Bats play host to a horde of nasty viruses — can studying their immunity help stop pandemics?

Randy Foo fills a pipette with orange juice from a bottle clearly labelled ‘For Bats ONLY’. He and his colleague Rommel Yroy are seated at a biological safety cabinet, wrapped in blue gowns and wearing face shields, gloves, scrub pants and shoe covers. Peeping out of Yroy’s clasped hands are two round, glossy black eyes, two slight, pointy ears and the furry snout of a young, male cave nectar bat (Eonycteris spelaea). It is wriggling and squealing, occasionally protruding its long, pink tongue to lick driblets of the sweet drink. This is a little treat for enduring a transfer in a blue cotton bag from its cage to the laboratory, followed by a quick weigh-in and inspection for injuries along its stretched-out wings and dense, fur coat. “The younger guys are generally a little bit more feisty,” says Foo.

The bat in Yroy’s hand is one of some 140 cave nectar bats housed in a research breeding colony in Singapore — the first in Asia. Foo, who manages the colony and is affiliated with Duke–National University of Singapore (Duke–NUS) Medical School, and Yroy, a veterinary technician at SingHealth Experimental Medicine Centre, have nurtured the bats for years. The original 19 members of the colony were caught using butterfly nets under highways around Singapore in 2015 and 2016; the first pups arrived a couple of years later.

The colony was set up by Lin-fa Wang, a virologist at Duke–NUS Medical School to create a controlled setting for studying bat biology, including the inner workings of their immune system.

For Wang, who has spent decades studying bats and infectious diseases, the colony has been a research boon, allowing him to ask questions about, for example, the cells that make up bat immune systems and how they respond to an infection. Now that the bats are breeding productively, the team’s research can be replicated more easily. They have shared bat tissue with about a dozen teams around the world. “Bats have become a hot topic,” says Wang.

Wang’s research niche has become more crowded since the emergence of SARS-CoV-2. Attendance at talks and conferences about bats is rising — at one symposium hosted last year in the United States, there were 30% more participants compared with the same event organized before the pandemic — and funders are ploughing money into studies of bats and infectious diseases: in 2021, for instance, both China and the United States announced specific funding pots for research into bats and viruses.

Of particular interest is the bat immune system, especially its ability to tolerate viruses that are deadly to people and other mammals — from Ebola to Nipah and severe acute respiratory syndrome (SARS). Although bat immunity is poorly understood, its consequences are clear: bats are thought to be the source of various catastrophic viral outbreaks in humans.

The field is now at an inflection point, fuelled partly by the pandemic. Researchers who have spent decades studying infections in bats, together with enthusiastic newcomers, are developing and applying new tools to the question of how bats can live with such dangerous pathogens. Some hope that those insights could one day lead to treatments for tackling infections in people and ways to prevent viruses spilling over from bats.

“There are going to be some huge steps forward in the next two or three years in terms of bat virology and bat immunology,” says Tony Schountz, an immunologist at Colorado State University in Fort Collins.

Mangoes and melons

The bats in Wang’s colony are a precious resource, and the researchers treat them accordingly. Over the years, they have tweaked the bats’ diet and environment to keep the animals healthy. The bats enjoy fresh chopped melon, papaya and mango, powdered milk and a sweet-smelling, nectar-like liquid. A burlap sack hangs from the top of each cage to give the bats — who live in groups of about 25 — some privacy and darkness. Foo plans to introduce further enrichments when the colony moves to a facility with larger cages later this year.

“The colony has given us everything we wanted,” says Wang. His office is adorned with souvenirs collected over the years — a ‘batman’ keychain, a bat-printed mug, resin-encased bat specimens, framed drawings of bats.

The researchers have studied the bats’ genomes and the diversity of viruses the animals host. They have also used bats’ cells to develop airway organoids — mini organs grown from stem cells. Their current experiments are focused on the bats’ response to infection, their ageing process and their very active metabolism during flight. The colony is a valuable research resource.

But studying bats and their viral tenants has been a hard grind, because the tools available to researchers are quite limited.

Why do bat viruses keep infecting people?

Colonies are expensive to establish and maintain; bats have longer pregnancies and fewer pups than the standard laboratory mouse. Only a handful of the more than 1,450 species of bat have been bred in research colonies around the world. These include Wang’s cave nectar bats, Jamaican fruit bats (Artibeus jamaicensis) in Fort Collins, Colorado, Egyptian fruit bats (Rousettus aegyptiacus) on the island of Riems in Germany and big brown bats (Eptesicus fuscus) in Hamilton, Canada. None of the colonies features the key coronavirus-hosting horseshoe bats (Rhinolophus spp.). “People have tried and failed,” to breed those bats, probably because researchers don’t know enough about their roosting preferences, says Aaron Irving, an infectious-diseases researcher at Zhejiang University in Haining, China.

Trapping wild bats comes with its own logistical and safety challenges, and bat cells are notoriously difficult to propagate in cell culture.

Some elements of the toolkit used for other lab animals have been lacking for bats. There is a dearth of monoclonal antibodies, which immunologists use to tag immune cells and proteins. For a long time, there was no high-quality genome for a bat species. Immunologists working with mice and human tissue have been “completely spoiled” by access to these tools, says cell biologist Thomas Zwaka at the Icahn School of Medicine at Mount Sinai, New York City.

The lack of tools means that researchers still don’t have a clear picture of the “basic architecture of the bat immune system”, says Peng Zhou, an infectious-diseases researcher at the Guangzhou Laboratory, China.

But years of work by established researchers and an influx of newcomers are yielding new tools and methods, including high-quality genomes and lab-made bat tissue. The next decade will see exciting insights, says Emma Teeling, a bat biologist at University College Dublin, Ireland. “And the only reason we’re going to be able to do this is due to this new generation of tools.”

There is more money for bat work too, and more research papers. References to bats in immunology articles have more than tripled, from about 400 in 2018 to 1,500 in 2021, according to the scholarly database Dimensions. A start-up has raised US$100 million in venture capital funding, hoping to use knowledge gained from bat research to develop therapies for conditions from cancer to inflammation and ageing.

The latest research is filling in details of the biological mechanisms underpinning the bat immune response, including the identification of cell types that are potentially unique to bats¹. Researchers are also unravelling the diverse ways in which bat species tolerate viral infections. This can help determine whether there is “one global mechanism that applies to all of the bats, and all of the viruses”, says Diane Bimczok, a mucosal immunologist at Montana State University in Bozeman, who has jumped on the bat bandwagon. “We don’t know if that’s the case.”

The flood of interest in this previously niche field has been somewhat overwhelming, says Hannah Frank, an evolutionary ecologist at Tulane University in New Orleans, Louisiana, who received a grant to study bat immunology from a fund created for this field by the US National Institutes of Health (NIH). “I’m really excited about where we are,” says Frank. But the more researchers dig into the bat immune response across species, the murkier their current conclusions will get, she says. “We’re also going to realize just how complex this question is.”

Viral vessels

Researchers find a lot to puzzle over in bats. They are “super cool animals” says Zwaka. They are the only mammals to have evolved flight, and they use sound waves to locate objects in the dark. They live exceptionally long lives for their small size, and have a low incidence of cancer.

But the trait that has brought bats into the spotlight in recent decades is their ability to host a rich collection of viruses. Certain species, especially horseshoe bats, accommodate an exceptional diversity of coronaviruses, which include those closely related to SARS-CoV-2. Some species also host viruses such as rabies, Ebola and Marburg. Bat genomes are studded with viral remnants².

What is it about bats that allows them to tolerate viruses without showing signs of infection? Over the years, researchers have come up with some theories, which the new tools could help to refine.

Studies reveal that some bat species mount a robust first defence against invaders³. Even in the absence of a foreign threat, some species maintain high levels of interferons — molecules that raise the alarm and ramp up efforts to disable viruses — which could allow the animals to quash viral replication quickly. Bats also have an expanded repertoire of genes encoding proteins that interfere with viral replication or stop viruses from leaving cells. Their cells are equipped with an efficient system for disposing of damaged cell components, known as autophagy, which has been shown to help clear viruses from human cells.

When pathogens do intrude, bats typically don’t overreact with an outsized inflammatory response, which is often responsible for much of the damage caused from an infection³. Bats have several ways to tame the inflammatory response, such as suppressing the activity of large multiprotein molecules known as inflammasomes. Instead of spending huge amounts of energy getting rid of a virus completely, they seem to tolerate low levels of its presence, says Irving. “There’s kind of a peace treaty,” between bats and the pathogens they host, says Joshua Hayward, a virologist at the Burnet Institute in Melbourne, Australia.

Both established researchers and newcomers to the field are now beginning to look beyond the swift and indiscriminate part of the bat’s defence — the innate immune response — and towards the slower, more targeted adaptive response that retains information about a pathogen and springs into action when it meets its foe again. Adaptive immunity is restricted to a few specific cell types and a “pain in the butt” to study, says Frank.

Some researchers are also studying the link between bats’ immune response and their ecology to better understand when and where they shed viruses, and the risk of spillover to other animals⁴. This work could illuminate the environmental factors that put bats under stress, and whether that increases shedding and spillover risk.

Into the bat cave

Some of these questions took Javier Juste to an abandoned dam in Cádiz, Spain, one night in May 2020. He crept into a concrete tunnel at the dam and collected two horseshoe bats from their roost.

The coronavirus pandemic was swirling — and scientists knew that the virus had probably originated in bats. Juste’s colleagues in the United States were keen to get their hands on bat tissue, hoping that they could grow the cells and use them to explore how deadly viruses can jump from bats to people.

Bats that can host coronaviruses are not commonly found in North America, but they are all over Europe. So Juste, who is based at the Doñana Biological Station in Seville, Spain, had agreed to net and send bats from a roost near Cádiz, to New York City — in the middle of one of the strictest COVID-19 lockdowns in the world, and the mass grounding of international flights.

Tens of thousands of people exposed to bat coronaviruses each year

Speeding down deserted highways with two bats, Juste and a colleague reached the airport in Madrid the next morning. Outside the FedEx warehouse, working in the boot of their car, the researchers euthanized the animals, sliced and squeezed the bones and organs into six specimen tubes, and stashed the tubes in a coolbox to keep the cells alive. They hauled their precious cargo to the counter, with minutes to spare before the plane door closed. “It was probably one of the longest days of my life,” says Juste, who had spent months acquiring permits for the journey that day.

Some 26 hours later, the samples arrived at Zwaka’s lab. The lab was nearly lifeless, and the package hadn’t fared much better; many of the cells had already died. Zwaka, who had never handled bat tissue before, rushed to extract marrow from the wing bones and cut squares of skin from the diaphanous, rubbery wings.

His team used the specimens to produce stem cells — a commonplace tool for studying biology and disease in other species but difficult in bats. These induced pluripotent stem (iPS) cells, described in a paper in Cell in February, have already revealed some intriguing insights about the close evolutionary ties between bats and viruses⁵. Zwaka says that the cells have led his team “down quite a rabbit hole in terms of biology”.

Together with Teeling and other colleagues, Zwaka sequenced the RNA expressed by these cells and found an abundance of sections that were essentially viral fragments, many of which were originally coronavirus genomes. The viral gene expression was higher and more diverse in pluripotent cells than in both bat skin cells and in pluripotent cells from mice and humans. What’s more, the pluripotent bat cells actually used the viral fragments to make what appeared to be virus-like particles.

“The results were extraordinary,” says Teeling. When you make bat stem cells, you essentially “wake up all the fossilized viruses that you find in the genomes”. The cells seem to systematically suck up viral information in their genomes — “almost like a sponge” — and then express it. This makes these bat cells an environment conducive to viruses, says Zwaka. But what exactly this means for how these bats have learned to coexist with viruses isn’t yet clear. One possibility is that the genetic inserts somehow protect the bats from the negative outcomes of a viral infection, just as a vaccine would, says Teeling.

The researchers now plan to use the stem cells to generate lung, gut and blood tissue, as well as infecting the cells with viruses. Zwaka hopes to use the tissue to better understand bat immunity, and eventually “to develop strategies for human health”. Other researchers are using bat organoids developed using stem cells extracted directly from bats to answer similar questions.

1,000 genomes

Cells and tissues are one thing, but one of the most important resources for cell biologists is a genome. Before 2020, there were about a dozen bat genomes, of varying quality. That year, Teeling and her colleagues described the first high-quality genomes for six bat species⁶, each belonging to a different genus and each with its protein-coding genes clearly tagged.

The project was part of a global genome consortium, called Bat1K, co-founded by Teeling and aiming to create high-quality genomes for every bat species. A surge of interest and funding since the pandemic, including from biotech companies, has resulted in the sequencing of some 80 bat genomes so far, says Teeling.

The availability of high-quality genomes has transformed the bat immunology field. It has facilitated large-scale studies of RNA molecules and proteins and provided a way to classify immune cells, to some extent overcoming the lack of monoclonal antibodies. The genomes will be the “foundation for many, many studies,” says Marcel Müller, a virologist at the Charité University Hospital in Berlin.

Closest known relatives of virus behind COVID-19 found in Laos

Irving is working with the Bat1K consortium to expand its collection of Chinese horseshoe bats (Rhinolophus sinicus), which are the hosts for the closest known relatives of SARS-CoV-2. They have sequenced 10 new genomes — including 4 from the horseshoe, or rhinolophid family. In a preprint posted in February⁷, Irving, Teeling and colleagues found that bat genomes had many more genes involved in immunity and metabolism under positive selection than do other mammals. They took a closer look at one gene, ISG15, which expresses an antiviral protein that plays an important part in hyperinflammation observed during SARS-CoV-2 infections in people.

In cell experiments, they found that the rhinolophid and hipposiderid versions of the protein lacked an amino acid found in most other mammals. The change seemed to keep it from leaving cells, and probably prevented the protein from triggering an inflammatory response. Such proteins could hold important clues for how bats live with deadly viruses and could inspire therapies in people, says Irving.

Among the trendiest techniques enabled by high-quality genomes is single-cell RNA sequencing, in which researchers take a cell of interest and analyse its RNA contents to explore the cell’s components and how they work.

Last November, Wang’s team published the results of its first single-cell sequencing foray⁸. The researchers infected cave nectar bats with Pteropine orthoreovirus, a virus commonly found in this species but which doesn’t make them sick. In the bats’ lung cells, the researchers identified the fingerprints of many familiar immune cells, including T cells, as well as some unfamiliar cells.

At present, most single-cell studies in bats are simply catalogues of immune-cell activity. In unpublished work, Schountz and his colleagues infected Jamaican fruit bats with H18N11 influenza A virus and looked at which cells it targeted. They found that the virus targets macrophages — immune cells that patrol the body and gobble up pathogens — which has not been seen before for influenza A viruses. The single-cell study gave the researchers a great head start for more detailed cell-culture experiments. “At the very least, it gives you some ideas of where you should start looking,” says Schountz.

Other scientists are using RNA sequencing to compare bat and human cells. For instance, Nolwenn Jouvenet, a virologist at the Pasteur Institute in Paris, and a new entrant to the field, is combining this technique with CRISPR gene editing in bat cell lines from a range of species to look for differences in the innate immune response of bat and human cells. Ultimately, Jouvenet hopes to identify the genes responsible for controlling viral replication.

For some questions, only a whole bat will do. At his colony, Schountz wanted to test whether fruit bats, which are not natural hosts of SARS-CoV-2, can be made susceptible to it. So his team used a viral vector to express the ACE2 receptor, which SARS-CoV-2 uses to enter cells, in the bats’ lungs, and then infected the bats with SARS-CoV-2. They found that the bats produce T-helper cells specific for the virus; these cells are key players in the adaptive, targeted immune response. Stimulating the T-helper cells produced small proteins known to regulate inflammation, which could explain the tempered inflammatory response in bats. The results were posted as a preprint in February⁹. Schountz is planning a new bat facility, with construction starting in June and to be completed by 2024, with flight rooms to house larger bats such as flying foxes (Pteropus). Other teams also plan to establish colonies, including one for Jamaican fruit bats at Montana State University.

Back in Singapore, it is hot and humid under the bright midday Sun, but inside the vivarium, the temperature is noticeably cooler. Yroy and Foo are speaking loudly above the noisy hum of a machine outside.

The bats seem unfazed, huddled together, upside-down, in a dark corner of their cage, letting out occasional squeaks. “They are used to people coming and walking around the cage,” says Foo. Earlier that morning, Yroy had laid new plastic sheets to catch their droppings, and hung fresh bowls of water. Soon, it will be feeding time. “So far, they are quite happy,” says Foo.