INSTITUTE OF PARASITOLOGY | Turning on the Bat Signal

The source of the article: www.the-scientist.com

Hannah Thomasy, PhD

Mar 15, 202

Bats are a deeply underappreciated order of mammals. They snap up enormous numbers of mosquitoes and crop pests, preventing more than a billion dollars in crop damage per year, according to estimates.1,2 They are important pollinators for hundreds of plant species, including Agave tequilana, which supports the multibillion dollar tequila industry.3

Bats are also infamous as carriers of deadly pathogens, a reputation which even bat enthusiasts admit is not entirely undeserved.

“Bats are reservoirs for viruses that cause the highest case fatality rates in human hosts—some of the most virulent zoonotic viruses that have been described,” said Cara Brook, a disease ecologist at the University of Chicago. “This includes viruses in the paramyxovirus family, such as Hendra and Nipah virus, lyssaviruses like rabies, filoviruses, which includes Ebola and Marburg, and then of course, coronaviruses.”

However, as Brook points out, eradicating bats won’t prevent zoonotic diseases. Devastating zoonotic pathogens are also spread by rodents, monkeys, dogs, cats, livestock, and more.4 Furthermore, even large-scale bat culls don’t reliably reduce viral spillover to other species.5

Instead, she said, “There is an immense opportunity to learn from bats. They’re extremely successful reservoirs of viral infection.” Studying the molecular mechanisms by which bats regulate antiviral and anti-inflammatory pathways may give scientists a preliminary blueprint to start building therapeutics for infectious and possibly even inflammatory diseases.

A brave new world

Cara Brook smiles while wearing a headlight and blue gloves to hold a small brown bat. Researchers set up a large net over the mouth of a cave.

TOP: Cara Brook holds a Hipposideros commersoni bat in Ankarana National Park in Madagascar. BOTTOM: Brook’s research team, called Ekipa Fanihy (Malagasy for “team fruit bat”) nets Eidolon dupreanum fruit bats on the west side of Ankarana National Park.

HAFALIANA CHRISTIAN RANAIVOSON; CARA BROOK

For decades, scientists have been fascinated by the unusual relationship between bats and viruses. As early as the 1930s, researchers remarked on bats’ ability to spread rabies while appearing healthy themselves.6 Scientists finally identified bats as reservoir species for Hendra, Nipah, SARS-like viruses, and potentially Ebola in the 2000s. 7–10 After that, the field of bat immunology really began to take flight.

Whether bats are truly natural reservoirs for Ebola is still a hot zone of debate. Multiple studies have found Ebola-specific antibodies in bats, but none found the actual virus.10 A widely publicized report of Zaire ebolavirus genetic material in a West African bat in 2019 hasn’t been published in a peer-reviewed journal.11 Many bat biologists still maintain that there is insufficient evidence to support bats as reservoirs for Ebola.12

This circumstantial link between bats and Ebola first drew Arinjay Banerjee, now a comparative immunologist at the University of Saskatchewan, to study these unusual mammals. In 2013, at the beginning of a major Ebola virus outbreak in West Africa, Banerjee watched a talk by University of Saskatchewan veterinary microbiologist Vikram Misra on bats as potential Ebola virus hosts. Banerjee was immediately intrigued; he walked up to Misra after the talk and explained that he wanted to study how bats coexisted with viruses.

When Misra asked him what he would need for such a project, Banerjee responded that they would need bat cell lines. There was just one problem: For the vast majority of bat species and tissue types, these cell lines did not exist.

Undaunted, Banerjee joined Misra’s research group at the University of Saskatchewan, where he devoted his master’s degree research to creating immortalized big brown bat cells, the first cell line of a bat species found in Canada.13

For bat researchers, said Banerjee, “It’s not as easy as just testing a hypothesis. We have to start by making the tools we need to test the hypothesis.”

In the last few years, however, the pace of research has accelerated. “It’s been very cool to see how this field has grown,” he said. “Especially after COVID-19, we’ve gotten so many people that are interested in doing this research. It’s a very rapidly growing field; I compare it to what CRISPR-Cas was ten years ago.”

Taking flight

Like many researchers in the field, Emma Teeling did not begin her scientific career intending to study bats. Teeling, now a geneticist at University College Dublin and one of the most recognizable faces in bat research, initially hoped to study the evolution of mate choice behavior in cats. When that didn’t work out, she applied for a position studying bat evolution. Teeling has never looked back.

“Within a month of deeply studying the biology of bats, I was completely hooked,” she recalled. “They’re the most amazing species.”

In Teeling’s early work, she played a key role in constructing the evolutionary history of bats, including how echolocation arose.14 But she soon realized that echolocation wasn’t the only unusual ability that bats possessed. “As you work with bats more and more, you realize how many cool adaptations they have,” she said. “I wanted to figure out how they are able to live as long as they do, and how they are able to tolerate all of these different viruses without getting sick. And I thought, ‘God, the secret’s probably in the bat genome.’”

To this end, Teeling and a colony of other bat biologists started the Bat1k project in 2016, with the ultimate goal of fully sequencing the genomes of all extant bat species.15 Although the project is far from complete, it is already yielding some interesting insights into bat immunity.

In a recent Bat1k project preprint that involved Teeling, Banerjee, and dozens of others, scientists examined signatures of selection in several bat genomes compared to nearly a hundred other mammals in ten different orders, from primates to rodents to carnivores.16 Signatures of selection can be detected by studying how organisms accumulate synonymous and nonsynonymous mutations, or changes in the DNA that don’t alter amino acids and those that do alter them, respectively.

“We expect both synonymous and nonsynonymous changes to occur,” said Michael Hiller, an evolutionary genomicist at the LOEWE Centre for Translational Biodiversity Genomics and an author of the preprint. However, if nonsynonymous mutations occur at a faster rate, this means that changes in the protein sequence were favored over the course of evolution. Often, he said, “this highlights genes that have functional differences.”

“The immune system generally evolves very fast, and in many mammalian orders, we see that immune system genes are under positive selection.” In this study, he said, “What was striking is that we saw that immune gene selection is most prevalent in bats.”

A black-gloved hand holds a fuzzy brown fruit bat.

Cara Brook studies viral transmission in fruit bats like this baby Pteropus rufus.

CARA BROOK

Using this genetic information, researchers also explored where in the tree of life this accelerated immune evolution took place. “A strong part of that signal came from the ancestral bat lineage,” he said. “This indicates that those immune gene changes started to happen around the time that flight also evolved in bats.”

This squares with the standing hypothesis that bats’ unusual tolerance of viruses is due, at least in part, to the evolution of flight. “Bats are the only mammals that have achieved true, self-powered flight,” said Teeling. “Flight is extremely metabolically costly. So, bats produce so much of these damaging free radicals, which break up DNA. [In other mammals], the immune system recognizes this as a pathogen and gets highly inflamed. So, the argument is that bats have had to evolve a mechanism to dampen the constant sterile inflammation they must experience from flight.”

For many viral infections, including cases of influenza and COVID-19, scientists think that excessive inflammation plays a major role in tissue damage and mortality of the host.17,18 Therefore, mechanisms that evolved to reduce flight-induced inflammation may have had an added benefit of increasing bats’ ability to tolerate viruses.

Bat Immune Systems: The Original Antivirus Programs

Bats have many adaptations, some of which are consistent across different species, that possibly contribute to their unusual resiliency to viral infections. However, bats are a heterogeneous group, with major lineages diverging more than 50 million years ago, so different evolutionary pressures may have given rise to important species-specific adaptations.

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A) DNA Sensors

Several species of bats have lost the entire PYHIN gene family. Pyrin and hematopoietic interferon-inducible nuclear domain (PYHIN) proteins sense DNA, either from pathogens or the host’s own damaged DNA, and trigger an inflammatory response. Loss of this gene family may explain bats’ ability to limit harmful virus-induced inflammation.

B) Escape From Viral Antagonism

While some pathogen sensors have been lost, others have diversified. Protein kinase R (PKR) proteins are activated by double-stranded RNA, which signals the presence of a viral infection, and can then initiate an antiviral response. Many viruses, including some poxviruses, herpesvirus, and influenza viruses, have evolved ways to interfere with PKR function. Researchers think that PKR gene diversification may help bats escape this viral interference.

C) Interferons

Interferons are key players in the antiviral response. Different bat species display differences in interferon genes and regulation of interferon expression. Some species show constitutive expression of interferon-α, while others have a notable expansion of the less studied interferon-ω genes.

D) Inflammasomes

Bat ASC2 (apoptosis-associated speck-like protein containing a CARD 2) inhibits activation of the inflammasome. While inflammasome signaling is important for the antiviral response, overactivation has also been implicated in mortality for certain viruses, including influenza. Indeed, researchers showed that bat ASC2 increased the survival of influenza-infected mice.

E) Possible Self-vaccination

A recent study on bat induced pluripotent stem cells revealed an unusually large number of endogenous viral sequences. While still unsubstantiated, the study’s authors suggested that future research should test whether these endogenous viral sequences serve to “defend against viruses and microbes and encode viral proteins in a self-vaccination scheme.”

F) Undiscovered Mechanisms

Bats are a diverse group with more than 1,400 species. According to approximate counts from the Bat1K gene sequencing initiative, very few species have fully sequenced genomes. Thus, many potential antiviral or anti-inflammatory mechanisms remain to be discovered.

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Turning down the volume of inflammation

Several genetic studies have suggested various mechanisms that might reduce inflammation in bats. For example, several species have completely lost a family of genes that triggers inflammation in response to cytosolic DNA, including viral DNA and self-DNA (potentially freed into the cytoplasm by flight stress).19 At least 30 species have a mutation in the gene coding for STING, an important protein in several DNA-sensing pathways.20

Translating these genetic findings into new therapies is the next hurdle. According to Banerjee, “We’re not making transgenic humans anytime soon.” However, he said that by understanding the molecular changes that drive specific immunological characteristics in bats, scientists could design small molecules or synthetic proteins to mimic those effects in humans.

A few years ago, this idea may have seemed far fetched, but a research team led by zoonotic disease researcher Linfa Wang at the Duke-National University of Singapore (NUS) Medical School is already taking steps to make this a reality. Wang, affectionately referred to as “The Batman,” has been studying bats and their viruses for more than 20 years.

Matae Ahn was a medical student at Duke-NUS when he first met Wang in 2013. Although Ahn originally intended to pursue a straightforward medical degree, he soon became interested in research as well, as he was drawn to the diseases and processes that doctors did not yet fully understand.

For a while, he wasn’t sure which area of research to pursue. But one day when Wang gave a talk for the medical students, everything fell into place. “I instantly knew it was something that I had long been waiting for,” said Ahn. “Almost from that moment on, I decided to really dive into bat research.”

Ahn joined the research team and prepared to begin exploring the bat immune system. But where exactly to begin was an important question. The immune system is one of the most complex systems in the body, with practically innumerable interlocking parts. “There’s millions of potential pathways and targets that we could study in bats,” said Ahn.

A researcher wears gloves and pipettes liquid in a fume hood.

A scientist on Arinjay Banerjee’s research team works with zoonotic viruses in the high containment facility.

VACCINE AND INFECTIOUS DISEASE ORGANIZATION

They began by looking at the big picture: What observable traits made bats unique? Bats can deal with the stress of flight, have high viral tolerance, and possess remarkable longevity. (The Brandt’s bat can live up to 41 years, about ten times as long as expected for a mammal of its size.)21

The researchers hypothesized that these traits were linked, so they began to look for molecular mechanisms that might underlie all of these abilities. They decided that the inflammasome, which is a multiprotein complex that plays an important role in response to metabolic stress, infection, and aging, was a good place to start their investigations.

Early observations showed that normally-potent inflammasome stimulators didn’t strongly activate bat inflammasomes. “For my entire PhD and research fellowship, I have been trying to figure out why,” said Ahn.

Wang, Ahn, and Aaron Irving, another immunologist on the research team, went on to discover several molecular tweaks in bats’ inflammasome signaling pathways, including changes in inflammasome activators, components of the inflammasome itself, and in downstream signaling molecules.19,22,23

In probing the dynamics of this pathway, they analyzed the transcriptomes of bat immune cells and found something surprising: high expression of ASC2, a protein previously thought to only exist in primates.24 It wasn’t present in just one species either; all thirteen bat genomes that the researchers examined contained the ASC2 gene. Because of ASC2’s high expression and ubiquity across bat species, said Ahn, “We believed that there might be something special about it.”

ASC2 was also interesting, said Irving, who is now at Zhejiang University, “because it’s a very small protein, so it’s easy to manipulate, and it’s possible to even make it into a drug.”

Even in humans, however, the role of ASC2 in immunity and inflammation is not well understood. To investigate the function of bat and human ASC2, researchers engineered different populations of human cells to stably express either bat ASC2 or human ASC2. They found that bat ASC2 was more effective at reducing the cells’ responses to inflammatory stimuli, inhibiting inflammasome activation, preventing cell death, and reducing the release of the inflammatory cytokine interleukin-1β.

Arinjay Banerjee and a fellow researcher wear white suits with clear face plates and hold test tubes.

Arinjay Banerjee and his research team carry out research on bat-virus relationships inside the high containment facility at the Vaccine and Infectious Disease Organization.

VACCINE AND INFECTIOUS DISEASE ORGANIZATION

To test the effects of this protein in vivo, researchers created the first mouse edited to harbor a bat gene. Researchers then infected the bat-mice with influenza A to determine how bat ASC2 affected the response to viral infection in a different species.

“What’s really striking is that this mouse line can now survive influenza infection,” said Hiller, who was not involved in the ASC2 research, but has collaborated with Wang on other projects. “Typically, influenza kills essentially all mice after a few days, but in this mouse line, about 50 percent of the individuals survived. And there’s good evidence that this is because excessive inflammation is being reduced.” Hiller said that this is one of the first promising targets for drug development to come out of the bat immunology field.

Researchers determined that just four bat ASC2 residues were responsible for its enhanced inflammasome inhibition. They then used site-directed mutagenesis to target these four regions in human ASC2, creating a bat-ified version that they called Hupa4 ASC2. In human cells, Hupa4 appeared to be about as effective as bat ASC2 at reducing the inflammatory response, suggesting the possibility of developing Hupa4 as a human therapy.

Because this peptide is not highly bioavailable, researchers are currently exploring how it could be applied to surface tissues like the skin and eyes or the lungs, which can be accessed via inhalation. For other tissues, they are studying how the same molecular effects could be achieved with small molecules or nucleic acids.

An evolutionary arms race

While limiting excessive inflammation can be key to survival, too little inflammation can also be dangerous. “You have to have some level of inflammation because otherwise you’re a sitting duck,” said Teeling.

“The inflammatory response is very important for a proper antiviral response,” agreed Hiller. “Inflammation tells cells and tissues that something is not normal, and it recruits additional factors and triggers repair mechanisms to help you get back to a normal state.”

“What I think is going on—and we still need to conclusively prove it—I think they’ve evolved a perfect balance,” said Teeling.

Adaptations related to interferons (IFN), a diverse group of proteins that are crucial components of a cell’s antiviral response, are thought to be part of bats’ defensive arsenals, although the particular adaptations may differ between different species.

An Australian bat called the black flying fox, for example, has an unusually small number of IFN-α genes for a mammal, but its cells show high IFN-α expression, even in the absence of viral infection.25 While this kind of sustained IFN upregulation is related to autoimmune disease in humans, researchers suggested that in the flying foxes, it might serve as an always-on antiviral defence system.26

In contrast, the Egyptian Roussette bat doesn’t display constitutive IFN expression, but it does have a greatly expanded set of the less well studied antiviral IFN-ω genes—22 genes compared to humans’ single copy—which might provide more flexibility in antiviral response.27

Indeed, as mammals evolve more effective antiviral IFN-mediated responses, “viruses coevolve proteins that can very efficiently suppress interferon responses,” said Banerjee. “It’s a constant battle.”

“I wanted to know if bat interferons could resist this shutdown by viral proteins,” he said. For certain bats with certain viruses, this seems to be the case. Middle-East respiratory syndrome coronavirus (MERS-CoV) has an estimated 35 percent mortality rate in humans, but when researchers at the National Institutes of Health experimentally infected a group of Jamaican fruit bats, not one showed any symptoms of illness.28

Banerjee’s research in another bat species suggests that interferons could play an important role in this disease resilience. He showed that while MERS-CoV suppressed the IFN-β response and reached higher viral loads in human cells, big brown bat cells had a stronger IFN-β response and lower viral loads, an effect that seemed to be mediated by bat interferon regulatory factor 3 (IRF3).29 Banerjee and his colleagues continue to explore the persistence of this virus in bat cells and tissues.

Michael Hiller wears a blue shirt and stands outside against a grey wall.

Michael Hiller is a member of the Bat1k initiative and studies the genetic underpinnings of bats’ extraordinary abilities.

SVEN TRÄNKNER

Lucie Etienne, who studies the evolution of host-virus interactions at the International Center for Infectious Disease Research, also explores the evolutionary arms race between bats and viruses.

Etienne and her colleagues showed that some species of mouse-eared bats have two copies of the gene encoding protein kinase R (PKR), which is highly unusual. In research to date, all other mammal species have only one copy.30

“We got interested in PKR because it’s a very broad antiviral factor,” said Etienne. “It can sense and carry out antiviral functions against a very diverse array of viruses, including herpes viruses, influenza viruses, retroviruses, flaviviruses, and poxviruses. We thought that studying this master antiviral factor could have an impact for many different viral infections.”

Many of these viruses, however, have evolved strategies to interfere with the PKR response. By having two different PKR genes, mouse-eared bats may be better equipped to evade these inhibitors, leading to better viral control.

Etienne noted that this could possibly explain bats’ high numbers of zoonotic viruses. “Modern viruses that are circulating in bats must be able to replicate in a host that may have a very expanded and diversified antiviral defense. So perhaps this means that those viruses are more able to jump the species barrier.”

Secrets in the stem cells

While many researchers were drawn to this field because of an initial interest in immunology or virology, others have taken less direct paths. Thomas Zwaka, a researcher at the Icahn School of Medicine at Mount Sinai, has long been focused on stem cell biology, especially understanding the molecular mechanisms that guide a stem cell toward its ultimate fate.

In January 2020, his research began to take a new direction when virologist Adolfo García-Sastre, a fellow Icahn School of Medicine researcher, suggested generating lung cell models to study the new respiratory virus that was spreading rapidly in China. Zwaka began working on these induced pluripotent stem (iPS) cell-derived models and reading more about SARS-CoV-2. In one of the first major papers characterizing the virus, researchers stated that it was probably of bat origin, which piqued Zwaka’s interest.31

“I thought, ‘Wouldn’t it be super cool to do the same experiment I was just doing with human cells, but instead use bat iPS cells; differentiate them into lung cells and really compare if bats mount a different immune response?’”

After that, he fell down the rabbit hole into the wonderland of bat biology. “When you look at these little creatures, you realize that they’re so sophisticated,” he said. “It’s like 80 million years of evolution are just yelling at you.”

“I read paper after paper looking at bats’ ability to coexist with viruses without getting sick,” said Zwaka. “I found this really fascinating; it indicates that we still don’t understand enough about viruses and why they make us sick.” Perhaps equally importantly, scientists also don’t fully understand why most viruses don’t make humans sick.

Obtaining the correct bat cells turned out to be tricky, however. SARS-like viruses are found in horseshoe bats, which are an Old World species that don’t exist in the Americas. By March 2020, labs were shuttered and international travel—not to mention international shipping of bat tissues—was difficult.

Javier Juste, an evolutionary biologist at the Doñana Biological Station, came to the rescue. He caught a few horseshoe bats near Seville, then drove through the night to Madrid to ensure that the bats could be on a flight to New York by the following morning.

While other scientists had unsuccessfully attempted to make bat stem cells, the Mount Sinai team had two major advantages. First, one of the bats they received had been pregnant, so the researchers could derive bat embryonic fibroblasts, which are easier to reprogram than adult cells, according to Zwaka.

Zwaka’s team also has years of experience with creating iPS cells. “It’s almost like cooking,” he said. “You start to develop a feel for it.” And so, after much trial and error, the researchers eventually succeeded in developing horseshoe bat iPS cells.32 To determine if the same protocol could work in other bat species, they teamed up with Teeling, who sent them wing biopsies from the mouse-eared bat colony she was studying.

Previous genomic studies by Teeling, Hiller, and others, had revealed a diverse array of endogenous viral sequences in bat genomes.33 While these sequences are generally silenced in fibroblasts, in the bat stem cells, some viral sequences were transcribed into mRNA, or even translated into proteins.32

To some extent, expression of viral sequences can occur in human cells as well, and some may even play important roles in cell function.34 However, the researchers noted that the bat cells seem to generate these viral products on a larger scale than cells from humans or other animals, leading them to wonder what kinds of functions they might serve.

“Does this have anything to do with the very strange relationship between bats and viruses?” asked Zwaka. “One idea was that it could function almost like a self-vaccination scheme: If you put a virus sequence in your genome and then express it at particular time points, it may train your immune system, [like the antigen in a vaccine].” Or it could be the opposite, he said: if these proteins were presented in a different context, they could instruct the immune system to tolerate the virus in the same way that the immune system is taught not to attack the body’s own tissues.

At the moment, all of this remains entirely speculative, but researchers are eager to investigate. “Everyone sees this excitement and the really novel biology of bats,” said Zwaka. “It’s almost a bit of a gold rush because any rock you turn over, there’s something really interesting there.”

Thomas Zwaka and Marion Dejosez wear white lab coats and examine a petri dish.

Thomas Zwaka and Marion Dejosez are bringing their expertise in stem cell biology to the field of bat research.

MOUNT SINAI DEVELOPMENT OFFICE

Awaiting discovery

What does the future hold for bat research? Zwaka, Wang, and Teeling, along with biologists Paul Matsudaira at the National University of Singapore, and Richard Young at the Massachusetts Institute of Technology, have founded Paratus Sciences, a biotechnology start-up with the aim of developing therapeutics inspired by bat biology. Even as translational efforts are beginning, researchers emphasize the enormous amount of basic research that remains to be done.

“When I started my PhD ten years ago, we knew about 1,200 bat species,” said Banerjee. “Since then, we have discovered more than 200 additional bat species, and we’re still discovering them.”

A greyscale microscopy image of a cluster of cells.

Bat induced pluripotent stem cells are helping researchers explore the unusual relationships between bats and viruses.

MARION DEJOSEZ, ICAHN SCHOOL OF MEDICINE

While some immune-modulating factors like ASC2 appear to be shared across many species, other factors are species-specific. For example, in the Bat1k project preprint that examined signatures of selection on immune genes, researchers also carried out detailed experiments to study the function of these genes, including interferon-stimulated gene 15 (ISG15), which plays a multifaceted role in the response to viral infections.16 Researchers tested the antiviral effects of human ISG15 against ISG15 from nine different bat species in a simplified cell model. Many bat ISG15s appreciably differed from human ISG15, and they also often differed from each other. Complicating the picture even further, the ISG15 from each bat species also varied in efficacy depending on the virus that infected the cells.

This isn’t an isolated case either. Etienne’s research also demonstrated that PKR from different bat species differed in their abilities to escape from K3s, PKR inhibitors encoded by various poxviruses.30

Thus, each species’ immune adaptations may be finetuned for the types of viruses with which they coevolved. Today’s adaptations may have been driven by ancient viral epidemics. Therefore, studies on one species or even a handful of species can’t necessarily be extrapolated to the hundreds of other species that exist, which is why researchers are arguing for a careful examination of each species.

“Not every candidate we find will be translatable,” said Hiller. “Some things will not be compatible with human life or too difficult to manipulate or have side effects that are too strong. But there is probably not just one master regulator. Evolution is probably using several different strategies. So, we hope that if we study not just one bat but many bats, we will find a number of interesting candidates, and maybe some of them can be translated, and improve the human condition.”

Furthermore, researchers like Teeling, Ahn, and others believe that studying bat immune systems could teach us much more than just how to survive viruses. Bats could provide insights into the many disorders related to immune dysregulation, including autoimmune diseases, age-related diseases, and cancers, some of humanity’s most pressing health problems.35

References