The mystery virus that protects against monkeypox

At the turn of the 19th Century, a bizarre medical panic swept across London. Informative pamphlets were distributed. Alarmist books were authored. Dubious treatments emerged. The public was warned, en masse, that it was in peril – at urgent risk of… morphing into cow-humans.

A small group of controversial doctors had been whipping up concerns about a pioneering medical procedure, which included taking a virus thought to infect cattle and using it to protect people against its cousin, smallpox.

The technique was named "vaccination", after the Latin "vaccinus", meaning "of a cow" – and the early evidence suggested that it was extraordinarily effective, protecting 95% of people from an infection which usually killed around 30% of its victims and permanently disfigured most of the rest. There was even an early hope that it might eventually vanquish the disease forever.

But it didn't take long for the first ever vaccine sceptics to pop up. In particular, these breakaway physicians were convinced that the "bestial humour" – the cowpox virus – had no place within the human body. Among the more ludicrous claims was the suggestion that vaccinated children had begun developing bovine features, such as the blotches on dairy cows, or that it risked them eventually having oxen-like thoughts. One prominent advocate suggested vaccinated women might start fancying bulls.

The thing is, the early vaccine sceptics had got it all wrong. Of course, the new technique did not convey the essence of bovine into innocent people – cowpox was just a normal virus, and over the coming centuries, it would drive smallpox to extinction. But it may also never have had anything to do with cows at all.

In fact, to this day no one knows where the virus that eradicated smallpox ultimately came from. And yet, this mystery microbe is still being used – including in the vaccines currently being deployed against monkeypox, which has now been declared a global health emergency by the World Health Organization.

After mostly being found in Africa for the past five decades, in May 2022 the monkeypox virus began spreading around the globe. To combat it, scientists have turned to two vaccines previously used against smallpox – ACAM2000 and JYNNEOS. These are the only ones licensed in the US to protect against the emerging virus (the EU also recently approved the JYNNEOS version). Both are exceptionally safe and thought to be highly effective, but they too are part of the puzzle.

For more than a century, the smallpox vaccine was widely assumed by the scientific community to be made from cowpox – this is the explanation still found in many websites and curriculums worldwide. But in 1939, nearly 150 years after vaccination was invented, molecular tests revealed that it's not. More recently, genetic sequencing has confirmed these findings. Instead, the vaccines that were used to eradicate smallpox, and those in use today against monkeypox, are based on an unknown virus that no one has been able to identify – a "ghost" pathogen that has only ever been found in vaccine form.

Despite an 83-year search, no one knows how, why or precisely when this imposter appeared in the smallpox vaccine, or whether it still exists in the wild. Only one thing is clear: millions who lived through the reign of smallpox owe their lives to its existence. Without it, our current monkeypox outbreak is likely to have spread even more rapidly.  

"For many years, until 1939, people assumed that what we call vaccinia, the smallpox vaccine, was the same as cowpox," says José Esparza, a virologist and fellow of the Robert Koch Institute, Germany. "And then it was discovered that they were different. And since then, we accept that cowpox is one virus, and vaccinia is another virus of unknown origin."

How has this happened? Where could this virus have come from? And will we ever be able to find it in its natural host?

A very English affliction  

The man universally credited with inventing vaccination is Edward Jenner, a silver-haired surgeon who announced his discovery in 1796. The usual story of how he did it is a compelling tale of beautiful milkmaids, eureka moments and ethically questionable experiments. But it turns out this is not entirely accurate.

The most familiar version goes something like this. Jenner noticed milkmaids often had unusually clear skin, without the pockmarks that afflicted a large proportion of the population – up to 85% of those who recovered from smallpox may have been left with a significant number of characteristic facial scars. He realised that those who had fallen ill with the milder illness of cowpox during the course of their work were less likely to go on to catch smallpox. To prove it, he infected an unknowing eight-year-old boy with the former, and then intentionally exposed him to the latter to see if he was still susceptible and would get sick (luckily, he wasn't, and lived).

In fact, the serendipitous discovery came nearly three decades earlier, with a country doctor who had just moved to the market town of Thornbury in Gloucestershire. There John Fewster set up a practice for "variolation" – an ancient method of protecting people from smallpox by rubbing a small amount of pus from a smallpox pustule into an incision in an uninfected person's arm.

The procedure had been used across Asia, from India to Tibet, for centuries, but was largely unknown to Europeans until Lady Mary Wortley Montagu learned about it in Istanbul – then known as Constantinople – and popularised it in 18th-Century Britain. If it went well, the technique usually produced a single smallpox mark at the site of the infection, which indicated that the person's immune system had learned to recognise the virus. If it went badly, and the infection spread – as in about 2-3% of cases – the patient often died.

But in Thornbury, a number of locals didn't react to variolation at all – no pock marks were produced, and despite repeated attempts, the procedure was unsuccessful. Fewster was baffled. Then one day, a farmer explained that he had recently been infected with cowpox – he already had immunity.

"Cowpox was largely confined in Britain to the south-west [at the time]," says Arthur Boylston, a retired pathologist and the author of Defying Providence: Smallpox and the Forgotten 18th-Century Medical Revolution. He says that it was never particularly common – outbreaks only occurred every few years – and though farmers in the area were generally aware that it existed, only the younger generation had linked it to protection from smallpox.

It's thought this folk wisdom eventually found its way to Jenner, who attended the same medical society as Fewster.

On 14 May 1796, Jenner took some pus from a cowpox mark on the hand of a dairy maid, who had caught it from a cow called Blossom. This was used to vaccinate an eight-year-old boy. Six weeks later, the child was variolated, and when he did not react by developing a pustule, Jenner realised that the pioneering technique had worked.  

"What they were observing is what we know today, that all poxviruses produce cross-species immunity," says Esparza.

But in 1939, even this version of events hit a snag.

When scientists tested cowpox antibodies on the smallpox vaccine that was supposedly made from it, they discovered that they were not the same after all – these were two totally different viruses.

A large group

As it happens, humans are not alone in their battle against poxviruses. This vast, encompassing family includes tens of viruses which each have their own niche in a wide variety of animals – even scarab beetles have their own versions. Within this there's the Orthopoxvirus group to which smallpox, which should arguably be called humanpox, belongs. Alongside it are other mammalian viruses including horsepox, camelpox, buffalopox, rabbitpox, mousepox, monkeypox and raccoonpox.

Vaccinia is just another member of this group – one that was used to inoculate nearly everyone born before the early 1970s against smallpox, before the vaccination was discontinued. But finding its wild ancestor from among the diverse Orthopoxviruses has proven to be tricky.  

One possible candidate is horsepox.

In Jenner's original paper on vaccination, he describes his suspicions that cowpox may actually originate in horses – in whom it was known as "grease".

In one case, the undergardener to the Earl of Berkeley noticed the horses he worked with were coming down with the disease, and later accidentally transferred it to the cow herd he was milking – and himself. Twenty-five years later, the same man turned up with his family at a variolation session run by Jenner. It didn't work – though the procedure was repeated many times, nothing happened. Later, when the man's entire family fell ill with smallpox, he "received no injury from exposure to the contagion".

Not knowing whether he was working with cowpox or horsepox, or a virus that routinely hops between both, Jenner pressed on. After inventing the vaccine, he devoted the rest of his life to distributing it and finessing the ways it was administered.

But "cowpox" outbreaks were rare, and as the technique exploded in popularity, finding enough infectious material became a major challenge. After Jenner's first ever vaccination experiment, he couldn't do any more research for two years, when the disease temporarily vanished from the area.

Jenner's subsequent early attempts at vaccination were based on transferring the protective virus from person to person – each new patient who was infected became a store of pus that could be used to vaccinate someone else. There were no purification steps, and no refrigerated supplies of neat vaccine ampules.

A more sophisticated method involved soaking pieces of thread in infectious material, then drying it out – this made it possible to rapidly spread the vaccine to the furthest corners of the globe. In 1800, Jenner sent a pus-coated thread on a 2,272-mile (3,656km) journey to Newfoundland, where it was successfully used to vaccinate hundreds of people.

Alas, these techniques were not entirely reliable – and if the relay chain was broken, the whole process had to start from the beginning. That meant finding a new cow with "cowpox".

One solution was to simply broaden the animal base involved, and horses were an obvious second choice. It soon became clear that poxvirus harvested directly from horses worked just as well as that from cows – so well, that by 1817, Jenner had abandoned "vaccination" and switched his full attention to "equination". And here his distribution networks – as well as those of others relying on pox viruses from horses – began to have an impact.

In 1817, Jenner sent a stock of lymph, infectious fluid taken from equinated individuals – which by then was being preserved in lancets of gold, rather than dried on threads – to the National Vaccine Establishment. From this central hub in London, it was sent on to many other doctors. Could this have been when the cowpox-based inoculation began to be replaced with one made from horsepox? Or was the virus always horsepox that had been transferred to cows?

An unexpected twist

Though it's been centuries since the early smallpox vaccinations, there are still relics of the old viruses used hiding in museums and collections around the globe – mostly in the form of scabs and lymph from vaccination kits. Back in 2017, an international team of scientists led by Esparza dug out one that had been manufactured in Philadelphia in 1902. 

Orthopoxviruses have unusually large genomes consisting of double-stranded DNA, and the researchers managed to piece together an almost complete genome from the historic sample they had. "These vaccines have been kept at room temperature for more than 100 years," says Esparza. It's only because of sophisticated modern techniques that sequencing the degraded genetic material was possible, he says.

What the scientists found added weight to the long-suspected vaccine virus mix-up: there was no evidence of cowpox in the strain they tested, and instead it was closely related to a horsepox virus identified in Mongolia in 1976. "This is the only sequence we have for horsepox – the only one," says Esparza. "…And it's very similar."

Since then, the team have sequenced many other historic vaccines. "In 31 samples, we have not found cowpox in any of them," says Esparza.  

Other work completed by a different team has found similar results. In addition to horsepox, their vaccines – from mid-to-late 19th-Century Philadelphia – were a good match for a virus endemic to Brazil, Cantaglo virus, which causes periodic outbreaks in cattle. Again, this is not cowpox – it is thought to have descended from a smallpox vaccine that escaped into the wild many years ago.

So, it looks like the majority of these 19th and early 20th-Century vaccines really were made from horsepox – either cowpox was never used, or it was replaced by its equine cousin remarkably quickly. However, this is not the end of the puzzle.

"There is a mystery that we have still not solved," says Esparza. His team has recently uncovered evidence – not yet published – for a radical shift in the vaccines used to prevent smallpox, which happened around 1930. "We're investigating that now," he says.

When the team sequenced more recent smallpox vaccines, they found that around this time they underwent a transformation. Rather than being composed primarily of horsepox, they were mostly based on the mystery virus found in vaccines today. "The core [sequence] that used to be horsepox until 1930 changed to modern vaccinia, which is also an Orthopoxvirus sequence but we don't know the origin of that virus. It's not cowpox," says Esparza.

How did it replace the previous vaccine? What might it be made from? And could it still exist out in the wild?

A vanished virus

In Esparza's view, the sudden jump from one kind of smallpox vaccine to another likely comes down to the way vaccines were distributed.

"For the first 100 years [of vaccines], they were kept from arm to arm in humans," says Esparza. "In 1860, scientists in Italy and France introduced the animal vaccine – instead of passing the virus from human to human to human, they found that they could put it back into cows, and maintain it in cows." Eventually, this system of mass production expanded to include other animals, including sheep, horses and donkeys.

At some point, a virus from an unknown animal started being used as a smallpox vaccine. There are no records of who did this, or when, why or how they went about it, but it's possible it was just an accident – someone harvested what they thought was horse or cowpox from a farm animal, when it was actually a random, unidentified imposter. It worked well, so no one noticed.

Sometime after 1930, this mystery virus became the most common vaccine – and by the mid-20th Century, there were hundreds of different versions circulating across the globe. Then in 1966, the WHO announced the smallpox eradication campaign, and chose just six vaccine strains that would be used to achieve this. With each passing decade, the dominance of the unknown virus became more entrenched.

But where is it now – and why has no one ever found vaccinia's natural host?

Though the emergence of monkeypox might seem to suggest that poxviruses are thriving, for a long time, many were highly endangered – and smallpox may not be the only one that has now vanished.

It's thought that horsepox once caused regular outbreaks in parts of Europe – it may even have been common – but it hasn't been identified in the wild since 1976, when horses began falling ill with lesions and fever-like symptoms in Mongolia. It's thought that improved husbandry practices and better diagnosis may have driven it to extinction.

"Horsepox basically disappeared from Europe at the beginning of the 20th Century," says Esparza, who explains that the mystery virus used in modern smallpox vaccines may have met the same fate. "We have speculated about that possibility."

However, Esparza laments that not enough research has been done. Once smallpox was eradicated, interest in studying its relatives dried up – and today there are very few research groups looking into identifying new poxviruses, such as the one that may be the ancestor of vaccinia.  

"So maybe the current [monkeypox] outbreak will stimulate more science… meaning more competition for work," laughs Esparza.    

A fresh use

In fact, today the mystery virus is more useful than ever.

Monkeypox is a close relative of smallpox usually found in tropical central Africa, where it tends to infect rodents and non-human primates. It's harder to catch than its cousin, and is mostly transferred by close contact with bodily fluids or contaminated objects, such as bedding. Unlike smallpox, monkeypox is rarely deadly, but there have been reports of more severe cases which resemble sexually transmitted infections. It usually causes a fever, followed by lesions which may be filled with pus and can be extremely painful.  

The monkeypox virus was first discovered in 1970, and until recently, infections were mostly confined to Africa. But in May 2022, it began to creep across the globe – an unprecedented spread. To slow it down, many countries have ordered millions of doses of two vaccines. Both are directly descended from the same enigmatic virus that became the dominant smallpox vaccine in the 1930s.

First up, there's the JYNNEOS vaccine, developed by biotechnology company Bavarian Nordic. This new, safer version of the old smallpox vaccine was developed by accident in the 1960s, when a scientist noticed that his stock of a Turkish strain of vaccinia – which he had been growing in chicken embryos for years – had mutated.

Modified Vaccinia virus Ankara (MVA), which was later developed into the JYNNEOS vaccine, had became so altered that though it could still make more copies of itself in chicken embryos, it had lost the ability to replicate in humans. Researchers quickly realised that this would make it safer to use for immunisations – the old version is thought to have saved 150-200 million lives between 1980 and 2018 alone, but in rare cases it can lead to infections that spread through the body. This new vaccine represented a less risky alternative. 

Initially, MVA was not widely used. In the 1960s it wasn't yet clear if the vaccine was as effective as the previous version, so it was mostly given to immunocompromised people as a bonus shot. But experiments in other animals and army personnel have since suggested that it's likely to work, so today it's in high demand.

The other vaccine, ACAM2000, is a less-favoured option in the current monkeypox outbreak. First developed in the early 2000s as an alternative to the vaccinia strains used to eradicate smallpox, it has been stockpiled by several countries around the globe, including the US and the UK, for emergencies, such as a smallpox attack by terrorists.

There are recent reports of ACAM2000 being used against monkeypox, but it's not yet licensed for this. Though it is safe in the vast majority of people, it does carry some risks – it can make copies of itself in the human body, so it's not suitable for those who are immunocompromised.

As of July 2022, the US government had ordered nearly seven million doses of both smallpox vaccines to arrive over the next year, and there is now a global shortage. The irony is, it's thought that the monkeypox outbreak may have only been possible because we ceased smallpox vaccinations in the first place.

"What we see now with monkeypox is very interesting," says Esparza. "Smallpox was declared eradicated in 1980. And since then, smallpox vaccination has stopped in most countries, and immunity in the population against all Orthopox[viruses] has decreased. And that's what is probably behind the emergence of monkeypox in the world."   

Other viruses might be seizing the same opportunity. Though cowpox – the real deal this time, not the mistaken-identity version used in vaccines – is now rare in cattle, it's still endemic in rodents throughout the globe. And since mass smallpox vaccination ceased in the early 1970s, more and more cases are being reported in children.  

Today people are most likely to catch cowpox from rats, or cats who pick it up from rodents in the wild – in one unusual case, it was acquired from a circus elephant. Most infections are mild, producing pox lesions on the hands or face, and unlike monkeypox they aren't yet being spread from person-to-person.

But there have been fatalities. And as with monkeypox, the rise in cases has been linked to the end of widespread smallpox vaccination. Some experts have even gone so far as to describe cowpox as an emerging health threat.

So, vaccinia is still very much in demand. But will we ever know where humanity's favourite poxvirus came from? Esparza is sceptical. "We still have more questions than answers," he says, though he hints that he and his colleagues have made some progress – and will be releasing more tantalising details about the mystery in the coming months.

Whatever it's made from, without the smallpox vaccine, there's little doubt that the world would be a radically different place – still grappling with an ancient plague that had been disfiguring and killing people for millennia. And just as in the early 19th Century, we have far more to fear from avoiding inoculation, than we do from turning into human-cows...

Source: BBC