In the summer of 2020, six months after the start of the coronavirus pandemic, scientists went to forests in northern Laos to catch bats that could harbor close relatives of the pathogen.
In the middle of the night, they used fog nets and tarpaulin traps to trap the animals as they emerged from nearby caves, collected samples of saliva, urine, and feces, and then released them back into the darkness.
The fecal samples turned out to contain coronaviruses, which scientists studied in high-security biosafety laboratories known as BSL-3 using special protective gear and air filters.
The three Lao coronaviruses were unusual: they carried a molecular hook on their surface, very similar to the one on the virus that causes Covid-19 called SARS-CoV-2. Like SARS-CoV-2, their hook allowed them to cling to human cells.
“It’s even better than the early strains of SARS-CoV-2,” said Mark Eloit, a virologist at the Pasteur Institute in Paris who led the study, keeping in mind how well the Lao coronavirus hook binds to human cells. The study was published online last month and has yet to be published in a scientific journal.
Virus experts are buzzing about the discovery. Some suspect that these viruses, like SARS-CoV-2, may already infect humans from time to time, causing only mild and limited outbreaks. But under the right circumstances, pathogens can cause a pandemic similar to Covid-19, they say.
The findings are also important for a tense debate about the origins of Covid, experts say. Some people speculate that SARS-CoV-2’s impressive ability to infect human cells could not have resulted from the natural spread of infection from an animal. But new data seems to suggest otherwise.
“It really disproves any notion that this virus had to be invented or somehow manipulated in the laboratory to infect humans so well,” said Michael Sparrow, a virologist at the University of Arizona who was not involved in the work.
These bat viruses, along with more than a dozen other viruses discovered in recent months in Laos, Cambodia, China and Thailand, could also help researchers better anticipate future pandemics. The virus family tree provides clues as to where potentially dangerous strains are hiding and which animals scientists should look at to find them.
Last week, the US government announced a $ 125 million project to identify thousands of wild viruses in Asia, Latin America and Africa to determine their risk of spread. Dr. Eloit predicted that many more relatives of SARS-CoV-2 remained to be found.
“I’m fly fishing,” he said. “When I can’t catch trout, it doesn’t mean that there is none in the river.”
When SARS-CoV-2 was first discovered, its closest known relative was the bat coronavirus, which Chinese researchers discovered in 2016 in a mine in Yunnan province in southern China. RaTG13 is known to share 96 percent of the genome with SARS-CoV-2. Based on the mutations carried by each virus, scientists estimate that RaTG13 and SARS-CoV-2 share a common ancestor that infected bats about 40 years ago.
Both viruses infect cells using a molecular hook called a “receptor-binding domain” to anchor to their surface. The RaTG13 hook, which is adapted to attach to bat cells, can only weakly cling to human cells. The SARS-CoV-2 hook, by contrast, can hijack cells in the human respiratory tract, which is the first step towards the potentially fatal Covid-19.
To find other close relatives of SARS-CoV-2, wildlife virus specialists checked their freezers full of old samples from around the world. They identified several similar coronaviruses from southern China, Cambodia and Thailand. Most of them are descended from bats, and a few are from scaly mammals known as pangolins. No one was more closely related than RaTG13.
Instead, Dr. Eloit and his colleagues set out in search of new coronaviruses.
They traveled to northern Laos, about 150 miles from the mine, where Chinese researchers discovered RaTG13. For six months, 645 bats of 45 different species were caught. Bats were carriers of two dozen coronavirus species, three of which were strikingly similar to SARS-CoV-2 – especially in the receptor-binding domain.
In RaTG13, 11 of the 17 key building blocks of the domain are identical to those in SARS-CoV-2. But in the three viruses from Laos, as many as 16 were identical – the closest match to date.
Dr. Eloit suggested that one or more coronaviruses could infect humans and cause mild illness. In a separate study, he and his colleagues took blood samples from people in Laos who make a living by collecting bat guano. Although the Lao showed no signs of being infected with SARS-CoV-2, they carried immune markers called antibodies that were apparently caused by a similar virus.
Linfa Wang, a molecular virologist at Duke-NUS School of Medicine in Singapore, who was not involved in the study, agreed that such an infection is possible because newly discovered viruses can firmly attach to a protein on human cells called ACE2.
“If the receptor-binding domain is ready to use ACE2, these guys are dangerous,” said Dr. Wang.
Paradoxically, some of the other genes of the three Lao viruses are more distantly related to SARS-CoV-2 than other bat viruses. The reason for this genetic patchwork is the complex evolution of coronaviruses.
If a bat infected with one coronavirus becomes infected with a second, two different viruses may end up in the same cell at the same time. When this cell begins to replicate each of these viruses, their genes shuffle, forming new viral hybrids.
In Lao coronaviruses, this gene shuffling gave them a receptor-binding domain very similar to the SARS-CoV-2 domain. According to preliminary analysis by Spyros Litras, a graduate student at the University of Glasgow in Scotland, the initial genetic exchange took place about ten years ago.
Litras and his colleagues are now comparing SARS-CoV-2 not only with new viruses from Laos, but also with other close relatives discovered in recent months. They find even more evidence of gene shuffling. This process, known as recombination, can change the shape of viruses from year to year.
“It is becoming increasingly clear how important recombination is,” said Mr Litras.
He and his colleagues are now drawing random evolutionary trees for viruses like SARS-CoV-2 based on these new discoveries. Looking for new viruses can help clarify the picture. But scientists disagree about where to look for them.
Dr. Eloit believes the Southeast Asia zone is best suited, which includes the site where his colleagues found coronaviruses, as well as the nearby Yunnan mine where RaTG13 was found.
“I think the main landscape corresponds to northern Vietnam, northern Laos and southern China,” said Dr. Eloit.
A new US government virus scan project called DEEP VZN could identify one or more SARS-CoV-2-like viruses in the region. A spokesman for USAID, the funding agency for the work, named Vietnam as one of the countries to look for researchers and said the new coronaviruses are one of their top priorities.
Other scientists believe that relatives of SARS-CoV-2 should look further away. Dr. Sparrow of the University of Arizona said that some bat coronaviruses carrying SARS-CoV-2-like segments have been found in eastern China and Thailand.
“It is clear that recombination shows us that these viruses are part of a single gene pool for hundreds and hundreds of miles, if not thousands of miles,” said Dr. Sparrow.
Colin Carlson, a biologist at Georgetown University, suspects that a virus that could trigger a Covid-like outbreak may be lurking even further. He noted that bats as far east as Indonesia and west as far as India share many biological characteristics with animals known to carry viruses like SARS-CoV-2.
“This is not only a problem in Southeast Asia,” said Dr. Carlson. “These viruses are diverse and more cosmopolitan than we thought.”
Interest in the origins of the pandemic has brought renewed attention to the security measures that researchers use when studying potentially dangerous viruses. To win the DEEP VZN grants, a USAID spokesperson said, scientists will need to provide a biosafety and biosecurity plan, including staff training, instructions for using protective equipment in the field, and laboratory safety measures.
If scientists find more close relatives of SARS-CoV-2, this does not necessarily mean that they are a deadly threat. They may not spread to humans or, as some scientists suggest, cause only small outbreaks. It is known that only seven coronaviruses have overcome the species barrier and have become recognized pathogens for humans.
“There are probably many other coronaviruses that will ultimately not go anywhere,” said Jessica Metcalfe, an evolutionary ecologist at Princeton University.
However, recombination can turn a stuck virus into a new threat. In May, researchers reported that Indonesia had recombined two coronaviruses in dogs. The result is a hybrid that infects eight children.
“When the coronavirus, which we have seen for decades and which we consider to be just something that our pets can get infected, can take a leap – we should have foreseen it, right?” – said Dr. Carlson.