, others seemed to be under strong positive selection. Specifically, genes responsible for DNA damage checkpoints and repair pathways seemed to be undergoing accelerated positive selection. These authors hypothesized that flight-induced adaptations had inadvertently also affected the bat immune system. The strenuous and prolonged physiological efforts exerted during flight impose oxidative stress, resulting in severe DNA damage and the release of self-DNA fragments into the cytoplasm (Barzilai et al., 2002), somewhat similar to the DNA damage caused by a viral infection. Consequently, evolving an efficient DNA repair mechanism aimed at dealing with flight-induced cellular damage might have also enabled bats to fight off viral infections. Zhang et al. further hypothesized that these mechanisms may also be involved in the unique longevity of bats.
Additional bat genomes have as been studied, revealing new insights into the bat immune system Seim et al., 2013). Interestingly, one of the most important viral defense lines, namely the interferon (IFN) system, has been shown to vary greatly among bat species (Clayton and Munir, 2020). Interferons (IFNs) are secreted cytokines that induce an antiviral response by the host and are primarily responsible for inhibiting viral replication. Signaling pathways of IFN were already found in bats in 1969 (Stewart et al., 1969). New research has revealed a species-specific gene length size in bats, with much variability in functional responses, including permanent vs. stimulation-dependent secretion of IFNs, with different effects on the immune response: 1. Type I IFN locus has shortened in Pteropus Alecto (Zhou et al., 2016), but expanded in Pteropus vampyrus and Myotis lucifugus (Pavlovich et al., 2018); 2. Zhou et al., 2016) found a contraction of the type I IFN locus in the Australian black flying fox (P. alecto) and an unusual constitutive expression of IFN-a in these bats. Moreover, IFN type 3 in the same bat was induced in response to a viral infection; 3. Pavlovich et al., 2018)  iScience Review that while poly I:C treatment (imitating dsRNA stimulus which is usually associated with viral infection) induces the secretion of type I IFNs in both human and Eptesicus fuscus bat cells, the bat cells express much lower levels of these inflammatory mediators; and 5. Sarkis (Sarkis et al., 2018) found the induction of selective IFN stimulated genes in the common vampire bat (Desmodus rotundus). Some of these versatile responses led to the realization that the antiviral state achieved by a variety of IFN phenotypes in bats is also related to an anti-inflammatory response (see more in this recent review (Clayton and Munir, 2020).
The IFN system has also been shown to vary at the genetic regulation level. Xie and Li (Xie and Yang Li, 2020) demonstrated that a variety of bat species have a dampened interferon response owing to the replacement of the highly conserved serine residue in STING (stimulator of interferon genes), an essential adaptor protein in multiple DNA sensing pathways. This means that, in these species, the IFN response has substantially diminished, resulting in a reduced inflammatory response. Via the IFN antiviral cascade, the balanced reduction of inflammasome has started to be discovered.

A restrained immune response serves better in contending with viruses
Recent findings suggest that a novel ''trick'' of the bat immune system might be that of the reduced inflammatory response that accompanies the antiviral response of the system. In recent years, evidence is accumulating that in addition to its antiviral abilities, the bat immune system is characterized by a general restrained response during inflammatory processes. One mechanism responsible for reducing the immune response is that of the complete and unique loss of the PYHIN gene that was found in P. alecto and M. davidii bats (Ahn et al., 2016). This family of proteins serves as important immune sensors of intracellular self and foreign DNA and as activators of the inflammasome and/or interferon pathways. This reduction aids in achieving a milder inflammatory response. Another example of a dampened pathway is related to the important inflammasome sensor NLR family pyrin domain-containing 3(NLRP3), which has been linked to both viral-induced and age-related inflammation. Ahn et al., 2019 found a dampened NLRP3mediated inflammation in P. alecto,with implications for longevity and unique viral reservoir status.
Recently, a diminished inflammatory signaling pathway was found in P. alecto and M. davidii bats (Goh et al., 2020).
As nicely summarized by Schneider et al. (Schneider and Ayres, 2008) there are two ways to survive infection: resistance and\or tolerance. It seems that bats have developed an excellent balance between the two: an enhanced host defense response, and immune tolerance through several different mechanisms (see (Irving et al., 2021) for a detailed review article). Suppressed inflammasome pathways-as noted abovecontribute to immune tolerance in bats and a well-balanced reaction. In humans, the dysregulation of the immune system seems to be responsible for increasing the severity of illness in the acute phase of viral disease (Hope and Bradley, 2021). Bats, in contrast, contend better with deadly viruses and, despite a longer or slower time of reaction, they eventually overcome these viruses to reach full recovery and elimination of the pathogen. Recent studies have focused on bats' ability to contend with some of the most notorious viruses, including Marburg virus (Guito et al., 2021), COVID-19 (Ruiz-Aravena et al., 2022), and others (Mandl et al., 2018). A restrained immune response has also been shown to be valuable regarding longevity (Kacprzyk et al., 2017;Gorbunova et al., 2020).

Conclusions
When considering the interaction of bats with viruses, the time seems right for a paradigm shift. Many bats contend with a variety of deadly viruses better than other mammals. This ability has evolved over nearly 60 million years of adaptation to powered flight. Bats balance their immune response in such a way that it is slow but highly efficient, making them seropositive and immune to viruses. Following immunity, their chance of relapse, to the point of becoming contagious, is low. This is evident from the numerous studies cited above, which have not managed to isolate a viable virus from antibody-seropositive bat individuals; and it is also evident from intentional bat infections in which the virus was shown to disappear after up to one month. In most cases, bats thus carry and spread infectious agents during the limited time frame of their sickness before they overcome it. A spillover of viral pathogens can only occur when bats harbor the identical human pathogenic virus. However, many viruses carried by bats cannot infect humans without first undergoing a natural process of evolution, meaning that bats carry the ancestral viruses and not the human pathogen (Forni et al., 2017;Clayton and Munir, 2020;Latinne et al., 2020). This is also what is known so far for COVID-19 (Poon et al., 2004;Boni et al., 2020;Ruiz-Aravena et al., 2022;Frutos et al., 2022). We should seek to avoid the disruption of their natural habitats that are resulting from rapid urbanization, iScience Review wildlife trade, and deforestation (Greger, 2007). This was neatly stated by Markotter et al. (2020), who wrote: ''It is important to recognize the role of bats in zoonotic disease outbreaks and implement mitigation strategies to prevent exposure to infectious agents including working safely with bats. Equally important is the crucial role of bats in various ecosystem services. This necessitates a multidisciplinary One Health approach to close knowledge gaps and ensure the development of responsible mitigation strategies to not only minimize the risk of infection but also ensure the conservation of the species'' .
Bats' antiviral immunological abilities should be studied in greater depth, so that we, humans, may learn more about efficiently combating viral disease, aging, and cancer. The immense diversity of species in the Chiroptera makes the information gathered highly species-specific and therefore quite complex. Differences in the biology, ecology, and physiology of the different species constitute important factors that must be considered. Fortunately, such understanding is now growing in the scientific community (Foley et al., 2018;Mollentze and Streicker, 2020;Cockrell and An, 2021;Irving et al., 2021). Despite all of the above, bats are nonetheless frequently blamed for being virus reservoirs with the scientific literature driving the popular opinion. In light of the complex immunological and ecological phenomena that we have highlighted in this review, scientists should refrain from using generalizations such as: ''. many of these terrible diseases are caused by viruses originated from bats'' (Han et al., 2015); or headlines such as ''Bats as reservoirs of severe emerging infectious diseases'' or ''Bats as vectors of diseases and parasites'' (Klimpel and Mehlhorn, 2014). Like all animals, bats deserve a more accurate and scientific approach to the terminology applied to them (Puechmaille et al., 2021;Shapiro et al., 2021).