How an Indian scientist couple worked to trace origin, course of COVID-19

The second wave of Covid-19 is sweeping through India, and we are expecting a third wave. More than 3.5 million people on the globe have perished in the pandemic. Yet, no one knows how exactly the deadly virus, Sars-CoV-2, emerged and spread to humans.

We need to know how it happened, so as to prevent the next pandemic. But, even a year after the outbreak, no robust process has been established for examining the origin of the virus.

Until last month, the possibility that the virus escaped from a lab was treated as a conspiracy theory. Today, things have changed, and a lab origin of the virus remains a possible theory. The US president and the former director of the Centers for Disease Control, besides many renowned scientists, have called for a proper investigation of the origin.

As a scientific couple, we were intrigued by the virus and started an online expedition at the end of March 2020. The journey led us to discover the link between a copper mine and the nearest relative of the virus.

Being scientists in biology, we were only a step ahead of laymen in understanding the pandemic. We began by reading scientific papers on coronaviruses and Covid-19. We were fixated on reading the work of Professor Ralph Baric of the University of North Carolina. Baric has been studying coronaviruses for decades and had created Sars-like chimeric viruses in the lab.

One of the papers he co-authored, with Vineet Menachery of the University of Texas, is known as the Menachery paper. Published in Nature Medicine in 2015, it was about creating a chimeric Sars-like coronavirus. Two of its 13 other co-authors were from the Wuhan Institute of Virology (WIV): Ge Xingyi and Shi Zhengli, the famous Bat Woman of China. It acknowledged that Shi provided spike sequences and plasmids.

WIV has the most advanced laboratories in coronaviruses, and Shi leads its researchers. Shi had an interview with Scientific American in 2020, which we read online. Simultaneously, Kristian Andersen from California’s Scripps Institute and four other authors published a note in Nature Medicine about the Proximal Origins of Sars-CoV-2. This paper argued that the virus was of entirely natural origin and that a lab origin was not possible.

We were not convinced—because we knew that Baric’s lab was working on building chimeric viruses and hence it was possible to make genetically engineered viruses, especially Sars-like coronaviruses. We also knew that Shi’s lab had collected a bat coronavirus, RaTG13, that was 96.2 per cent similar to Sars-CoV-2.

Shi had described Sars-CoV-2 for the first time in a paper in Nature in 2020. But it did not mention RaTG13 as an actual virus, and it seemed more like a bat fecal sample. No detailed information of this first cousin of Sars-CoV-2 was given—except that it was a sample that her group had collected in Yunnan in 2013.

We spent two weeks getting more information about RaTG13. A preprint published by Dean Bengston in April 2020 mentioned that this virus had an older name (Ra4991), and therefore, a reference. We found the reference where Ra4991 was mentioned.

In 2013, WIV made an expedition to an abandoned mineshaft in Mojiang, Yunnan. The mine had several types of bats in large numbers, especially horseshoe bats. Horseshoe bats are the natural reservoir of Sars and Sars-CoV-2.

WIV had found that only one beta coronavirus, Ra4991, came from the intermediate horseshoe bat, whose scientific name is Rhinolophus affinis. “That’s how the Ra in RaTG13 could have come from!” we guessed.

And we recollected that, in her interview, the Bat Woman had talked about a mineshaft in Mojiang where a lethal pneumonia-like disease had occurred in six miners in 2012. A diverse group of coronaviruses was discovered in the mine following the outbreak.

We found a third reference for the Mojiang mine in the Science Magazine news published in 2014. It said that in April 2012, a pneumonia-like illness had occurred in six miners who were cleaning bat faeces from a copper mineshaft in Mojiang, killing three of them. The last clue confirmed what we thought: Yes! This could be the same mine where WIV had picked up RaTG13.

Let us turn to a page in Monali’s diary: It was an unforgettable summer evening in early May (2020). Thunder and wind rumbled and, after a while, there was thudding rain. We were opening several links on the laptop, reading and tallying some references. The last fifteen days had been a nonstop hunt of a clue about the nearest cousin of Sars2. And there it came. We found the reference confirming that RaTG13 came from the same mine—an abandoned copper mine in Mojiang. The mine where six miners took ill; three died of severe pneumonia. On reading the word pneumonia, we thought of Covid. We found that the nearest cousin of the Covid virus was from the same mine.

The electricity went out. Rain was pouring. And there we were exclaiming with excitement and a shade of sadness: “It is the same mine.” There was a straight connection—samples were taken from Yunnan, the hotbed of coronaviruses, to Wuhan, where the pandemic started. The feeling was as if we had just seen a horror movie.

Miners of Mojiang

When we found the connection between the Mojiang mine, the miners' pneumonia and RaTG13, we decided to publish a small preprint to disclose this discovery to the scientific world. We also communicated our findings to Nature in the ‘Matters Arising’ category.

The day we published our preprint, an email popped up from an unknown person. His Twitter name was @TheSeeker268. The Seeker’s email said: "I also wish to bring your kind attention to the post-analysis on the causes, diagnosis, and treatment process of the six Mojiang mineshaft patients." And he attached a tweet with a link to a master's thesis in the Chinese language and a crude translation of the same.

The Seeker had found the thesis on the cnki.net website, which is the official website for master’s and PhD theses in China and, therefore, considered to be a valid source. We translated the thesis using google translation. A professional translation is now available online thanks to a research agency (bioscienceresource.org).

We collected the following information from the thesis: In early April 2012, in a place called Tongguan in Mojiang, four men were hired for cleaning bat faeces in an abandoned copper mine. The layer of bat waste must have been thick, and probably the workers worked without any protection such as gloves or masks. The oldest of them was 62, and the others were in their forties.

After working for about ten days, they had cough, fever and breathlessness. And, after 14 days, they were so sick that they could not work anymore. All of them consulted local doctors or hospitals but eventually landed up in a sophisticated hospital in Kunming. All had raging fevers, cough, body pain and breathing difficulty. Eventually, most of them needed ventilators.

As this batch of four became ill, two younger men were hired for the same job. But, after working for four or five days, they came down with a running fever and cough. These two also landed in the same hospital.

After ten days, the oldest man breathed his last. He had severe pneumonia and acute respiratory stress, and his condition degraded day by day. The reason for the death was cardiac arrest. The second man died in June 2012. He had the same symptoms. The two young men recovered and were discharged.

The doctors were puzzled by this mysterious illness. China already had the experience of Sars. They knew Sars had originated in bats, passed through the intermediate hosts, civet cats, and then jumped to people. Did this illness, too, do so?

The doctors may have suspected that it was Sars as it was a bat-infested mine, and consulted Zhong Nanshan, the Sars doctor of China, who commanded enormous respect.

The consultation was through video conferencing. Observing the two severely ill but alive patients, he made a diagnosis: it was a case of primary viral pneumonia with a secondary fungal infection.

A lengthy treatment of antivirals, antibiotics, antifungals and even blood thinners followed. A third miner died after a hundred days in the hospital ICU. The fourth miner was discharged after four months.

The thesis featured medical reports, radiological images such as CT scans, and detailed information about the diagnosis and treatment of the miners. We read the thesis several times to understand it. A radiologist in Pune confirmed that the CT scans of the miners were very similar to those of Covid-19 patients.

We then wrote a paper, ‘Lethal pneumonia cases in Mojiang miners and the mineshaft could provide important clues to the origin of Sars-CoV-2’. The connection was clear. WIV had analysed the miners’ samples, according to the thesis. WIV had also studied the bat faecal samples from the distant mine, 1,800km from Wuhan.

The next question in our minds was: “Could any of these viruses, either from the patient’s samples or from the bat faecal samples, have resulted in or were Sars-CoV-2? We left the paper with open questions for the Wuhan lab, as there were no further publications by WIV on the miners’ case or the samples.

It was said the RaTG13 genome was sequenced in 2020. But, later on, Shi said it had, in fact, been sequenced in 2018. We wondered: Why did they not publish about the viruses from the Mojiang mine? Why did Shi say that the miners had died of fungal disease when the evidence and Zhong Nanshan’s diagnosis pointed to a primary viral illness, most probably due to Sars-like coronaviruses from the horseshoe bats in the mine? Why were the cases not reported to the WHO or to the rest of the world?

A PhD thesis, also found by The Seeker, mentioned that the four miners were tested for Sars antibodies, and they had IgG antibodies. But later on, in November 2020, Shi denied this fact.

The miners’ pneumonia was very similar to Covid-19 but was not transmitted further. And hence it probably needed genetic manipulation to cause a transmissible illness.

Our research on the miners’ disease, including the fact that they had Sars-like antibodies of RaTG13 from the same mine, was published in the journal Frontiers in Public Health on October 20, 2020.

We had sent our paper to top journals like Science and New England Journal of Medicine, but they did not find these cases important and did not review the paper. Today, our paper is widely read (50,000 reads) and cited in some crucial documents, including a letter by the Republicans to the New York-based EcoHealth Alliance and an open letter to the WHO.

Later on, it became clear that Shi and her WIV lab had collected eight more Sars-like coronavirus samples from the same mine between 2012 and 2015. And still, at the beginning of the pandemic, when they published their first paper about the outbreak, they mentioned RaTG13 but neither its old name, Ra4991, nor its connection to the Mojiang mineshaft and the pneumonia outbreak was mentioned.

Were the Chinese scientists afraid of getting traced to the mine, which was the birthplace of a Covid-like illness in the miners? In a recent interaction session in a webinar, Shi replied that the miners’ samples did not have any Sars-like coronaviruses. Surprisingly, it seems that they had retained these samples, and tested them against Covid-19 and found them negative.

China’s CDC director, George Gao, was asked the same question in a documentary by France2 television. He replied that the miners had antibodies to Sars. But these could have been from an earlier infection or by an unknown virus. George Gao’s student Canping Huang had mentioned positive antibodies in the four miners.

_{Chimeric viruses}

The Wuhan Institute of Virology has thousands of bat coronavirus samples. These were mainly from Yunnan and Guangdong provinces. Shi and her lab collected all these samples and have developed techniques for creating recombinant or chimeric viruses. They also use reverse genetics and create pseudo-viruses.

They have done experiments to insert spike genes into the backbone of many viral genomes for creating chimeric viruses. These chimeric viruses are then tested for their ability to infect human cell cultures and humanised mice. According to them, this information would help predict the likelihood of spillover, the jump of a coronavirus from bats to people.

Shi has collaborated with Peter Daszak, who heads the NGO EcoHealth Alliance. They have received several grants, for bat coronavirus research, from America’s National Institutes of Health (NIH) and National Institute of Allergy and Infectious Diseases (NIAID), of which Dr Anthony Fauci is the director. Though WIV has denied that it had a Sars-CoV-2 virus before the pandemic, the kind of samples it collected and the kind of experimentation it did, especially in creating chimeric viruses, could have resulted in the creation of Sars-CoV-2 like viruses.

There is a possibility that the virus was either modified or synthesised using a RaTG13 or a similar backbone or a genome. Utilising bioinformatics tools and modelling, with a RaTG13 or a similar virus as a reference, a sequence can be created, which can be further used to create an actual virus. RaTG13 was one such candidate virus or a genetic progenitor. Additionally, the RaTG13 sequence was the most exclusive one from the WIV collection of Sars-like coronaviruses and still remains the closest one to Sars-CoV-2.

Peter Daszak had tweeted in November 2019 that they had discovered several of such Sars-like coronaviruses and that testing work was going on using humanised mice. The humanised mice expressed human ACE-2 receptors in their respiratory systems. This receptor is a protein (angiotensin-converting enzyme 2) that is an entry point for the coronavirus to infect human cells.

Mysteriously, several databases of WIV which were online are now offline. The most important of all was a viral database which had information about 22,000 coronavirus samples, 16,000 of them from bats! This database went offline in September 2019. The reason, according to Shi’s statement to a WHO-China joint investigation team, was that there were more than 3,000 hackers. The question is, what were the hackers seeking in September 2019, when there was no trace of the pandemic? This fact casts doubts on the whole thing.

The Mojiang mine is now inaccessible. Reporters from the Associated Press, BBC, France2 and many other media groups could not reach the area because of roadblocks and secret police. If there is nothing to hide, why close the mine to reporters and scientists? Also, why didn’t the WHO team visit the mine?

_{Low-safety level labs}

Shi mentioned in a Q&A session published in Science magazine in July 2020 that they used Bio-Safety Level 2 and 3 labs for coronavirus research. BSL-2 is a very low-level security for experiments with bat coronaviruses. A recently found PhD thesis, which was done under Shi’s guidance in WIV, stated that the proliferation and cell infection experiments of live viruses (including recombinant viruses) were performed in a BSL-2 laboratory.

In November 2019, WIV published an article about a security exercise in which all the safety staff and department heads participated. During the meeting, the deputy director of security at WIV presented “a number of common problems that were discovered during the security check in the last year.” He stressed the danger of potential security breaches and “that the correction of hidden dangers must take place in a thorough manner.”

The collection of bat coronavirus samples from the caves was often done with the least protection. Several photos are available on the internet where scientists are seen without PPE kits and with the least protection.

_{Why lab leak theory remains a likely scenario}

The world’s leading coronavirus scholar Ralph Baric told the Italian television programme Presa Diretta: “You can engineer a virus without leaving any trace. The answers you are looking for, however, can only be found in the archives of the Wuhan laboratory.”

Not all manipulation of viruses in the lab can be seen. For at least ten years, in the two most advanced labs in the world, in North Carolina and Wuhan, researchers have used seamless techniques to combine genetic material of different types of viruses without leaving scars in the joints between one piece and another.

And the Wuhan databases have disappeared: in the archives of the network, we discovered that Shi had made available to the scientific community a very rich database in bat and rodent viruses. It contained data on more than 20,000 samples and viruses collected over the years in different parts of China. It reported very detailed information: the GPS coordinates of the sampling location, the type of virus found, whether the virus had been sequenced or isolated (that is, grown in cell cultures).

The database provided for password access to consult data relating to viruses not yet published, with the only obligation not to disclose the information until the date of publication. Since June, however, the entire page has been missing from the web. According to a portal that monitors China’s science databases, the data was inaccessible as early as September 12, 2019. “Why? What experiments were done in Wuhan?" asked the scientist Ralph Baric.

To summarise, considering all the facts mentioned earlier, it is a highly likely scenario that Sars-CoV-2 might have come from a lab. Though natural origin still remains a possibility, the circumstantial evidence points towards a lab leak scenario, which should be investigated.

The lab leak hypothesis also includes several possibilities, such as a lab staff getting infected while directly working with the virus or a sample; exposure of discarded lab material to humans; exposure of animals which were used for experimentation to humans; discharge of the material in the sewage without proper disinfection; and infection during sampling. Wuhan had several animal experimentation labs which were scattered over different campuses. Animal carcasses, discarded after experimentation with coronaviruses, could also be one of the sources of infection.

DRASTIC group

During our journey, we met an informal group of scientists, analysts and sleuths on Twitter. It is called DRASTIC. Its prominent members at that time were: TheSeeker, Rossana Segreto, Billy Bostickson, KevinMcH, Francisco and Luigi Warren. Some of them had nicknames and preferred to remain anonymous. Anonymity gives protection from possible reprisals and helps carry out potentially controversial research without compromising their careers.

DRASTIC (drasticresearch.org) is interested only in Covid origins research, and its findings have been mentioned in international newspapers. Even as questions about Covid origins are becoming part of an increasingly political fight, with bias on both sides, DRASTIC has remained science-based. It does not bend to pressure and is not hierarchical; we regularly challenge each other.

We also challenged the WHO-China joint investigation team’s statement in February that a lab-derived Covid virus was unlikely. Led by Jamie Metzl, 26 scientists signed an open letter calling for a full and unrestricted international forensic investigation into the origins of the virus. We pointed out that half of the joint team was Chinese and that the international members had to rely on information that the Chinese authorities chose to share with them.

Two more open letters followed. In the final letter, we demanded a full investigation which includes access to all the records, samples, personnel and facilities at the Wuhan labs.

It is of utmost importance that we investigate the lab leak theory as best as we can.

Dr Rahalkar is scientist, Bioenergy Group, Agharkar Research Institute, Pune. Dr Bahulikar is scientist, BAIF Development and Research Foundation, Pune.