Searching for animal zero: The detective work of tracing the origins of a virus

by David

The first mission to Wuhan in search of the source of SARS-CoV-2 — the virus that causes COVID-19 — wrapped up this week. More than a dozen experts from around the world, including epidemiologists, veterinarians, medical doctors and virologists, spent the past fortnight looking for clues to the virus's origins. Last night, at a press conference, World Health Organization food safety and animal diseases expert Peter Ben Embarek said the coronavirus is unlikely to have leaked from a Chinese lab, and most probably jumped to humans via an intermediary species. And that fact-finding mission will be the first of quite a few, if previous outbreaks are anything to go by. COVID-19 is the latest in a long line of zoonotic diseases — infections that started in animals, but spilled into the human population. Typically, identifying the animals in which a virus normally lives — called the primary reservoir — takes many years, says Hume Field, a veterinary epidemiologist who has helped trace the source of a number of viruses, including the first SARS virus in China and Hendra in Australia. The animal origins of the first SARS virus SARS-CoV-1, which made to jump to humans in 2002, wasn't nailed until 2017, when Chinese scientists reported that they'd found all the genetic building blocks of SARS in a single population of horseshoe bats.

Sometimes scientists can search for years and still not find a definitive primary reservoir, as has been the case with Ebola (so far). Other times, though, it can happen relatively quickly, says Dr Field, who is now Science and Policy Adviser for China and South-East Asia with US-based NGO EcoHealth Alliance. "The case of flying foxes in Australia and the Hendra virus was probably one of the shortest periods of time — it only took a couple of years," he said. In search of Hendra's origins When searching for the animal — or animals — involved in zoonotic diseases, Dr Field says there's no general playbook to follow. Instead, it's a matter of starting with whatever information you can find, which is usually the time and place of an event such as the first known human case, and try to trace forwards and backwards from that point. Bats, including flying foxes, can live with viruses that cause disease and death in other animals.(Pixabay: shellandshilo) The first recorded outbreak of Hendra, for instance, occurred in the Brisbane suburb of Hendra in 1994, when more than a dozen horses in a stable died from an acute respiratory disease within a week.

The disease spread to a trainer and the stable foreman, who were nursing the horses, and they both came down with a flu-like illness days after the first horse died. The trainer subsequently died of the disease. At that point, no-one knew where the mystery disease came from. To find out, the tracing team identified the first horse to get sick, a mare named Drama Series, and looked to the paddocks she'd lived in for the months prior to her arrival at the Hendra stables. Once diagnostic tests confirmed the horses and humans became sick from an as-yet-undescribed virus, and not something like a toxin, the next question to answer was: where did that virus come from? Dr Field was brought in to explore the possibility that the virus came from a source in the paddock. He and colleagues trapped live animals and took blood samples to look for signs of the virus in the form of antibodies. "We got rodents of various types, possums, a couple of feral cats, lizards, the odd bird and so on," Dr Field said.

"I can't remember how many samples we ended up with, but it was hundreds." But none of the blood samples contained antibodies for the Hendra virus. Next, they drove around the paddock at dusk, dragging a giant net behind them to catch insects — maybe the virus was carried by mosquitoes. Again, no luck. By this point, it was getting to October, 1995. A year had passed since the Hendra stables trainer died, and health authorities still didn't know where the virus came from. But then a crucial piece of the puzzle slotted into place.

"We became aware of a case that occurred in northern Queensland, just a little bit earlier than the Brisbane case," Dr Field said. The spotlight swings to bats In the month before the Hendra outbreak, a horse owner in Mackay assisted in autopsies after two of his horses died. He was admitted to hospital with meningitis three weeks later. And while he eventually recovered, he fell ill again and died 14 months later. Brain samples from the patient confirmed the presence of Hendra virus. "So then the question was: how can a mammalian virus have the ability to be in Far North Queensland and Brisbane at effectively the same time?" Dr Field said. And so the spotlight swung to bats.

The tracing team also considered other animals, such as feral cats, which could become infected by the disease. But after taking blood samples and swabs from captive populations of flying foxes in a variety of locations in Queensland, they found groups that had high levels of antibodies to the Hendra virus. The clincher was isolating the Hendra virus from flying foxes in early 1996. "And this is very much a jigsaw kind of approach," Dr Field said. "In my experience, you get small pieces that add up to make the big picture. "Then you can stand back and say, 'On the weight of evidence here, this species is a primary reservoir for the virus.' "But this doesn't mean there's not another primary reservoir for the virus, though.

" Source of the first SARS virus Dr Field was also involved in an early mission to look for epidemiological clues behind the source of the SARS-CoV-1 virus, which emerged in China's Guangdong province in November, 2002. Back then, as is the case today, Chinese centres for disease control were not centralised like the US version — instead, they're at all levels, from local to national. This means information can be collected quickly, funnelled up the chain and analysed to look for these clues. "And what was common was that the vast majority of early cases had direct or indirect contact with wildlife markets in the Pearl River Delta region of Guangdong province." The Pearl River Delta is a vast trade area, famous for its wildlife markets. But unlike SARS-CoV-2, which appears to have emerged from one market in Wuhan, the SARS outbreak saw a cluster of cases, in multiple markets in the area, appear more or less simultaneously. "So it became increasingly evident that this was sort of like a cluster source," Dr Field said.

"There was something about these markets and the people there that was resulting in infection." Unlike the Hendra virus, where the intermediate animal was clearly horses, the animal that gave the SARS virus to humans was still a mystery. So a Chinese team started taking blood tests from animals in the wildlife markets and testing them for SARS antibodies. Among the animals that carried antibodies, the team managed to isolate the SARS virus from masked palm civets (Paguma larvata), cat-like mammals farmed for food. And in 2005, two studies published days apart by different groups found wild bats were natural hosts of SARS-like coronaviruses. A good decade would pass before the specific bat population — a species of horseshoe bat called Rhinolophus sinicus — was identified as a primary reservoir for SARS-CoV-1. The team investigating the origins of the COVID-19 pandemic started their work in Wuhan, but whether it's really the first infection location is yet to be determined.

(Getty Images: Hector Retamal) What about SARS round two? As the WHO-led investigative team made the rounds of Wuhan and surrounding areas, they searched for clues that point to the source of SARS-CoV-2. Unlike the teams that looked for the source of SARS and Hendra, the WHO crew had a bit of a leg-up. Most virologists and biologists agree that the virus probably came from a bat, says Michelle Baker, a bat immunologist at CSIRO's Australian Centre for Disease Preparedness. "Bats are known to be reservoirs for coronaviruses, and many other viruses," she said. "Now, I think it's pretty good agreement that this [SARS-CoV-2] virus is likely to have come from a bat." Despite reporting last night from China that thousands of swabs taken from bats had, so far, tested negative to SARS-CoV-2, the WHO-China investigative team said work to identify the origins of the coronavirus pointed to a natural reservoir in bats. The flying mammals probably weren't in Wuhan, though.

Something else we don't know, Dr Baker added, is whether there was an intermediate animal involved before it spilled over into humans — although it is likely. In the case of Middle East respiratory syndrome (MERS), the primary reservoir was bats, with camels the link between them and humans. And unlike bats, which seem to have a super-charged immune system, intermediate hosts do often show signs of the disease. "For instance, for horses infected with Hendra, they get quite sick, and the virus amplifies in horses, and then it's infectious to humans," Dr Baker said. Technology, too, has made significant advances since Hendra and SARS emerged. Genome sequencing, which maps out an organism's entire genetic code, has become faster, cheaper and more portable in the past few years. Techniques that once involved samples taken to a lab for analysis can now be done in the field, Dr Baker said.

This means scientists who isolate coronaviruses from animals can quickly compare them to the coronavirus that made the jump to humans. Even before the current pandemic was declared, scientists reported a coronavirus in bats that's around 96 per cent similar to SARS-CoV-2. Virus 'likely hiding in reservoir species for years' Another aspect to consider is the specific parts of the genome where the viruses differ, says Sebastian Duchene, a genomic epidemiologist at the Doherty Institute. The 96-per-cent-similar bat coronavirus may be, for the most part, genetically identical to SARS-CoV-2. Yet their differences, while seemingly small, are significant. "Those key differences are in a region of the ACE2 receptor binding domain, and that's really what determines the host range," Dr Duchene said. ACE2 receptors on human cells, like the ones that line our nose, are SARS-CoV-2's way in.

The virus's spike protein latches onto the ACE2 receptor, like a lock in a key, to gain entry to the cell. Once inside, the virus replicates and busts out to infect more cells. Differences in the DNA that codes for the spike protein mean the key won't fit the human ACE2 lock, and the virus can't infect humans. Family tree analysis can also tell us how long a virus has been circulating in an animal reservoir before infecting humans. This was done in camels to estimate how many times the MERS coronavirus jumped from camels to humans. (Lots, it turns out — more than 50, according to some estimates.) Last year, a team traced the genetic ancestry of viruses most closely related to SARS-CoV-2, and calculated how long ago they split on different evolutionary branches.

"And what we do know is SARS-CoV-2 diverged from the closest known virus probably decades ago," Dr Duchene said. So while there's no way of saying what the animal reservoir was, he adds, "It's been hiding in the reservoir species, probably for many years, before it actually had the exact right mutations to jump in humans and lead to more and more transmission." In any case, we may be waiting a while yet before we know where SARS-CoV-2 originated and exactly how humans became infected. It'll be worth the time and effort, though, Dr Baker said. "The main thing is to understand how it happened. That's the primary goal, so we can avoid it happening again."