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DRASTIC - An Analysis of Project DEFUSE

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An Analysis of the Project DEFUSE proposal submitted by EcoHealth Alliance [EHA] to the Defense Advanced Research Projects Agency [DARPA] on 3/27/2018; DARPA rejected the proposal, ultimately choosing 5 other groups for its PREEMPT initiative. The proposal describes, in great detail, experiments which could have directly resulted in the SARS-CoV-2 virus that emerged in Wuhan in the fall of 2019; elements of particular concern include the introduction of human proteolytic cleavage sites into SARS-like coronaviruses, the expressed interest in identifying viral strains which could exploit the DC-SIGN pathway, introduction of "key RBD residues" to test ACE2 affinity in humanized mice, and targeted "boosting" [suppression] of bat interferon responses. Given that the initial wild-type SARS-Cov-2 genome contained a Furin Cleavage Site, high hACE2 affinity, DC-SIGN receptors and several ORF components which degrade interferon responses, the existence of the DEFUSE proposal documents raises important questions about the origin of the SARS-CoV-2 virus and the COVID-19 pandemic.
Project DEFUSE
DARPA - PREEMPT (HR001118S0017)
Documents made available by an anonymous source
EcoHealth Alliance (EHA) Tried To Bypass The P3CO/DURC Frameworks 7
EHA Would Have Used US Taxpayer Money To Pay Peng Zhou At Half Time And Shi Zhengli And Ben Hu At
Quarter Time 8
EHA Would Have Invited Shi Zheng Li To A Project Kickoff At DARPA Headquarters 8
EHA ‘had’ 3 Key Cave Sites In Yunnan For SARS-r CoV Collection 8
EHA Planned To Inoculate Wild Bats With Aerosolized Vaccines 9
The Proposal Does Not Properly Discuss Ethical, Legal And Social Issue 10
EHA Wanted To Oversee All Work In China 10
Live Bats Were Meant To Be Used At The WIV And Various International Labs For Infection Experiments, Often
Using Captive Bat Colonies 10
EHA Proposed Multiple, Regular Visits To 3 Yunnan Cave Sites 12
EHA Planned To Send Samples To Duke University (Singapore) And UNC Chapel Hill 12
The Proposal Set A Clear Pathway For Chimeric Virus Construction 13
EHA Has 180 Unpublished SARS-r-CoV Strains 13
All Coronaviruses Were To Be Screened At The WIV 14
Three To Five Chimeric Coronaviruses Were To Be Created Per Year 14
The Proposal Planned To Identify “Key Minor Deletions” In The Receptor Binding Domain (RBD) To Alter Human
Pathogenicity 14
The Proposal Includes The Introduction Of “Human-specific Cleavage Sites” 14
The Proposal Planned To “Introduce” Naturally Occurring Proteolytic Cleavage Sites To Create Novel
Coronaviruses 16
The Proposal Planned To Research Alternate Receptors To ACE2 16
The Proposal Planned To Introduce “Key RBD Residues” Into Low Risk Strains To Test Pathogenicity In Human
Airway-cells And In hACE2 Mice 16
The Proposal “Spillover Probability” Assessment Does Not Include Lab-research Related Risks 17
A Potentially Highly Important “Spike Protein Dataset” Was Not Public 17
EHA Proposed MERS-coronavirus Experiments And Had Already Introduced SARS And MERS Into Bat Cell
Lines 18
EHA Proposed A Database Of All Field, Lab And Modelling Work 18
EHA Proposed Industrial Scale Bat Sampling 19
EHA Misled DARPA About Risks To General Public 19
EHA Proposed To Generate “Batified Mouse Models” 19
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These leaked documents describing bat research proposed by EcoHealth Alliance should be
considered in light of the following context:
On August 27th, 2021, the US intelligence community issued a 502-word summary of the conclusions
drawn up by the joint investigation ordered by President Biden in late May. Conspicuously absent
from the brief statement were any indications that the evidence presented in testimony to Congress
had been part of the intelligence community analysis - at least not in the unclassified version that
was released.
The lifting of the gain-of-function (GOF) moratorium in late 2017, via the Potential Pandemic
Pathogen Care and Oversight framework (P3CO), has allowed GOF research with SARS-like
coronaviruses to resume with very few practical limits. In particular the absence of clear definitions of
GoF, creative interpretations of the guidelines, and rather discretionary decisions to refer research
projects or not, all contributed to reducing the effectiveness of the P3CO framework - despite the fact
that other agencies of the US federal government actively maintained the GOF standards.
DRASTIC recently became aware of documents which show that EcoHealth Alliance (EHA) in
concert wIth the WIV were looking towards implementing an advanced human pathogenicity BatCoV
research project that clearly qualifies as GoF, in a grant proposal submitted to a funding proposal call
by the Defense Advanced Research Projects Agency (DARPA) in the spring of 2018. The EHA / WIV
proposal (named ‘DEFUSE’) was ultimately rejected for full funding (but leaving open the door for
partial funding), in part because it skirted the GOF guidelines.
In other words, a branch of the federal government had already judged aspects of EHA’s research,
and the corresponding shared research plan with the WIV, as falling under the definition of GOF, only
for HHS to approve similar work without P3CO review in 2018 and 2019. In particular, the P3CO
framework was designed to allow greater flexibility for vaccine development, and in June of 2018 the
NIH’s Vaccine Research Center (VRC) expanded its existing partnership with Moderna to include
full-scale research into a pan-coronavirus (CoV) vaccine platform. EcoHealth Alliance repeatedly
took advantage of this flexibility to continue their work with the Wuhan Institute of Virology
DRASTIC has reviewed the contents of these documents. They detail past achievements and
planned experiments in collaboration with researchers from the Wuhan Institute of Virology (WIV),
East China Normal University (ECNU), UNC-Chapel Hill, Duke-National University in Singapore, the
USGS National Wildlife Health Center (NWHC) and Palo Alto Research Center (PARC).
The grant proposal includes some elements of research that are already public via scientific papers,
as well as other elements that have never been made public; these include vaccinating wild bats
using aerosolized recombinant SARSr CoV spike proteins in nanoparticles or in orthopoxviral
vectors, and further work on published and unpublished CoV strains that could fill the extant gaps in
our understanding of the origins of SARS-CoV-2.
These grant proposal documents also show a staggeringly deep level of involvement of EHA with
the WIV, on matters of national interest (such as DURC), for instance by proposing that the DARPA
grant pays a good chunk of key WIV researchers salaries, or that some of these WIV researchers
should be invited to DARPA headquarters in Arlington. All the while without proper risk assessment
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and considerations for ethical and social issues and with an incorrect evaluation of what constitutes
GoF research.
DARPA PREEMPT program (HR00111880017):
- Brief introduction’:
- Press release: (archived version)
- Grant Opportunity:
- Copies of some of the main Grant Opportunity files:
- Description of the selected teams:
The teams are led by (1) Autonomous Therapeutics, Inc., (2) Institut Pasteur, (3) Montana
State University, (4) The Pirbright Institute and (5) The University of California, Davis.
Most of the teams are made of US, UK or Australian partners, plus one partner in Estonia
(Tartu) and the Institut Pasteur network in Asia. None of the selected teams include Chinese
- Examples of funded PREEMPT projects:
$9.37mln award for UCDavis team PREEMPT project
Montana State University team PREEMPT project.
-D1: 75 page ‘PROPOSAL: Volume I’ - link
-D2: 8-page budget - link
-D3: Summary of Rejection Letter (DARPA) - link
-D4: Executive Slides - link
Note: page numbers are given as the nth page in the corresponding PDF document. Hence (D1,
p.10) means 10th PDF page of document D1.
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2014 May 27 Award to EHA of NIH R01Al110964 grant (‘‘Understanding the Risk of Bat
Coronavirus Emergence’’) for 5 years (June 2014 - May 2019) - with subgrants to
WIV, East China Normal University (Shanghai) and (starting year 3) Wuhan
WIV: ~$695,000
East China N. Uni: ~$259,000
Wuhan University: ~$442,000
(all direct costs over 5 years, see pages 5 and 189 of grant)
Early 2017: China sets up its own version of DARPA, under the Central Military Commission.
2018 Jan 4 Launch of DARPA Preventing Emerging Pathogenic Threats (PREEMPT)
2018 Jan 19 Notice (BAA) for proposals for PREEMPT Program HR001118S0017, with a span
of 3.5 years (Dec 18 - May 22).
2018 Jan 19 Cables from U.S. Embassy health and science officials about lack of trained
personnel as the WIV P4 and the potential danger of newly found BatCoVs that
can directly infect humans.
2018 Mar 27 EHA fills a proposal for DARPA PREEMPT project under HR001118S0017 by
stated deadline: Project DEFUSE (Defusing the Threat of Bat-Bome
Coronaviruses) for a requested amount of $14,209,245 (over 3.5 years).
2018 ?? Rejection of EHA project DEFUSE due to important concerns
2018 Nov Application for renewal of NIH R01Al110964 grant ‘‘Understanding the Risk of Bat
Coronavirus Emergence’ (p. 316 of grant) - with subgrants to WIV (inc. Wuhan
University work), Institute of Pathogen Biology (IPB, Beijing), East China Normal
University (Shanghai)
WIV: ~$353,000
IPB: ~$350,000
East China N. Uni: ~$368,000
(all direct costs over 5 years - in particular see p. 420 for East China Normal
University via the consultancy of Dr Guangjian Zhu)
2019 Jul 24 Fast renewal of NIH R01Al110964 grant grant for 5 years (June 2019 - May 2024)
2020 Apr 24 Suspension of NIH R01Al110964 grant
2020 Jul 8 Reinstantiation of NIH R01Al110964 grant - but all activities are suspended until
NIH conditions are satisfied (see also p 320 of grant).
2020 Aug 20 New 5-year grant for ‘Understanding Risk of Zoonotic Virus Emergence in EID
Hotspots of Southeast Asia’ (excluding China
1As noted in the Reinstantation letter sub-wards were not (and are likely still not) correctly reported
by EHA in the Federal Subaward Reporting System. The numbers above are compiled from a
careful reading of the grant documents recently disclosed via an FOI request.
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EHA confidently assessed in its proposal that the work to be carried was neither subject to P3CO
(GoF) nor DURC (Dual Use Research of Concern) restrictions:
“These QSo strain viral spike glycoproteins will be synthesized, and those binding to human
cell receptor ACE2 will be inserted into SARSr-CoV backbones (non-DURC, non-GoF).” (D1,
The paragraph above actually contains the only mentions of GoF and DURC in the whole DEFUSE
project proposal - and it dismisses them.
Nevertheless the DARPA review of the DEFUSE project concluded that the project potentially
involved GoF. This was part of the reasons for the rejection of the project as such, and of a
qualification for any partial funding:
“Given the team's approach does potentially involve GoF/DURC research (they aim to
synthesize spike glycoproteins that may bind to human cell receptors and insert them into
SARSr-CoV backbones to assess capacity to cause SARS-like disease), if selected for
funding an appropriate DURC risk mitigation plan should be incorporated into contracting
language that includes a responsible communications plan”
Effectively EHA unsuccessfully proposed the use of bat-SARSr-CoV backbones and not the human
evolved SARS-CoV in what looks like a deliberate attempt at circumnavigating the restrictions of the
P3CO framework and related DURC restrictions.
“An enhanced PPP is a PPP resulting from the enhancement of a pathogen’s transmissibility
and/or virulence. Wild-type pathogens that are circulating in or have January 9, 2017 2 been
recovered from nature are not enhanced PPPs, regardless of their pandemic potential.
We also know from emails FOI’d by USRTK that Jonathan Epstein asked on the 23rd March for help
from Ralph Baric for some wording for a section in the proposal that would address ‘communicating
dual-use information’:
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That email request for help was sent 4 days before the deadline for submitting the proposals (27th
Mar 2018), which suggests that little attention was being paid to this question. Not surprisingly, the
review of the proposal effectively concluded than no satisfying DURC language had been included:
“.. if selected for funding an appropriate DURC risk mitigation plan should be incorporated
into contracting language that includes a responsible communications plan”
EHA DEFUSE proposed to have Peng Zhou work half time (8h/day, 22d/month) for the two first
years on the project, with Shi Zheng Li and Ben Hu as 1/4th of their time, all drawing salaries through
the DARPA grant (D2, p.8).
We note that, by contrast, the previous grant NIH R01Al110964 did not provide for a salary to Shi
Zheng Li. See p. 71 of the previous grant): “Dr. Shi will not take salary on this grant and is funded by
discretionary sources at her Institute.”
As per D2, top of page 4, the budget of EHA DEFUSE contains an entry for an invitation of Shi
Zheng Li and one key WIV personnel (likely Peng Zhou or Ben Hu) to a kickoff meeting at the
DARPA headquarters in Arlington, VA.
Three caves in Yunnan Province are specified as of particular importance:
“Our strategy begins by a complete inventory of bats and their SARSr-CoVs at our
intervention test site cave complex in Yunnan, China that harbours bats with high-risk
SARSr-CoVs. We will collect data from three caves in that system (one is our intervention
test site and two control sites) on: monthly bat abundance and diversity, viral prevalence and
diversity, individual bat viral load and host physiological markers; and genomic
characterization of low- and high-risk SARSr-CoV strains among bat species, sexes, and age
classes; satellite telemetry and mark-recapture data on bat home range and inter-cave
movement; and monitoring of daily, weekly and seasonal changes in bat populations.” (D1,
“However, our test cave site in Yunnan Province, harbours a quasispecies (QS)
population assemblage that contains all the genetic components of epidemic SARS-CoV34,
We have isolated three strains there (WIV1, WIV16 and SHCO14) that unlike other
SARSr-CoVs, do not contain two deletions in the receptor-binding domain (RBD) of the
spike, have far higher sequence identity to SARS-CoV (Fig. 1), use human ACE2 receptor
for cell entry, as SARS-CoV does (Fig. 2), and replicate efficiently in various animal and
human cells.” (D1, p.7-8)
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The proposal for wide scale inoculation of bats in the wild using aerosolized inoculum delivery
has never been publicly released or opened to the wider scientific community for discussion as to
potential risks associated with this plan.
This is a specialist area of research of Dr Rocke, Dr. Ainslie and Dr. Unidad (PARC) who have
previously researched and developed the technological solutions necessary to make this possible:
Dr. Jerome Unidad is a researcher PARC, (2018a) at PARC (owned by Xerox) (PARC, 2018d), who
developed the Filament Extension Atomizer (FEA) (PARC, 2019). This technology is used to spray
bats with scalable high viscosity mists that stick to their skin or that are edible (PARC, 2018c).
PARC previously partnered with NHWC to develop a vaccine for White Nose Syndrome (WNS) for
US bats, using FEA as the technological solution to administer vaccines via aerosol delivery (PARC,
Dr. Tonie Rocke is a researcher at USGS National Wildlife Health Centre (NWHC in the DEFUSE
proposal). She has previously worked on transdermal application of vaccines against rabies in
vampire bats ``The feasibility of controlling rabies in vampire bats through topical application of
vaccines (USFWS, 2019), also Infectivity of attenuated poxvirus vaccine vectors and
immunogenicity of a raccoon pox vectored rabies vaccine in the Brazilian Free-tailed bat (Stading et
al., 2016). There were doubts and concerns about her work:
These vaccine candidates use a viral vector (attenuated raccoon poxvirus, RCN) genetically
modified to express highly-conserved fungal and specific Pd antigens. While these vaccines
and other potential treatments continue to be developed, there is a need for safe and
effective methods of treatment delivery” (USFWS, 2019).
Another similar project:
We recently developed a new recombinant rabies vaccine specifically for bats with available
sequences from the rabies Phylogroup I glycoprotein. This sequence was cloned into
raccoon pox virus (RCN) and the efficacy of this novel RCN-MoG vaccine was tested in big
brown bats. Field studies are currently being conducted in Peru and Mexico to test the
feasibility of oral and topical delivery of vaccine and transfer rates between vampire bats
using biomarker-labelled jelly (without vaccine)” EEFMVZ (2021).
Dr. Ainslie is a Professor at the UNC Department of Biomedical Engineering and the UNC
Department of Microbiology and Immunology (Pharmacy UNC, 2021), who works on new polymers
for vaccines and electrospray for fabrication of immune targeting microparticles (nanoparticles).
Her publications include Historical Perspective of Clinical Nano and Microparticle Formulations for
Delivery of Therapeutics'' (Batty et al., 2021), Electrospray for generation of drug delivery and
vaccine particles applied in vitro and in vivo(Steipel et .,2019). Injectable, Ribbon-Like
Microconfetti Biopolymer Platform for Vaccine APPLICATIONS'' (Moore et al., 2020),
Considerations for Size, Surface Charge, Polymer Degradation, CoDelivery, and Manufacturability
in the Development of Polymeric Particle Vaccines for Infectious Diseases” (Genito et al.,2020).
One may contrast this fairly aggressive approach with the one described in a recent paper on
Self-disseminating vaccines to suppress zoonoses’. There the recommended approach is to start
with captive animals then carefully “perform releases within carefully isolated populations in
semi-natural enclosures or on small islands”. More generally concerns have been raised about such
self-disseminating vaccines.
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The proposal has about 22 lines on Ethical, Legal and Social Issues (ELSI), most rather vague. Or
even rather odd, such as when mentioning ‘common practice of bat-consumption’ in Yunnan when
bat-consumption is actually not common at all in Yunnan (if it ever occurs), with also a mention of
‘cultural leaders’, which may suggest some hasty editing based on a the text for a similar project in
South-East Asia:
“We will conduct educational outreach to local wildlife authorities and cultural leaders so that
there is a public understanding of what we are doing and why we are doing it, particularly
because of the common practice of bat-consumption in the region.” (D1, p,36)
Also worth noting is the mention that ‘The broader societal impact of this project could be significant,
as wildlife immunization against viral zoonoses has been limited to date’ without further proper
consideration (D1, p.36).
The ‘PREEMPT Risk Mitigation Plan’ section seems to suffer from another bad case of hasty editing
with a minimalistic 2-line ‘Risks to the general public section’ interrupted in mid-air:
Full extent of ‘Risk to general public’ section (D1, p.34)
The PREEMPT proposal to DARPA relied on trusting EHA (a private NGO) for oversight of high risk
pathogen research:
“The lead organisation, EcoHealth, Alliance will oversee all work.” (D1, p.3)
“Dr. Shi, Wuhan Institute of Virology will conduct viral testing on all collected samples,
binding assays and some humanized mouse work.” (D1, p.3)
WIV (Shi) was to work on Rhinolophus bats:
“At WIV, 20 adult wild Rhinolophus spp. bats (10 of each sex) will be captured at our test
cave site, housed within ABSL3, ACE2 receptor genes sequenced and used to pre-screen
spikes as above, then bats will be tested using PCR and serology for current and prior
exposure to SARSr-CoVs, and inoculated with WIV1, WIV16 or SHC014.” (D1, p.20).
“to Dr. Shi, Wuhan Inst. Virol., to conduct PCR testing, viral discovery and isolation from bat
samples collected in China, spike protein binding.assays, humanized mouse work, and
experimental trials on Rhinolophus bats.” (D1, p.25).
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“Subtask 7.5 Test targeted immune boosting in wild-caught captive Rhinolophus spp: (WIV).”
(D1, p.30).
The WIV was not the only institution meant to work with live bats for infection experiments within its
labs. As the proposal explains:
“Experimental work using bats and or transgenic mice will be conducted at the BSL-3 lab in
WIV, Duke-NUS, UNC, or NWHC. Each partner institute will apply for and procure animal
research approval from its respective IACUC. All anima! work conducted by EcoHealth
Alliance in China will be overseen by both the IACUC at WIV and the IACUC at Tufts” (D1,
Duke-NUS (Linfa Wang) has an Asian cave bat (Eonycteris spelaea) breeding colony:
“Our E. spelaea colony has now reached a sustainable population for infection experiments
and the ABSL3 facility has been outfitted with bat-specific cages.” (D1, p.20).
We will use the unique Duke-NUS Asian cave bat (Eonycteris spelaea) breeding colony to
conduct initial proof-of-concept tests, extended to small groups of wild-caught Rhinolophus
sinicus bats at WIV.” (D1, p.6-7)
Subtask 7.4.Test immune modulation in ‘captive Eonycteris sp. colony, using Malaka virus
and SARSr-CoV infections. (Duke-NUS).” (D1, p.30)
NWHC (Rocke) has a captive bat colony colony:
“We will use the NWHC captive bat colony and wild bats in US caves to trial delivery vehicles
using the biomarker rhodamine B (which fluorescently marks hair on consumption) to assess
uptake.” (D1, p.7)
CSIRO (Australia, with Linfa Wang at the time) and University of Queensland were already using
or are planning to be using live bats for experiments
“Previous infection studies were completed in Pteropus and Rhinolophus bats in Australia
by L-F Wang at CSIRO, AAHL and an additional Pteropus infection trial is currently planned
through the University of Queensland in Australia.” (D1, p.20)
As DRASTIC has previously discovered, the WIV was already keeping wild-caught bats more than a
decade ago, and established a colony around 2017 - as has been attested by patents, official
records and videos (see These revelations have been regularly characterised
as ‘conspiracy theories’ by Peter Daszak (see examples in Bostickson & Ghannam, 2021c; Taiwan
News, 2021; Sky News, 2021).
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Screenshot: Peter Daszak Denies WIV keeps
Live Bats. Source: Twitter via Taiwan News
Screenshot: Peter Daszak Denies WIV keeps
Live Bats. Source: Twitter via Taiwan News
In phase I will sample 60 bats each of R. sinicus, R. ferrumequinum, and R. affinis, (180
bats per cave) every three months non-destructively for 18 months from our three cave
sites.“ (D1, p.9)
“We will conduct pre- and post-intervention sampling (biweekly faecal pellet sampling for 4
months, and 10 male and 10 female bats per species tested every 2 weeks post-intervention
for 4 months, prior to- and post-deployment) to monitor SARSr-CoV QS and bat immune
status changes in test and control site bats during Phase I (TA2).” (D1, p.9)
The proposal states that:
Samples will be preserved in viral transport medium, immediately frozen in liquid nitrogen
dry shippers, and transported to partner laboratories with a maintained cold chain and under
strict biosafety protocols.” (D1, p.9)
This is further confirmed by items 37 and 38 on page 5 of D2 (Budget).
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Incidentally we also know from the recently released documents for the NIH R01Al110964 grant
(‘‘Understanding the Risk of Bat Coronavirus Emergence’’) that EHA has plenty of experience
shipping samples from and to China.
grant, p. 15:
This gives us unique access to working on-the- ground in countries where surveillance is
difficult, such as China, where our group has proven capacity to export samples from.
p. 141:
“Drs Shi, Zhang, and Daszak have collaborated together since 2002 and have been involved
in running joint conferences, and shipping samples into and out of China.
This practically means that it is likely that Duke (Singapore) and UNC Chapel Hill have
undocumented samples that could help trace the origins of SARS-CoV-2.
The use of known backbones is specified in the proposal:
Synthesis of Chimeric Novel SARSr-CoV QS: We will commercially synthesize SARSr-CoV
S glycoprotein genes, designed for insertion into SHC014 or WIV16 molecular clone
backbones (88% and 97% S-protein identity to epidemic SARS-Urbani). These are BSL-3,
not select agents or subject to P3CO (they use bat SARSr-CoV backbones which are
exempt) and are pathogenic to hACE2 transgenic mice. (D1, p.9)
However we do not know what additional, unpublished SARS-r CoV and MERS-r CoV research was
conducted by the WIV, Wuhan University and other Chinese institutions. Indeed, using analysis of
raw metagenomic datasets, unpublished MERS-r CoV infectious clone research in Wuhan has
recently been documented (Zhang et al. 2021).
This will be supplemented by characterization of isolated viruses under DEFUSE (at WIV),
approximately 15-20 bat SARSr-CoV spike proteins/year (at UNC, WIV), and >180 bat
SARSr-CoV strains sequenced in our prior work and not yet examined for spillover potential.
(D1, p.12)
Very little of this planned work has been published.
2As per budget (D2, p.5, item 13) the primer synthesis was to be done by Sangon Biological Engineering
Technology & Services (Shanghai).
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We will conduct in vitro pseudovirus binding assays, using established techniques2, and live
virus binding assays (at WIV to prevent delays and unnecessary dissemination of viral
cultures) for isolated strains.” (D1, p.12)
We will validate results from chimeric viruses by re-characterizing full-length genome
versions, testing whether backbone genome sequence alters full length SARSr-CoV spillover
potential. QS for full-genome characterization will be selected to reflect strain differences in
antigenicity, receptor usage, growth in human cells and pathogenesis.” (D1, p.13)
“We will test growth in primary HAE cultures and in vivo in hACE2 transgenic mice. We
anticipate recovering ~3-5 full length genome viruses/year. (D1, p.13)
“Testing Synthetic Modifications:
“We will synthesize QS with novel combinations of mutations to determine the effects of
specific genetic traits and the jump potential of future and unknown recombinants.
RBD deletions:
Small deletions at specific sites in the SARSr-CoV RBD alter risk of human infection. We will
analyze the functional consequences of these RBD deletions on SARSr-CoV hACE2
receptor usage, growth in HAE cultures and in vivo pathogenesis.”
(D1, p.13)
Human protease-specific site insertion was proposed. The proposal does not specify exactly which
protease, but does discuss Furin in the preceding text.
We will analyze all SARSr-CoV S gene sequences for appropriately conserved proteolytic
cleavage sites in S2 and for the presence of potential Furin cleavage sites74,75”.
SARSr-CoV S with mismatches in proteolytic cleavage sites can be activated by exogenous
Trypsin or Cathepsin L.
Where clear mismatches occur, we will introduce appropriate human-specific cleavage sites
and evaluate growth potential in Vero cells and HAE cultures.
(D1, p.13)
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Furin recognition cleavage motifs are widely used in laboratory research. Furin is an endoprotease
which cleaves proteins at a specific motif (RxxR|x) which for virus envelope glycoproteins, can
enhance viral fusion with host cell membranes (Coutard et al., 2020).
For SARS-CoV-2 the Furin cleavage site (FCS) has been shown to be key for pathogenicity (Bestle
et al. 2020; Hoffmann et al. 2020; Johnson et al., 2021).
No other sarbecovirus subgenus CoV including SARS-CoV possesses a Furin cleavage site, and as
Furin cleavage sites have previously be inserted into coronaviruses in laboratories to increase
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tropism and pathogenicity (Cheng et al. 2019), the origin of the FCS has been widely debated (Wade
2021). Wu and Zhao (2021) propose the FCS arose through natural insertion.
Segreto and Deigin (2020) note the “CGGCGG” coding for the two leading arginines is rare for bat
origin coronaviruses and note a FauI restriction site just upstream of a leading proline and propose
the fcs could have been inserted in a laboratory.
Kaina (2021) proposes that in vitro recombination in human cell culture of a SARS-CoV-2 progenitor
with a virus containing the Furin cleavage site as a possible source; Segreto et al. (2021) propose
laboratory insertion of a more potent motif and insect cell culture passage to generate the “RRAR”
FCS sequence.
Given that we find in this EHA proposal, a discussion of the planned introduction of human-specific
cleavage sites into novel SARS-r CoVs, a review by the wider scientific community of the plausibility
of artificial insertion of an FCS into SARS-CoV-2 or a progenitor is warranted.
The proposal planned to introduce ‘wild type’ proteolytic cleavage sites from high risk strains into
more abundant low risk strains, presumably to increase the pathogenicity of the low risk strains:
We will also review deep sequence data for low abundant high risk SARSr-CoV that encode
functional proteolytic cleavage sites, and if so, introduce these changes into the appropriate
high abundant, low risk parental strain.” (D1, p.13)
To evaluate this, we will sequentially introduce clade 2 disrupting residues of SARS-CoV
and SHCO14 and evaluate virus growth in Vero cells, non-permissive cells ectopically
expressing DC-SIGN, and in human monocytes and macrophages anticipating reduced virus
growth efficiency.“ (D1, p.13)
We note that while SARS-CoV was documented to use DC-SIGN as an attachment receptor (Marzi
et al. 2004), L-SIGN and DC-SIGN act as entry receptors for SARS-CoV-2 (Amraei et al. 2020;
Thépaut et al. 2021).
Low abundance micro-variations:
We will structurally model and identify highly variable residue changes in the SARSr-CoV S
RBD, use commercial gene blocks to introduce these changes singly and in combination into
the S glycoprotein gene of the low risk, parental strain and test ACE2 receptor usage, growth
in HAE and in-vivo pathogenesis”.
(D1, p.13)
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Figure. 7 below (D1, p.14) documents an extensive sampling, and lab experimentation plan. It is
quite astounding that the spillover probability calculation incorporates multiple ‘processes
contributing to spillover’, all of which require extraction of viruses from bats and experimentation in
laboratories, yet does not incorporate a risk factor for field sampling and laboratory experimentation
to deliberately increase the pathogenicity of SARS-r CoVs.
It is not clear what that ‘dataset of S protein sequences from prior work’ refers to and whether EHA
has ever made it public (as is generally required under its grant conditions).
We will use a large dataset of S protein sequences and full-length genomes generated from
prior work and DEFUSE fieldwork to estimate SARSr-CoV substitution rate and its
genome-wide variation.” (D1, p.15)
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First, we will take wing punch biopsies from 3 individuals to sequence their ACE2 receptor
gene. This will be inserted into human cell lines to pre-screen viral strains for binding. Those
that bind will be used for in vivo experiments. We will use two coronaviruses (SARSr-CoV
WIV1 and MERS-CoV) in ABSL3. SARS and MERS infection studies are already underway
in Eonycteris and Pteropus cell lines and primary immune cells .“ (D1, p.20)
Primary cells can also harbour latent viruses that can become reactivated during in vitro cultivation
when the cells are outside the host and isolated from other components of the immune system that
would otherwise control virus replication” Banerjee et al. (2018).
In fact, experiments using non-immune bat cell lines were hallmarked by “subversion of the bat
immune system” which contrasts with the effective clearance of bat viruses shown by in vivo captive
bat studies into Marburg and Ebola Viruses carried out at Atlanta CDC BSL4, for example (Jones et
al., 2015; Schuh et al., 2017a & 2017b).
Research into bat immune systems using bat cell lines at WIV however did not mirror results from in
vivo studies, and it was precisely this feature of non-immune bat cell lines that led researchers to
create the first bat bone marrow-derived dendritic immune cells (Zhou et al., 2016), bat-mouse bone
marrow chimera (Yong et al., 2018) and IFNAR2 knockout bat cell lines using CRISPR/Cas9
technology (Zhang et al., 2017).
These novel cell lines and bat immune system mice were proposed to ensure that in vivo bat cell line
and in vivo mouse experiments effectively mirrored in vivo bat immune system response to clearing
of viruses (Zhou et al., 2016; Yong et al., 2018).
However, this also suggests that unknown or undetected highly pathogenic bat viruses would have
been able to replicate clandestinely in WIV bat cell lines as they would not have been constrained by
a bat immune system, and in turn, this may have led to contamination of operators and equipment at
the WIV BSL2 Laboratories (Bostickson & Ghannam, 2021c).
“Data Management and Sharing:
EcoHealth Alliance will maintain a central database of data collected and generated via all
project field, laboratory, and modelling work.” (D1, p.25)
This would indicate that EHA has similar databases relating to earlier projects - data that it would
have not shared publicly.
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“Sub-Task 1.2
Collect monthly specimens from bats at cave sites in Yunnan, China for SARSr-CoV
screening and sequencing. Oral, fecal, and blood sample collected from 360 Rhinolophus
spp. bats per month using live- capture and non-invasive sampling. Specimens shipped to
laboratory for analysis. Associated morphological, demographic, and physiological data for
individual bats collected (EHA, consultant Zhu).” (D1, p.25)
Specimens from 3,240 bats and fecal pellets collected from high-risk reservoir
populations which have been obtained with all proper permits and permissions and shipped
to WIV for analysis; real-time telemetry and mark-recapture data uploaded and made
available to DARPA collaborators; completed database maintained.” (D1, p.25)
EHA writes about ‘Risks to general public’ section:
Risks to general public:
The proposed work has minimal risk to the general public, as sampling will be done near the
cave sites and not in populous areas. Our team has extensive experience.
(D1, p.35)
That EHA could propose the identification and selection of highest risk SARS-r CoV’s, chimeric CoV
construction, serial passage using transgenic hACE2 mice and insertion of human adapted cleavage
sites (presumably furin cleavage sites), yet totally ignore the risks of laboratory escape is
inconceivable and shows a total lack of understanding of the risks of SARS-r CoV (and MERS-r
CoV) laboratory research (Demaneuf 2020).
We have shown efficient reconstitution of irradiated mice using bat bone marrow from
multiple species, including E. spelaea (Fig. 10), including reconstitution of bat PBMC’s in
the mouse, presence of circulating bat cells and generation of bat-specific antibodies in mice
incapable of producing an antibody response.
This ‘batified’ mouse model can be utilized for both circulating infection of SARS-CoV (in
the immune compartment only) and as a model for generating bat-specific antibodies against
CoV proteins.
(D1, p.18)
PI-TA-02 Task 7: Experimental testing. of ‘Broadscale Immune Boosting’ using-batified
mice and captive bat colonies (Duke-NUS).”
(D1, p.30)
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Here are some questions that journalists may like to ask EcoHealth Alliance and Peter Daszak.
These questions could also be asked under a subpoena process instigated by Congress.
1. Why does EHA refer to one test-site cave and two control caves as “our cave test sites” (D1, p.3
& 5), our cave complex (D1, p.32; D2, p.3), as “our three test cave sites” (D4, slide 2) in
2. Do EHA and the WIV actually own these cave sites or control access to them?
3. Is EHA trying to suggest that DARPA has to fund EHA if they want to do research that includes
these promising sites?
4. Does EHA have an actual evaluation of the ‘clear and present danger to US defense forces
defenses in the region’ represented by these Yunnan viruses it is focussing on in the proposal
(D4, slide 3)?
5. In particular, can EHA explain under which scenarios this may affect “US warfighters”, given that
“Security concerns across Asia make the region a potential deployment site for US warfighters.”?
(D4, slide 3).
6. Was this valuable aspect of the work clearly understood by the Chinese parties to the DEFUSE
7. Why did Peter Daszak firmly deny WIV kept live bats when the DEFUSE proposal requires the
WIV to do so, and to have the necessary experience to do so - in full agreement with the ample
evidence provided independently by DRASTIC?
8. Why did Peter Daszak deny that keeping live bats was common practice in laboratories, when
the DEFUSE proposal shows that many of EHA partner labs keep bat colonies or wild-caught
9. EHA referred to Eco Health Alliance (EHA) databases in the Proposal with obligation of
submission by their Chinese partners. Can EHA share those databases publicly?
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10. What does the proposal contain no risk mitigation program at all for DURC (Dual Use Research
of Concern) despite aiming to address ‘clear and present dangers’ to the US ‘war-fighters’
deployed in Asia, while proposing to hire and pay Shi Zheng Li a well as Ben Hu and Peng Zhou
and other WIV laboratory technicians and employees?
11. Did Scientists at WIV have or see a copy of this DEFUSE proposal and if so, what was the
extent of the contribution of the WIV in drafting this proposal?
12. Did Peter Daszak discuss the Proposal with Scientists at WIV, when, who, how, where?
13. Please summarise any discussions with WIV Scientists, ZLS, PZ, BH, regarding proposed
experiments and bat sampling.
14. Did you discuss the question of sending live Rhinolophus bats to WIV with Dr. Shi Zheng Li or
any other WIV Scientists?
15. Page 38 of the DEFUSE Proposal (D1) refers to the introduction of "appropriate human specific
cleavage sites". Can you explain that in more detail?
16. Can you confirm whether or not any Chinese Scientists have done the sampling / protein S /
recombination and animal testing work mentioned in the DEFUSE Project proposal?
17. What are the 180 Coronavirus sequences that resulted from the mentioned prior work by
EcoHealth Alliance?
“180 bat SARSr-CoV strains sequenced in our prior work and not yet examined for spillover
(D1, p.7)
18. Why was the research-related accident risk (in a foreign country where EHA could only have
limited oversight at best) totally ignored in the proposal?
19. What previous experiments did EHA or its collaborators carry out on irradiated “batified mice
models” (D1, p.18)? When, where and for what purpose?
20. The proposal reviewer wrote that there are several components of great interest in this
proposed effort that are potentially fundable should additional funding become available”.
Did additional funding become available?
21. Can Dr. Rocke, Dr. Unidad and Dr. Ainslie confirm that they were aware of the DEFUSE project
proposal and did they help draft the proposal?
22. Can Dr. Rocke, Dr. Unidad and Dr. Ainslie guarantee that no partial funding was made available
after rejection of the DEFUSE proposal, and none of their technology was subsequently used by
EHA or WIV or other collaborators in this proposal?
23. Outside of NIH/NIAID the WIV with CAS funding conducted a 2018.01-2021.12 project studying
the evolutionary mechanisms for SARSr-CoV host receptor adaptations and cross-species
infection risk (another link here and here). Does EHA have any further information as to what
work was conducted and any results that could help shed light on the origin of SARS-CoV-2?.
DRASTIC - Project DEFUSE HR001118S0017-PREEMPT-FP-019 v1.b - p. 21/25
24. Why were the 2012 cases of the Yunnan Mojiang’s mine workers - whose illness was consistent
with infection with a SARS-like illness (Rahalkar and Bahulikar, 2020) - not discussed in this
proposal, especially given the mention of ‘Test previously-collected human sera from Yunnan
Province to assess SARSr-CoV QS spillover’ (D2, p.3)?
25. Was the Mojiang mine part of ‘your’ Yunnan cave complex that DEFUSE proposed to use for
26. Did EHA know of the theses by Xu and Huang (see Rahalkar and Bahulikar, 2020) and the
conclusions therein that the miners were likely infected with a SARS-r CoV?
27. If so, did EHA deliberately withhold this information so that the SARS-r CoV’s in the Mojiang
mine would not be subject to P3CO restrictions?
28. Why does the proposal fail to contain any reference to Regulatory and ELSI (Ethical, Legal,
Social) issues, especially given its real-life deployment on Yunnan bat colonies?
29. Would EHA have also ignored Regulatory and ELSI issues if they had planned to deploy immune
boosting solutions on bat colonies in Texas?
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ResearchGate has not been able to resolve any citations for this publication.
Full-text available
The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLR S ) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2 ⁺ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.
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We are currently in a rapidly expanding pandemic of the SARS-CoV-2 virus, which originated in the city of Wuhan in central China. The disease COVID-19 is now spread worldwide and has tremendous socio-economic consequences. The origin of the virus can be reconstructed through epidemiological studies and, even more so, from genome comparisons. How the evolution of the virus and the transition to humans might have happened is the subject of much speculation. It is considered certain that the virus is of animal origin and very likely passed from bats to humans in a zoonotic event. An intermediate host was postulated, but many SARS-like bat viruses have the ability to infect human cells directly, which has been shown experimentally by scientists in the Wuhan Institute of Virology using collected specimens containing virus material from horseshoe bats. The propagation of SARS-like bat viruses in cell culture allowed experiments aimed at increasing the infectivity of the virus and adaptation to human cells. This article summarizes the unique properties of SARS-CoV-2 and focusses on a specific sequence encoding the spike protein. Possible scenarios of virus evolution are discussed, with particular emphasis on the hypothesis that the virus could have emerged unintentionally through routine culture or gain-of-function experiments in a laboratory, where it was optimally adapted to human cells and caused cryptic infections among workers who finally spread the virus causing the pandemic.
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The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2′ site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.
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Previously, high aspect ratio ribbon-like microconfetti (MC) composed of acetalated dextran (Ac-DEX) have been shown to form a subcutaneous depot for sustained drug release. In this study, MC were explored as an injectable vaccine platform. Production of MC by electrospinning followed by high-shear homogenization allowed for precise control over MC fabrication. Three distinct sizes of MC, small (0.67 x 10.2 µm), medium (1.28 x 20.7 µm), and large (5.67 x 90.2 µm), were fabricated and loaded with the adjuvant, resiquimod. Steady release rates of resiquimod were observed from MC, indicating their ability to create an immunostimulatory depot in vivo. Resiquimod-loaded MC stimulated inflammatory cytokine production in bone marrow derived dendritic cells without incurring additional cytotoxicity in vitro. Interestingly, even medium and large MC were able to be internalized by antigen presenting cells and facilitate antigen presentation when ovalbumin was adsorbed onto their surface. After subcutaneous injection in vivo with adsorbed ovalbumin, blank MC of all sizes were found to stimulate a humoral response. Adjuvant activity of resiquimod was enhanced by loading it into MC and small and medium sized MC effectively induced a Th1-skewed immune response. Antigen co-delivered with adjuvant-loaded MC of various sizes illustrate a new potential vaccine platform.
The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site containing multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.
Also known as electrospray, electrohydrodynamic atomization has been used extensively in the last 15 years to develop polymer-based particles for drug delivery in cell and animal models. More recently, novel core-shell, multi-axial, and other electrospray particles have been developed from an array of polymers for a variety of biomedical applications. This review focuses on electrospray as a novel method of particle fabrication for drug delivery, specifically highlighting the applications of these particle systems in cell culture and animal models while also discussing polymers used for particle fabrication. Applications of electrospray particles to treat glioma, ovarian cancer, and breast cancer are reviewed. Additionally, delivery of antibiotics, gene therapy, and bacterial cells formulated in electrospray particles is discussed. Finally, vaccines as well as drug eluting particles for differentiation of stem cells and tissue engineering are highlighted. The article concludes with a discussion of where the future of electrospray technology can go to strengthen its foothold in the biomedical field.