Critique to the Addendum (Zhou et al 2020) and other contradictions in reporting the facts about RaTG13 and its history

Dr. Monali C. Rahalkar, Scientist D, MACS Agharkar Research Institute, Pune, India

Zhou et al. 2020 (https://www.nature.com/articles/s41586-020-2012-7) described for the first time a novel coronavirus/ SARS-COV-2, which is causing the current pandemic COVID-19, killing one and half million people.

The authors had reported that the closest virus/sample was the one they had collected in 2013 from horseshoe bat feces, RaTG13. There were no details given about RaTG13 in the article, except its whole genome sequence. The Addendum https://www.nature.com/articles/s41586-020-2951-z#article-comments, appears to be the first correction in the form of an addition to the report.

The starting sentence of the addendum is, ‘Here we provide further information about the bat SARS-related coronavirus SARSr-CoV) strain RaTG13 reported in our Article’.

Three of the six miners had died cleaning a mine in Tongguan, Mojiang, in 2012, by a pneumonia-like disease. Researchers and other Twitter users had found out about this connection in early May, including us. However, in their publications or interviews, the WIV group had not mentioned this fact in conjunction with each other. To date, the scientific group from WIV had never mentioned RaTG13 in context to the miner’s illness. These two facts were mentioned separately by Zhengli Shi, the corresponding author, in two separate communications or interviews. In an interview published in Scientific American, she talked about the mine where miners got a pneumonia-like illness and two died https://www.scientificamerican.com/article/how-chinas-bat-woman-hunted-down-viruses-from-sars-to-the-new-coronavirus1/. However, she did not mention that RaTG13 sample was from the same mine, and RaTG13 being synonymous to 4991. This interview in SA was first published in February 2020.

In July 2020, Zhengli Shi gave a rather detailed explanation about RaTG13 in Science Magazine and answered many scientific questions. Here, she gave the details about the Tongguan mineshaft, the place where RaTG13 or 4991 was sampled. She did not say anything that the miners suffered from a lethal pneumonia and WIV analyzed the miners’ serum samples https://www.sciencemag.org/sites/default/files/Shi%20Zhengli%20Q%26A.pdf

Also, ZL Shi did not mention why they did the bat surveillance in the TG mine.

In contrast, in the scientific American interview, she does describe that the miners had a pneumonia-like disease, and their team did a surveillance of the mine for ‘a year’. Three years earlier Shi’s team had been called in to investigate the virus profile of a mine shaft in Yunnan’s mountainous Mojiang County—famous for its fermented Pu’er tea—where six miners suffered from pneumonialike diseases, and two died. After sampling the cave for a year, the researchers discovered a diverse group of coronaviruses in six bat species. In many cases, multiple viral strains had infected a single animal, turning it into a flying factory for new viruses.

This article seems to be linked to the Ge et al 2016 article (not sure if recently linked), which documented the Bt/CoV4991. But in February 2020, the fact that Bt/CoV 4991 and RaTG13 were the same was not known to the world or clarified in Zhou et al 2020, and hence, it was not clear that the next relative of SARS-CoV-2 was collected from the same mine where miners suffered pneumonia.

Most importantly, none of this information was given as a piece of supplementary information in Zhou et al 2020, or in the last 9 months, though all of the information mentioned above is eight years old.

The Bt/CoV4991 and RaTG13 could be the same samples, this fact was already being discussed on scientific forums and published in blogs and preprints at that time, e.g., Rossana Segreto  who wrote a comment on Virology Blog on 16 March, and a preprint mentioning that 4991 and RaTG13 could be the same https://osf.io/wy89d/ (a preprint by Dean Bengston). On 16 May 2020, we (I and Dr. Rahul Bahulikar) submitted a Matters Arising manuscript against Zhou et al 2020, where we stated the facts that 4991 could be the same as RaTG13 and that RaTG13 coming from the same mine where the miners suffered fatal pneumonia were given in our submitted article.

Subsequently, we published two pre-print versions (https://www.preprints.org/manuscript/202005.0322/v2) describing the same. In a peer reviewed paper, we described how the miners’ pneumonia resembled COVID-19https://www.frontiersin.org/articles/10.3389/fpubh.2020.581569/full. In our paper, we had asked many questions to the WIV authors, including the fact that according to the Master’s thesis by Li Xu and Ph.D. thesis by Canping Huang, samples of the miners were sent to WIV for analyses of the SARS or SARS-like Ab. Questions asked in our paper,

As has been stated, the miners’ samples were sent to WIV for SARS Ab testing (7, 9), the same institute that also conducted surveillance of the bat coronaviruses in the Mojiang mineshaft (10). The link between the SARS-like CoV (4991/RaTG13) from mine where lethal pneumonia cases occurred, has not yet been discussed in scientific papers by the WIV laboratory before February 2020. We are curious to know what kind of samples the WIV received from the Mojiang miners, along with other questions, such as whether the samples are still stored in WIV, and whether they are available for study by other researchers. It would also be of particular value to know whether any viruses were isolated and if there is any DNA/RNA available from these samples. It would also be useful to know if PCR was performed on the miners’ samples and available sequences. According to Huang’s Ph.D. thesis, four miners tested positive in an Ab test against SARS-like CoV. However, further questions remain as to which antigen was used for the Ab detection in the pneumonia patients and what was the exact protocol used. Why is this information not available in any of the seroprevalence studies by WIV? Why were the severe pneumonia cases in 2012 not mentioned in any of the WIV publications before 2020? Were any SARS-like CoV isolated from the bat fecal samples collected in 2012–13? Why were the Mojiang miners pneumonia cases in 2012 not reported to any public health agency like the WHO? Why did programs like PREDICT not mention the lethal pneumonia cases as a mini-outbreak? Was the mineshaft in Mojiang closed, when? According to the literature, three research groups went to the Mojiang mine to collect samples between 2012 and October 2014 (5, 9, 10). The mine was promptly closed as per the (2). Why was the Mojiang mine being visited by researchers until October 2014? Questions also remain as to why Dr. Shi attributed the outbreak in Mojiang to a fungus in the interview with Scientific American. Was the mine open for researchers and were any samples brought after 2014? Did any of the researchers who visited the Mojiang mineshaft get infected by any coronavirus between 2012 and 2019? Are there any whole genome sequences available for SARS-like CoV originating from this mine? Why is the pathogen database (http://www.viruses.nsdc.cn/chinavpi/) associated with the project (2013FY113500) (10) not accessible anymore?

The addendum appears to have been written after our paper was published, though our paper has not been mentioned directly. The addendum also could be as a result of many scientists demanding to Nature journal and on various forum about the facts associated with RaTG13.

However, the addendum, neither described this 8-year-old important story in detail, where six miners went to clean the mineshaft of which three died, nor the reference for it when it appeared in 2014 Science magazine https://www.sciencemag.org/news/2014/03/new-killer-virus-china. They only mention analysing 4 samples of miners of which one was deceased. They completely avoid to share any details of the miners’ illness, which has been documented in the Masters’ thesis by Li Xu, 2013 and PhD thesis by Canping Huang, 2016, available in the Chinese language, and these references could have been cited. The Master thesis in particular, gives all the details of the miners’ illness, including the CT scans, blood analysis, temperature, oxygen, and various investigations were done on them. The thesis also documented that Dr. Zhong Nanshan had remotely observed these cases and asked to check them for SARS-like coronaviruses, which were the suspected pathogens. The addendum also does not mention our paper, which was published a month before the addendum was published, https://www.frontiersin.org/articles/10.3389/fpubh.2020.581569/full which discussed all these facts in details.

Looking at the gravity of the current pandemic, it is positively not understandable why the authors failed to give any details of the pneumonia-like illness in the miners, which had so many similarities to COVID-19. Li Xu Masters’2013 http://eng.oversea.cnki.net/Kcms/detail/detail.aspx?filename=1013327523.nh&dbcode=CMFD&dbname=CMFD2014 and Canping Huang 2016 PhD thesis http://eng.oversea.cnki.net/kcms/detail/detail.aspx?dbcode=CDFD&QueryID=11&CurRec=1&dbname=CDFDLAST2018&filename=1017118517.nh&UID=WEEvREcwSlJHSldTTEYzWEpEZktmRXB3Sm9JeHRKZExVOG5ySkJjK0xHMD0%3d%249A4hF_YAuvQ5obgVAqNKPCYcEjKensW4IQMovwHtwkF4VYPoHbKxJw!!&autoLogin=0 (Chapter 3) were based on this important case study of the miners getting ill with this fatal illness.

The complete absence of description of the illness, also imposes a question that whether they do not want to discuss this, as it was a suspected viral illness, and the team found not only one but eight other SARS-like CoVs in the mine? And later, the pandemic started right in Wuhan, where they analyzed and worked with these samples. For the first time the WIV authors admit that the Mojiang mineshaft miners had severe respiratory disease. Further they also tell us that they collected the sample which they renamed RaTG13 from the same mineshaft. This is the first written evidence that WIV agrees that they collected a virus or a sample which is till now the closest neighbour of SARS-CoV-2 from a mineshaft. And that, the same mineshaft was the reason, why the miners or people working in cleaning the mine got ill.

They clearly say in the addendum, ‘We suspected that the patients had been infected by an unknown virus’. However, in the Scientific American, interview, the same corresponding author, who is the chief scientist working on those samples and now on SARs-CoV-2, Zhengli Shi, replied that fungus was the killer of those miners. Why did she say that in Feb 2020, whereas, there is no mention of this fact or reasoning in the addendum! Also, there is no mention that WIV did any test for detecting the fungus, etc.

The addendum appears to answer some of the questions asked in our paper starting with:  they received 13 serum samples, Between 1 July and 1 October 2012, we received 13 serum samples collected from 4 patients (one of whom was deceased) who showed severe respiratory disease.

To investigate the cause of the respiratory disease, we tested the samples using PCR methods developed in our laboratory targeting the RNA-dependent RNA

polymerases (RdRp) of Ebola virus, Nipah virus and bat SARSr-CoV Rp3, and all of the samples were negative for the presence of these viruses.

Usually, for testing SARS-like coronaviruses, throat swabs or nasal swabs or bronchoalveolar lavage fluids are the samples that would give sufficient RNA and positive results. Serum samples are not the right samples to detect the virus by RdRp PCR, as the viral amount in the blood would be much lower, as it is a respiratory illness. Secondly, the time at which the sample was collected for doing a direct RdRp PCR is incorrect. The sample would not have the viral RNA after 2 months of the primary illness, in this case, if it was a SARS-like infection which they could have suffered in April-May, the samples collected in July to October 2012 would not anyway give positive results. It is clear that after 2 months, the RdRp PCR for the virus would be anyway negative. And therefore, the negative PCR does not actually mean that the samples were negative for SARS-like viruses.

Further, they give an account of the tests they performed (in 2012?) and then in 2020 on these 13 samples.

We also tested the serum samples for the presence of antibodies against the nucleocapsid proteins of these three viruses, and none of the samples gave a positive result. Recently, we retested the samples with our validated enzyme-linked immunosorbent assay (ELISA) against the SARS coronavirus 2 (SARS-CoV-2) nucleocapsid protein—which has greater than 90%

amino acid sequence identity with bat SARSr-CoV Rp3—and confirmed that these patients were not infected by SARS-CoV-2′.

It is highly unclear what they tested in 2012 and in 2020. The question is did they test SARS-Antigen or Ab against the Ag by ELISA? The fact that they tested the patient’s serum samples after eight years and got negative results is not useful and perhaps do not mean anything.

Additionally, they have ignored and their results contradict to the fact that, Canping Huang, a PhD student of George Gao, had mentioned that four of the four samples from the miners had showed a positive IgG Ab test for SARS or SARs-like viruses, in his PhD thesis. He even mentions that out the four, who were still in the hospital (meaning patient 3 and 4) had lower Ab titre compared to the discharged ones (meaning patient 5 and 6).

If we go through the Masters’ thesis by Li Xu, several patients’ samples were taken. Also, as mentioned in the thesis, Dr. Zhong Nanshan had ordered to take swabs for testing for the coronaviruses, but there is no mention of swab samples being tested by WIV in the addendum. Also, why did they test for Ebola? Ebola was not in the picture that time and never found in China. The major recent outbreak had happened in Dec 2013 in Africa.

In both the references, Ge et al 2016, and the Scientific American interview, the team just says that they did the mine surveillance for a year (2012-2013).  But, suddenly, in November 2020, they disclose another old fact, that the mine was being surveyed for coronaviruses for 3 years, 2012-2015. On the contrary, in the Scientific American article, Zhengli Shi says that the mine was ‘promptly closed’!

The last paragraph in the Addendum states:

‘Between 2012 and 2015, our group sampled bats once or twice a year in this cave and collected a total of 1,322 samples. From these samples, we detected 293 highly diverse coronaviruses, of which 284 were designated alphacoronaviruses and 9 were designated beta coronaviruses on the basis of partial RdRp sequences.’ All of the nine betacoronaviruses are SARSr-CoVs, one of which (sample ID4991; renamed RaTG13 in our article to reflect the bat species, the location and the sampling year) was described in a 2016 publication. The partial RdRp sequence (370 bp) of ID4991 was deposited in GenBank in 2016 under accession number KP876546. All of the identified bat SARSr-CoVs are distantly related to SARS-CoV based on partial RdRp sequences. In 2018, as the next-generation sequencing technology and capability in our laboratory had improved, we performed further sequencing of these bat viruses and obtained almost the full-length genome sequence (without the 5′ and 3′ ends) of RaTG13. In 2020, we compared the sequence of SARS-CoV-2 with our unpublished bat coronavirus sequences and found that it shared a 96.2% identity with RaTG13′.

The world was unaware of the fact that the WIV team was visiting the mine for 3 years, and procured samples of 9 betacoronaviruses all which were SARSr-CoVs. Also, the authors say that they performed further sequencing of these bat ‘viruses’. The term in pleural clearly denotes that more than one of them was sequenced for their whole genome. However, they have just submitted RaTG13, and there is no mention, no names or even partial RdRp sequence has been given as a supplementary information. The authors have not even mentioned the sample ids or sampling years of these remaining eight viruses. In fact, their statement, ‘All of the identified bat SARSr-CoVs are distantly related to SARS-CoV based on partial RdRp sequences’, gives us a hint that these could be closer to SARS-CoV-2, as SARS-CoV-2 is in the distant most clade from SARS-CoV.

To summarize, the Addendum, rather than answering questions, give facts that are not complete. It does not provide details of the eight viruses they procured in the mine. Moreover, all of this information comes very late and is in-complete in terms of details and methodology.

Looking at the pandemic situation’s gravity, each person on this planet has the right that they get to know correct information about the virus and its closest relatives. However, the authors of the Addendum provide an incomplete and may be inaccurate information here. Astonishingly, none of the expert virologists have expressed these points or raised any criticism. Hence, although I work more on bacteria and archaea and am a molecular biologist, not precisely a virologist, I had to put these facts together on a paper and present these. It was an Addendum to the original Nature paper and should have been complete with supplementary information, such as the methodology used and the sequence of the other SARS-like eight coronaviruses. Unfortunately, in my opinion, this addendum does not do so.

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