Since the identification of theSARS-CoV-2 in Wuhan, China, in January 2020 (1),
the origin of the virus has been a topic of intense scientific debate
and public speculation. The two main hypotheses are that the virus
emerged from human exposure to an infected animal [“zoonosis” (2)] or that it emerged in a research-related incident (3).
The investigation into the origin of the virus has been made difficult
by the lack of key evidence from the earliest days of the
outbreak—there’s no doubt that greater transparency on the part of
Chinese authorities would be enormously helpful. Nevertheless, we argue
here that there is much important information that can be gleaned from
US-based research institutions, information not yet made available for
independent, transparent, and scientific scrutiny.
The
data available within the United States would explicitly include, but
are not limited to, viral sequences gathered and held as part of the
PREDICT project and other funded programs, as well as sequencing data
and laboratory notebooks from US laboratories. We call on US government
scientific agencies, most notably the NIH, to support a full,
independent, and transparent investigation of the origins of SARS-CoV-2.
This should take place, for example, within a tightly focused
science-based bipartisan Congressional inquiry with full investigative
powers, which would be able to ask important questions—but avoid
misguided witch-hunts governed more by politics than by science.
Essential US Investigations
The US intelligence community (IC) was tasked, in 2021 by President Joe Biden (4),
with investigating the origin of the virus. In their summary public
statement, the IC writes that “all agencies assess that two hypotheses
are plausible: natural exposure to an infected animal and a
laboratory-associated incident” (4).
The IC further writes that “China’s cooperation most likely would be
needed to reach a conclusive assessment of the origins of COVID-19
[coronavirus disease 2019].” Of course, such cooperation is highly
warranted and should be pursued by the US Government and the US
scientific community. Yet, as outlined below, much could be learned by
investigating US-supported and US-based work that was underway in
collaboration with Wuhan-based institutions, including the Wuhan
Institute of Virology (WIV), China. It is still not clear whether the IC
investigated these US-supported and US-based activities. If it did, it
has yet to make any of its findings available to the US scientific
community for independent and transparent analysis and assessment. If,
on the other hand, the IC did not investigate these US-supported and
US-based activities, then it has fallen far short of conducting a
comprehensive investigation.
This lack of an
independent and transparent US-based scientific investigation has had
four highly adverse consequences. First, public trust in the ability of
US scientific institutions to govern the activities of US science in a
responsible manner has been shaken. Second, the investigation of the
origin of SARS-CoV-2 has become politicized within the US Congress (5);
as a result, the inception of an independent and transparent
investigation has been obstructed and delayed. Third, US researchers
with deep knowledge of the possibilities of a laboratory-associated
incident have not been enabled to share their expertise effectively.
Fourth, the failure of NIH, one of the main funders of the US–China
collaborative work, to facilitate the investigation into the origins of
SARS-CoV-2 (4) has fostered distrust regarding US biodefense research activities.
Much
of the work on SARS-like CoVs performed in Wuhan was part of an active
and highly collaborative US–China scientific research program funded by
the US Government (NIH, Defense Threat Reduction Agency [DTRA], and US
Agency for International Development [USAID]), coordinated by
researchers at EcoHealth Alliance (EHA), but involving researchers at
several other US institutions. For this reason, it is important that US
institutions be transparent about any knowledge of the detailed
activities that were underway in Wuhan and in the United States. The
evidence may also suggest that research institutions in other countries
were involved, and those too should be asked to submit relevant
information (e.g., with respect to unpublished sequences).
Participating
US institutions include the EHA, the University of North Carolina
(UNC), the University of California at Davis (UCD), the NIH, and the
USAID. Under a series of NIH grants and USAID contracts, EHA coordinated
the collection of SARS-like bat CoVs from the field in southwest China
and southeast Asia, the sequencing of these viruses, the archiving of
these sequences (involving UCD), and the analysis and manipulation of
these viruses (notably at UNC). A broad spectrum of coronavirus research
work was done not only in Wuhan (including groups at Wuhan University
and the Wuhan CDC, as well as WIV) but also in the United States. The
exact details of the fieldwork and laboratory work of the EHA-WIV-UNC
partnership, and the engagement of other institutions in the United
States and China, has not been disclosed for independent analysis. The
precise nature of the experiments that were conducted, including the
full array of viruses collected from the field and the subsequent
sequencing and manipulation of those viruses, remains unknown.
EHA,
UNC, NIH, USAID, and other research partners have failed to disclose
their activities to the US scientific community and the US public,
instead declaring that they were not involved in any experiments that
could have resulted in the emergence of SARS-CoV-2. The NIH has
specifically stated (6)
that there is a significant evolutionary distance between the published
viral sequences and that of SARS-CoV-2 and that the pandemic virus
could not have resulted from the work sponsored by NIH. Of course, this
statement is only as good as the limited data on which it is based, and
verification of this claim is dependent on gaining access to any other
unpublished viral sequences that are deposited in relevant US and
Chinese databases (7,8).
On May 11, 2022, Acting NIH Director Lawrence Tabak testified before
Congress that several such sequences in a US database were removed from
public view, and that this was done at the request of both Chinese and
US investigators.
Blanket denials from the
NIH are no longer good enough. Although the NIH and USAID have
strenuously resisted full disclosure of the details of the EHA-WIV-UNC
work program, several documents leaked to the public or released through
the Freedom of Information Act (FOIA) have raised concerns. These
research proposals make clear that the EHA-WIV-UNC collaboration was
involved in the collection of a large number of so-far undocumented
SARS-like viruses and was engaged in their manipulation within
biological safety level (BSL)-2 and BSL-3 laboratory facilities, raising
concerns that an airborne virus might have infected a laboratory worker
(9).
A variety of scenarios have been discussed by others, including an
infection that involved a natural virus collected from the field or
perhaps an engineered virus manipulated in one of the laboratories (3).
Overlooked Details
Special concerns surround the presence of an unusual furin cleavage site (FCS) in SARS-CoV-2 (10) that augments the pathogenicity and transmissibility of the virus relative to related viruses like SARS-CoV-1 (11, 12). SARS-CoV-2 is, to date, the only identified member of the subgenus sarbecovirus that contains an FCS, although these are present in other coronaviruses (13, 14). A portion of the sequence of the spike protein of some of these viruses is illustrated in the alignment shown in Fig. 1, illustrating the unusual nature of the FCS and its apparent insertion in SARS-CoV-2 (15).
From the first weeks after the genome sequence of SARS-CoV-2 became
available, researchers have commented on the unexpected presence of the
FCS within SARS-CoV-2—the implication being that SARS-CoV-2 might be a
product of laboratory manipulation. In a review piece arguing against
this possibility, it was asserted that the amino acid sequence of the
FCS in SARS-CoV-2 is an unusual, nonstandard sequence for an FCS and
that nobody in a laboratory would design such a novel FCS (13).
Fig. 1.
In
fact, the assertion that the FCS in SARS-CoV-2 has an unusual,
nonstandard amino acid sequence is false. The amino acid sequence of the
FCS in SARS-CoV-2 also exists in the human ENaC α subunit (16), where it is known to be functional and has been extensively studied (17, 18). The FCS of human ENaC α has the amino acid sequence RRAR'SVAS (Fig. 2), an eight–amino-acid sequence that is perfectly identical with the FCS of SARS-CoV-2 (16). ENaC is an epithelial sodium channel, expressed on the apical surface of epithelial cells in the kidney, colon, and airways (19, 20), that plays a critical role in controlling fluid exchange. The ENaC α subunit has a functional FCS (17, 18) that is essential for ion channel function (19) and has been characterized in a variety of species. The FCS sequence of human ENaC α (20) is identical in chimpanzee, bonobo, orangutan, and gorilla (SI Appendix, Fig. 1),
but diverges in all other species, even primates, except one. (The one
non-human non-great ape species with the same sequence is Pipistrellus kuhlii, a bat species found in Europe and Western Asia; other bat species, including Rhinolophus ferrumequinem, have a different FCS sequence in ENaC α [RKAR'SAAS]).
Fig. 2.
One
consequence of this “molecular mimicry” between the FCS of SARS CoV-2
spike and the FCS of human ENaC is competition for host furin in the
lumen of the Golgi apparatus, where the SARS-CoV-2 spike is processed.
This results in a decrease in human ENaC expression (21).
A decrease in human ENaC expression compromises airway function and has
been implicated as a contributing factor in the pathogenesis of
COVID-19 (22).
Another consequence of this astonishing molecular mimicry is evidenced
by apparent cross-reactivity with human ENaC of antibodies from COVID-19
patients, with the highest levels of cross-reacting antibodies directed
against this epitope being associated with most severe disease (23).
We do not know whether the insertion of the FCS was the result of natural evolution (2, 13)—perhaps via a recombination event in an intermediate mammal or a human (13, 24)—or
was the result of a deliberate introduction of the FCS into a SARS-like
virus as part of a laboratory experiment. We do know that the insertion
of such FCS sequences into SARS-like viruses was a specific goal of
work proposed by the EHA-WIV-UNC partnership within a 2018 grant
proposal (“DEFUSE”) that was submitted to the US Defense Advanced
Research Projects Agency (DARPA) (25).
The 2018 proposal to DARPA was not funded, but we do not know whether
some of the proposed work was subsequently carried out in 2018 or 2019,
perhaps using another source of funding.
We
also know that that this research team would be familiar with several
previous experiments involving the successful insertion of an FCS
sequence into SARS-CoV-1 (26) and other coronaviruses, and they had a lot of experience in construction of chimeric SARS-like viruses (27–29).
In addition, the research team would also have some familiarity with
the FCS sequence and the FCS-dependent activation mechanism of human
ENaC α (19), which was extensively characterized at UNC (17, 18).
For a research team assessing the pandemic potential of SARS-related
coronaviruses, the FCS of human ENaC—an FCS known to be efficiently
cleaved by host furin present in the target location (epithelial cells)
of an important target organ (lung), of the target organism
(human)—might be a rational, if not obvious, choice of FCS to introduce
into a virus to alter its infectivity, in line with other work performed
previously.
Of course, the molecular mimicry
of ENaC within the SARS-CoV-2 spike protein might be a mere
coincidence, although one with a very low probability. The exact FCS
sequence present in SARS-CoV-2 has recently been introduced into the
spike protein of SARS-CoV-1 in the laboratory, in an elegant series of
experiments (12, 30),
with predictable consequences in terms of enhanced viral
transmissibility and pathogenicity. Obviously, the creation of such
SARS-1/2 “chimeras” is an area of some concern for those responsible for
present and future regulation of this area of biology. [Note that these
experiments in ref. 30
were done in the context of a safe “pseudotyped” virus and thus posed
no danger of producing or releasing a novel pathogen.] These simple
experiments show that the introduction of the 12 nucleotides that
constitute the FCS insertion in SARS-CoV-2 would not be difficult to
achieve in a lab. It would therefore seem reasonable to ask that
electronic communications and other relevant data from US groups should
be made available for scrutiny.
Seeking Transparency
To
date, the federal government, including the NIH, has not done enough to
promote public trust and transparency in the science surrounding
SARS-CoV-2. A steady trickle of disquieting information has cast a
darkening cloud over the agency. The NIH could say more about the
possible role of its grantees in the emergence of SARS-CoV-2, yet the
agency has failed to reveal to the public the possibility that
SARS-CoV-2 emerged from a research-associated event, even though several
researchers raised that concern on February 1, 2020, in a phone
conversation that was documented by email (5).
Those emails were released to the public only through FOIA, and they
suggest that the NIH leadership took an early and active role in
promoting the “zoonotic hypothesis” and the rejection of the
laboratory-associated hypothesis (5).
The NIH has resisted the release of important evidence, such as the
grant proposals and project reports of EHA, and has continued to redact
materials released under FOIA, including a remarkable 290-page redaction
in a recent FOIA release.
Information now held by the research team headed by EHA (7),
as well as the communications of that research team with US research
funding agencies, including NIH, USAID, DARPA, DTRA, and the Department
of Homeland Security, could shed considerable light on the experiments
undertaken by the US-funded research team and on the possible
relationship, if any, between those experiments and the emergence of
SARS-CoV-2. We do not assert that laboratory manipulation was involved
in the emergence of SARS-CoV-2, although it is apparent that it could
have been. However, we do assert that there has been no independent and
transparent scientific scrutiny to date of the full scope of the
US-based evidence.
The relevant US-based
evidence would include the following information: laboratory notebooks,
virus databases, electronic media (emails, other communications),
biological samples, viral sequences gathered and held as part of the
PREDICT project (7)
and other funded programs, and interviews of the EHA-led research team
by independent researchers, together with a full record of US agency
involvement in funding the research on SARS-like viruses, especially
with regard to projects in collaboration with Wuhan-based institutions.
We suggest that a bipartisan inquiry should also follow up on the
tentative conclusion of the IC (4)
that the initial outbreak in Wuhan may have occurred no later than
November 2019 and that therefore the virus was circulating before the
cluster of known clinical cases in December. The IC did not reveal the
evidence for this statement, nor when parts of the US Government or
US-based researchers first became aware of a potential new outbreak. Any
available information and knowledge of the earliest days of the
outbreak, including viral sequences (8), could shed considerable light on the origins question.
We
continue to recognize the tremendous value of US–China cooperation in
ongoing efforts to uncover the proximal origins of the pandemic. Much
vital information still resides in China, in the laboratories, hospital
samples, and early epidemiological information not yet available to the
scientific community. Yet a US-based investigation need not wait—there
is much to learn from the US institutions that were extensively involved
in research that may have contributed to, or documented the emergence
of, the SARS-CoV-2 virus. Only an independent and transparent
investigation, perhaps as a bipartisan Congressional inquiry, will
reveal the information that is needed to enable a thorough scientific
process of scrutiny and evaluation.
Supporting Information
Appendix 01 (PDF)
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