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BACKGROUND “speculation about accidental laboratory escape will likely persist, given the large collections of bat virome samples stored in labs in the Wuhan Institute of Virology, the facility’s proximity to the early outbreak, and the operating procedures at the facility (Zeng et al., 2016). Transparency and open scientific investigation will be essential to resolve this issue, noting that forensic evidence of natural escape is currently lacking, and other explanations remain reasonable” SARS-CoV-2: Combating Coronavirus Emergence (Graham & Baric, 2020)
Nevertheless, speculation about accidental laboratory escape will likely
persist, given the large collections of bat virome samples stored in labs in the
Wuhan Institute of Virology, the facility’s proximity to the early outbreak,
and the operating procedures at the facility (Zeng et al., 2016). Transparency
and open scientific investigation will be essential to resolve this issue, noting
that forensic evidence of natural escape is currently lacking, and other
explanations remain reasonable
Reference: SARS-CoV-2: Combating Coronavirus Emergence
(Graham & Baric, 2020)
1. Do you agree with Ian Lipkin's recent emailed statement to El Pais?
"I am concerned that scientists at the Wuhan Institute of Virology may not have
exercised appropriate safety precautions in growing bat viruses. An example is
papers describing work done in BSL-2"
2. Why did you say in the Italian RAI interview in September 2020 ( at 38:00)
that SARS-COV-2 could have been assembled using 3 or 4 approaches/techniques
for coronaviruses without leaving a signature to distinguish it from a natural a
3. Why did you in the same interview that there was no proof that it wasn't from
lab with respect to the genomic data analysed so far, and that the answer to
whether it was leaked from a lab could only be found at WIV?
4. Why did you choose to reveal this information to an Italian TV station (RAI)
rather than to the US media? Did you have a goal for this?
5. On May 22, 2020 why did you add an author correction to your 2015 original
article “A SARS-like cluster of circulating bat coronaviruses shows potential for
human emergence.”
6. Why did you perform the following study?
"Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as a
highly transmissible pathogenic human Betacoronavirus”
“We recently developed stabilizing mutations for coronavirus spikes that prevent
the transition from the pre-fusion to post-fusion states. Here, we present cryo-EM
analyses of a stabilized trimeric SARS-CoV S, as well as the trypsin-cleaved,
stabilized S, and its interactions with ACE2”
“Neither binding to ACE2 nor cleavage by trypsin at the S1/S2 cleavage site impart
large conformational changes within stabilized SARS-CoV S or expose the
secondary cleavage site, S2′."
7. Why did you examine the effect of trypsinization on the stability of the
immunogen in vivo (mostly presented in muscle cells and immune cells....not the
GI tract where trypsin is abundant)?
8. Was this study geared towards understanding the stability of the prefusion
trimer of an S1/S2 cleaved SARS-like coronavirus (SL-CoV)?
9. Did this research aim to ensure that cleavage didn't alter conformation or lead
to S1 breaking off, so that a 2-proline modified stabilized prefusion protein would
be a good vaccine immunogen even if furin cleaved the S1/S2 site and the S1/S2
cleaved SARS protein still was stable in the desired conformation?
10. Was this research useful for developing a SARS-CoV-2 vaccine?
11. You declared that it is impossible that a Pangolin is the intermediate host for
SARS-COV-2, so how do you explain the close match between GX/GD Pangolin
Coronavirus RBDs and SARS-COV-2 RBD?
12. If SARS-COV-2 acquired part of its RBD via recombination with GX/GD
Pangolin Coronaviruses, is this more likely to have taken place "naturally" or via
contaminated cell lines in a laboratory setting?
13. Do you know if WIV had access to Guangxi Pangolin Coronavirus isolates or
Pangolin Samples before 2020?
14. What is your opinion of the involvement of the Beijing State Key Laboratory
of Pathogens and Biosecurity, Professors Yang Ruifu, Yigang Tong and Colonel Wu
Chan Cao in the Pangolin Coronavirus (GX and GD) papers and research?
15. Do you communicate or have you communicated in the past with the three
Chinese researchers mentioned above? If so, in what context?
16. Did you know that the State Key Laboratory of Pathogens and Biosecurity in
Beijing isolated Pangolin GX_P2 coronavirus series in 2017 and sent samples to
BSL2 Labs in China?
17. Do you have a copy of any WIV viral pathogen databases or access to them or
a copy of the 61.5 MB SQL zip folder containing one database? How about the
password protected section?
18. More generally, which experiment did you perform in collaboration with Dr
Shi during the US moratorium 2014-2017?
19. What exemptions did you ask to NIH/NIAID during the moratorium and
what was the motivation for requesting them?
20. Why were your research proposals not considered “GOFROC” by grant
agencies and why did they escape the attention of the ethics committees in charge
of GOF regulation?
21. How did you manage to continue your GOF research in agreement with the
attached document, 2016?
22. Have you ever been bitten by a humanised mouse?
23. What specific virus was carried by the mice that bit UNC researchers?"
24. What contacts have you had since 2018 with William Karesh, Jonathan
Epstein and Kevin Olival from EcoHealth Alliance?"
25. How many viral pathogens have you received via EcoHealth from foreign
26. What is the exact nature of your work with DTRA and DARPA?"
27. Do you have or have you had any communication with Fang Li?
28. Do you have or have you had any communication with Sina Bavari?
29. Do you have or have you had any communication with Jens Kuhn?
30. Do you have or have you had any communication with David Franz?
31. Do you have or have you had any communication with George Gao Fu?
32. How many post-doctoral students from China have studied in your
33. Have any of these students gone on to work at Chinese BSL Laboratories, such
as WIV?
34. Have you heard of the DRASTIC research collective or read any of their
published papers?
35. If so, do you agree with their proposal for an independent forensic
investigation of Wuhan Laboratories engaged in bat coronavirus sampling and
36. Did the WIV request any more BALB/C hACE 2 mice? If so, how many and
37. Does WIV use human lung xenograft mice in experiments?
38. Do you think the two unpublished live isolates (WIV6 and WIV15) may be
SARS-like viruses?
39. Why do you think WIV does not want to disclose the sequences of clade
40. Do you think WIV may have continued the experiments of Hu et al. (2017) of
recombinant viruses doing some passages in hACE2 transgenic mice?
41. Does the WIV have the knowledge and technology to obtain a result similar
to what SARS-CoV-2 has?
42. Is it difficult to obtain in the lab a virus as transmissible as SARS-CoV-2
starting from a bat virus?
43. Is it possible that SARS-COV-2 was made using synthesis of fragments and
cloning, for example using 4991, RaTG13 or 7896 Clade bat betacoronaviruses and a
pangolin GX/GD coronavirus RBD?
44. In such a scenario, how would a FCS Site be inserted or selected for in cell
45. Did WIV experiment with coronaviruses on non-human primate models at
WIV BSL3 or BSL4 before 2020? Were you involved in any of such research?
46. In your considered opinion, is RaTG13 partly an artificial laboratory construct
or a valid natural virus sequenced without manipulation from fecal swabs held by
the WIV?
47. Given the strong evidence of faulty biosafety issues involving Wuhan BSL
Laboratories and lack of PPE worn by Chinese bat researchers while sampling bats,
do you consider a laboratory leak of SARS-COV-2 via contaminated cell lines a
48. Do you know why UNC chose to withhold all your email records between
March20, 2019 and January 9, 2020 against the FOIA request?
49. Would you be willing to supply these email records for any investigation?
50. Would you be willing to ask UNC to reconsider their decision?
51. Was WIV involved in any research into a pan Betacoronavirus vaccine with
the Wuhan Institute of Biological Products pre-2020 (Disease X)?
Reference: Wuhan Institute of Biological Products Co
52. If so, were you consulted in any way about such a project (Disease X)?
53. Which two novel bat coronaviruses were researched by Ben Hu at WIV?
Reference: "Pathogenicity of 2 new bat SARS-related CoVs to transgenic mice
expressing human ACE2"
54. During your interview with the Italian TV program Presa Diretta you
mentioned that researchers often add short sequences to their viral mutants to
show that they were made in their labs. Are you aware of any of those signatures in
the SARS-COV-2 genome?
55. Could the sequence TGGTCGC which starts at nucleotide 1465 and deletes part
of the nsp2 protein in the ORF 1ab of SARS-COV-2 be a “signature” of genetic
56. Are you aware of any modification of the TRN (transcript regulatory network)
of SARS-COV-2 which might explain its genomic stability?
57. It has been suggested that the FCS of SARS-CoV-2 shares 19 nucleotides (1nt
before and 6nt after) with a Moderna patent sequence, and that this shared
sequence is not found in either mammals or viruses. Would you agree that this is a
misleading claim?
Reference: Sequence 11652 from patent US 9587003 GenBank: KH664781.1
Modified polynucleotides for the production of oncology-related proteins and
58. Would you also disagree that it is perfectly placed for a single template
switching recombination to paste the CTCCTCGGCGGGCACGTAG into the
SARS-CoV-2 genome?
59. Do you consider it significant that SARS-CoV-2 has evolved a unique S1/S2
cleavage site resulting in the striking mimicry of an identical FURIN-cleavable
peptide on the human epithelial sodium channel α-subunit (ENaC-α)?
60. Would you agree that the location of this SARS-CoV-2 mimicked peptide in the
ENaC-ɑ structure in the extracellular domain suggests that SARS-CoV-2 may have
specifically evolved to mimic a human protease substrate?
61. As ACE2 and ENaC-ɑ are expressed generally in the apical membranes of
polarized epithelial cells such as primary lung epithelial alveolar type II cells, do
you consider it significant that these cell lines were also used in chimeric bat virus
research at the Wuhan Institute of Virology?
62. Do you agree that similar polybasic cleavage sites to SARS--COV-2 also seem to
be present in feline viruses (FIPV) and that a recombination event between FIPV
and CoV in a feline intermediate host cannot be ruled out?
63. Could you briefly describe what makes a live-attenuated vaccine different from
an inactivated vaccine?
64. Why was a live-attenuated vaccine necessary for Yellow Fever?
65. What were the difficulties making a vaccine for Yellow Fever?
66. Could you describe the chimeric coronavirus work your team was doing at UNC
in 2015?
67. Is there any functional difference between that chimera and the chimera
created for attenuation while making the Yellow Fever live-attenuated vaccine?
68. If you weren’t making a chimera for vaccine attenuation, what was the goal of
your research at UNC in 2015?
69. Press reports indicate several possible viral exposures at UNC in 2015 and later,
is that correct?
70. The viruses involved in these leaks have never been disclosed, however since
they are described as not being a threat to human life – isn’t that the description
you would provide in the case of a vaccine-related variant that leaked?
71. Which elements of COVID-19’s emergence are not consistent with the
emergence of a vaccine-derived variant?
72. What is your scientific explanation for why SARS-CoV-2 elicits the Original
Antigenic Sin of the common cold, a virus that has nothing to do with bats, when
SARS-CoV-2 seems to have derived the majority of its genome from bats?
73. Does SARS-COV-2 show signs of attenuations in its genome which might hint
at it being in the process or being synthesized as live attenuated vaccine?"
74. Can you kindly explain (at least) 3 of (potentially) 7 sequence alterations at the
positions noted below in the SARS-COV-2 genome that show signs of cloning using
the "seamless method" on BglI sites to assemble the viral genome from 6-8
contiguous cDNA fragments?
75. Would you agree that WIV Researchers were capable of carrying out these
methods in the years leading up to 2019 in line with published papers and
unpublished theses? For example:
this study, we established a reverse genetics system for coronaviruses, and based
on the genomic backbone of WIV1, we established a scheme to replace the S gene
without traces, constructed infectious BAC clones of 12 S-gene chimeric
recombinant viruses, and successfully construct an S gene chimeric
recombinant viral infectious BAC clone with WIV1 as the backbone and without
leaving any trace sequences (e.g. incorporated enzymatic sites) in the recombinant
viral genome
Reference: "Reverse Genetic System of Bat SARS-like Coronaviruses and Function
of ORFX" by Lei-Ping Zeng (2017)
Figure 3.1 Scheme for constructing S-gene chimeric recombinant viruses (Hu Ben
et al., unpublished results)
Reference: "Reverse Genetic System of Bat SARS-like Coronaviruses and Function
of ORFX" by Lei-Ping Zeng (2017)
Also published in Hu et al (2017): "Discovery of a rich gene pool of bat SARS-
related coronaviruses provides new insights into the origin of SARS coronavirus"
76. Would you agree that the following background summaries are accurate?
Bat CoVs have been assembled from viral nucleic acid fragments in the
past by WIV, where replication capable viruses could not be isolated
(from faeces, BALF, anal swabs etc.) Most CoV had been "recovered" by
reverse transcribing viral RNA fragments into cDNA, amplification and
cloning into vectors (concomitant sequencing by Sanger - later NGS).
Complete viral genomes were re-assembled using "seamless“ or "no see’m“
cloning technology originally developed by you at UNC, based on taking
6-8 contiguous cDNA strands (covering the complete virus genome) and
ligating them together using adapters at the 5’ and 3’ ends, with restriction
enzyme digestion, ligation and transfection into permissive cell lines (such
as Vero E6). Infectious clones were thus recovered and passaged, and since
the overlaps of the adapters are cut off by the restriction enzymes, the
exact cloning sites of the ligated construct can normally not be seen after
sequencing of the virus genome (no see’m cloning).
However, sometimes it is necessary to first modify the 5’ and 3’ ends in
order to generate a suitable restriction site. Therefore just a few bp (in the
cDNA) need to be changed by SDM (site directed mutagenesis) by
preserving the amino acid sequence of the ligated virus. This is done by
modifying only the wobble bases (introducing synonymous mutations) at
the respective cloning sites. The same technology can be used to swap
genes, e.g. exchange the spike proteins in a viral backbone. WIV has used
exactly this technology (described in a recently discovered 2018 doctoral
thesis) to "recover“ the Bat CoV named WIV1 (and possibly many more
Source: Strategy for construction of an infectious WIV1 BAC clone
Bat Severe Acute Respiratory Syndrome-Like Coronavirus WIV1 Encodes an Extra
Accessory Protein, ORFX, Involved in Modulation of the Host Immune Response
(Zeng et al, 2016)
77. Would you also agree that the following analysis is accurate?
8 contiguous cDNA fragments of the virus were ligated together using
predominantly BglI restriction sites that were generated by SDM. In the spike gene
(S1-domain) a short 12 nt sequence had to be modified in two bases: TGT CCT TTT
GGA had to be modified into TGC CCT TTT GGC, and both translate into the aa
sequence C P F G, since only the wobble bases (3rd positions of the codon) were
modified. The result is a perfect BglI palindromic site for joining the two fragments
(named E and F) 5’ - GCCNNNNNGGC - 3’ using your UNC based cloning
78. Do you agree that it is curious that we can observe exactly this BglI site starting
at position 22.568 in all SARS-COV-2 genomes, but not in (natural) SARS-1
79. Would you agree that from a virus evolution perspective GGC is not an optimal
codon (immunogenic), so that after passage it results in a codon optimized GGT,
when there is no other fitness advantage to the virus?
80. Would you agree that in the second of the two mutations, instead of the
consensus codon GGA there is either GGC or GGT and that we can observe GGC in
only a few virus isolates instead of GGT?
81. Although a natural evolution of the TGC CCT TTT GGC sequence in spike
cannot be ruled out entirely, would you agree that it is rather unlikely because it
would create additional GpC content, which is a codon deoptimization?
82. Do you think it makes sense for a virus to acquire additional CpG/GpC unless it
leads to a fitness advantage (e.g. increased tropism of the polybasic cleavage site
FCS in spike)?
82. Finally, would you agree that if the modified CPNG site had a fitness advantage,
this would be observed in other bat SARS-CoVs?
83. Can you explain why the above has not been observed in other bat SARS-CoVs?
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