Controversies in Annex D7 (about WIV) of the WHO report

Dr. Monali C. Rahalkar

 1. The  WHO joint team report assumes and documented that all the research in Wuhan laboratories including WIV was done in P3 and P4 probably because of the mere fact that such laboratories existed.

On the contrary, Zhengli Shi, the principal scientist of the coronavirus research in WIV has said earlier that they did all the coronavirus research in P2 and P3 labs (Science, July 2020 Q and A) [1].

2. The Mojiang mine was associate with deaths of three miners in 2012 due to a pneumonia like illness. From the same mine, WIV had picked up a bat fecal sample (CoV 4991) and renamed it as RaTG13 after the outbreak. We found this connection that the same mine in Mojiang, Yunnan was associated with both RaTG13 and three miners death and had documented earlier in a preprint and later in a peer-reviewed paper [2, 3]. WIV was involved in both, the Ab test of the pneumonia patients and had received the blood samples of the miners in 2012, and they also collected bat fecal samples for not just one but three years from 2012-2015. This information was not disclosed by WIV in any of their publications immediately after the pandemic, but they disclosed (it partially) after 11 months in the form of an Addendum to their original article (Zhou, P., Yang, XL., Wang, XG. et al. Addendum: A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 588, E6 (2020). https://doi.org/10.1038/s41586-020-2951-z). They did not give any details of the pneumonia in miners, although two thesis were available [4,5]

Instead, in the Annex D7, they put all the questions which were asked to them pertaining to the Mojiang mineshaft incident and its connection to their institute under the category, ‘conspiracy theories’. It is stated there on page 131 of the Annex:

With regard to the matter of morbidity and mortality in miners in a mine in Mojiang, Yunnan Province, where bats were present, Professor Shi said that the events had been clarified in an addendum to her Nature article. The reported illnesses associated with the miners, according to the WIV experts, were more likely explained by fungal infections acquired when removing a thick layer of guano”.

What happened in the Mojiang mine to the miners could be in fact a direct infection from bats to humans, also supported by several facts: several horse shoe bats including Rhinolophus affinis in the mine, Zhong Nanshan remotely observing the cases and diagnosing the disease as primary viral pneumonia with probable secondary infections [4]. This was a piece of good evidence to the whole mystery of how the virus could have come to a Wuhan laboratory.

We have written a perspective article which was peer reviewed and published in Frontiers in Public Health [3]. Our article discusses various points associated with the illness and after we compared it to COVID-19, it matched in most of the cases!

The conclusions in our paper were:

The striking similarities between the Mojiang pneumonia cases and COVID-19 are noteworthy, as is the fact that RaTG13/CoV4991, the next genomic relative of SARS-CoV-2 was found in the same mineshaft. The Master’s thesis by Li Xu concludes that the pneumonia-illness in the miners was due to a SARS-like CoV from horseshoe bats. The remote consultation and diagnosis by a prominent pulmonologist in China, Dr. Nanshan, adds credibility to the diagnosis of the pneumonia cases in 2012. Although we cannot say that RaTG13 or SARS-CoV-2 infected the miners, there is a high chance that it could be a virus quite similar in genetic composition to these two. The coincidence between the 2012 illness in Mojiang miners, the subsequent samplings, and finding the nearest SARS-CoV-2 relative from this single mine warrants further inquiry, and the data along with the full history of this incident would be invaluable in the context of the current pandemic.

Additionally, the miners in TG mine (nearest relative RaTG13 picked up) showed positive Ab to SARS (like disease) and the test was done in WIV (Canping Huang, student of George Gao, PhD Thesis, Chapter 3. 3, 2016) [3,4].

The relevant paragraph (Chapter 3.3, Canping Huang, 2016 PhD thesis says:

Blood test results of 4 cases showed that: 4 people carried SARS virus IgG antibodies, of which 2 were discharged with higher antibody levels and 2 were hospitalized with lower antibody levels (Wuhan, Chinese Academy of Sciences) Virology Institute).

WIV says it was a fungus (Annex D7 report), with no further support (medical or other documents).

3. WHO Report Annex D7 states: Miners had been to the cave 2-3 times and it was 1 meter thick with bat faeces.

Fact: Miners worked 12 hours a day for 14 days in 4 cases and 4-5 days in other two cases [5]. (Li Xu Master Thesis 2013).

5. The reported illnesses associated with the miners, according to the WIV experts, were more likely explained by fungal infections acquired when removing a thick layer of guano, as per the Annex. WIV is experts in viruses not fungi. They disregard what Zhong Nanshan said (The SARS doctor of China) that the miners had primary viral infection most likely due to the bat coronaviruses and the fungal infections were secondary. WIV skilfully does not mention either thesis by Li Xu 2013 [5] or the PhD thesis by Canping Huang 2016 [4]. Also, if fungus was the primary cause, it has to be proven by aspiration of the fluid and cultivation of the causative fungus and its identification (I talked to medical doctors). No such work has been demonstrated by WIV, supporting the statements that a fungus was identified in this case.

6. Zhengli Shi says: Clade 4 SARSr-CoVs only found in Yunnan. The clade 4 was described only in her webinars. There is no supporting literature for explaining what is clade 4. And if this is from Yunnan, then why does the WHO report mention that they should explore the bat coronaviruses from out of China? And then if the clade 4 is the clade where SARS-CoV-2 lies, why didn’t they allow people to visit the mine? Recently, more than eight news reporters were blocked from visiting the TG mine. Why didn’t the team visit TG and recommend to resample?

7. WHO joint team has completely overlooked that the nearest neighbour RaTG13 was collected from a mine where miners got a COVID-19 like illness [2,3]. Some conclusions for our paper: 1. The blood biochemical analysis from the pneumonia patients indicated elevated markers such as Serum Amyloid A (SAA) with a normal range of PCT (procalcitonin), which suggested that the patients had a viral infection. 2. The treatment given to the pneumonia patients included antivirals (ganciclovir, acyclovir injections), steroids (methylprednisolone), antibiotics (meropenem, vancomycin, etc.), antifungals (caspofungin, fluconazole), and anti-thrombotic medicines (warfarin, lmw heparin). 3. The thesis concludes that severe pneumonia in miners was due to SARS-like CoV from horseshoe bats. Dr. Nanshan’s conclusion that the Mojiang miner’s pneumonia appeared to be primarily viral and that it was most probably due to bat-related coronaviruses (from horse shoe bats).

8. In Jan 2020, WIV isolated the SARS-CoV-2 in no time! Whereas in the Annex they say: the limited amount of live virus available for research. If they have so many difficulties in isolation of viruses, how did they do from the patients in record time? Also, it is hard to imagine that they did not isolate any virus from the TG mineshaft after they monitored it for getting novel viruses (as stated in the Annex D7 of the report:

Professor Shi’s team went there in 2012-15 about seven times to look for novel viruses

9. One virus strain with high homology with SARS-CoV-2, was renamed as RaTG13, and the information published in Nature (Annex D7).

Why was RaTG13 renamed? As the other samples or virus strains are named as numbers!

The sequence of RaTG13 looks problematic as the spike region does not bind strongly to bat ACE-2 (Zhengli Shi, webinar, Rutgers Uni, March 2021). Other issues with the raw data of RaTG13 are documented earlier [8,9, 10]. This also includes the fact that the coverage of RaTG13 assembly is low, de-novo assembly could not be done and there are gaps. Also, the raw data shows: strong evidence for DNA contamination in the sample, the presence of non-adapter related repetitive sequences in majority of reads, reported by two preprints [9,10].

10. Other controversies in the Annex D7: all fieldwork is done with full PPE (numerous pictures of WIV lab members shows that this was not the case!).

Also, in our publication [3], certain questions were openly asked to WIV, and we still have almost no answers, neither by WIV nor in the report.

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 (Supplementary Material). 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?

References:

1.       Cohen, J. Wuhan coronavirus hunter Shi Zhengli speaks out. Science 2020, 369, 487-488.

2. Rahalkar, M.C.; Bahulikar, R.A. Understanding the Origin of ‘BatCoVRaTG13’, a Virus Closest to SARS-CoV-2. Preprints 2020, 2020050322 https://www.preprints.org/manuscript/202005.0322/v2

3.       Rahalkar, M.C.; Bahulikar, R.A. Lethal Pneumonia Cases in Mojiang Miners (2012) and the Mineshaft Could Provide Important Clues to the Origin of SARS-CoV-2. Frontiers in Public Health 2020, 8, doi:10.3389/fpubh.2020.581569.

4.       Huang, C. Novel virus discovery in bat and the exploration of receptor of bat coronavirus HKU9. China, 2016 PhD Thesis.

5.       Xu, L. ‘The analysis of 6 patients with severe pneumonia caused by unknown viruses’ original thesis in Chinese language, Kunming Medical University, China, China, 2013 Master’s Thesis.

6.       Ge, X.Y.; Wang, N.; Zhang, W.; Hu, B.; Li, B.; Zhang, Y.Z.; Zhou, J.H.; Luo, C.M.; Yang, X.L.; Wu, L.J.; et al. Coexistence of multiple coronaviruses in several bat colonies in an abandoned mineshaft. Virol Sin 2016, 31, 31-40, doi:10.1007/s12250-016-3713-9.

7.       Zhou, P.; Yang, X.L.; Wang, X.G.; Hu, B.; Zhang, L.; Zhang, W.; Si, H.R.; Zhu, Y.; Li, B.; Huang, C.L.; et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020, 579, 270-273, doi:10.1038/s41586-020-2012-7.

8.       Rahalkar, M.; Bahulikar, R.A. The Anomalous Nature of the Fecal Swab Data, Receptor Binding Domain and Other Questions in RaTG13 Genome Preprints 2020, doi: 10.20944/preprints202008.0205.v3.

9.       Singla, M.; Ahmad, S.; Gupta, C.; Sethi, T. De-novo Assembly of RaTG13 Genome Reveals Inconsistencies Further Obscuring SARS-CoV-2 Origins. Preprints 2020, doi: 10.20944/preprints202008.0595.v1.

10.       Zhang, D. Anomalies in BatCoV/RaTG13 sequencing and provenance. https://zenodo.org/record/3969272#.Xy0m5jVS_IX 2020.

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