Information

Is the receptor of SARS-CoV-like viruses in bats still unknown?


A fairly detailed 2012 review (Whittaker et al.) on the Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein noted that

SARS-CoV-like viruses have been isolated in bats. In this case, entry does not occur via ACE2 and their receptor(s) is/are unknown; however, replacement of the amino acid sequence found between residues 323 and 505 with the corresponding sequence of the SARS-CoV RBD is sufficient to allow human ACE2 receptor usage [46].

Is the actual (bat) receptor that allows SARS-CoV-like viruses to replicate in bats still unknown?


Abstract

An ongoing outbreak of pneumonia caused by a novel coronavirus, currently designated as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), was reported recently. However, as SARS-CoV-2 is an emerging virus, we know little about it. In this review, we summarize the key events occurred during the early stage of SARS-CoV-2 outbreak, the basic characteristics of the pathogen, the signs and symptoms of the infected patients as well as the possible transmission pathways of the virus. Furthermore, we also review the current knowledge on the origin and evolution of the SARS-CoV-2. We highlight bats as the potential natural reservoir and pangolins as the possible intermediate host of the virus, but their roles are waiting for further investigation. Finally, the advances in the development of chemotherapeutic options are also briefly summarized.

Keywords: Coronavirus, Novel coronavirus, pneumonia, SARS-CoV-2, COVID-19


Spike Structure Gives Insight into SARS-CoV-2 Evolution

Abby Olena
Jul 16, 2020

ABOVE: Two models of the SARS-CoV-2 spike protein show the closed receptor binding domain (tan, left) and the open receptor binding domain (tan, right).
ADAPTED FROM A VIDEO BY DONALD BENTON

I t’s clear that SARS-CoV-2, the coronavirus behind the COVID-19 pandemic, is most closely related to a group of viruses that usually infect bats. But exactly how and where it evolved to become such an efficient respiratory pathogen remains to be seen. Now, in a study published July 9 in Nature Structural & Molecular Biology, researchers have determined that the spike proteins of SARS-CoV-2 and of the closely related bat coronavirus RaTG13—while similarly structured overall—differ in their stability and affinity for binding ACE2, the receptor that SARS-CoV-2 uses to infect human cells.

The substantial difference in the spike protein of the closest viral relative “tells you that this was not a direct jump from this virus into humans,” says Amesh Adalja, a physician who studies emerging infectious diseases at the Johns Hopkins Bloomberg School of Public Health and was not involved in the work. It’s likely that SARS-CoV-2 “had been evolving in some other species—possibly an intermediate species—before it acquired the ability to be this human pathogen of such a degree that it is today.”

A group of researchers in structural biologist Steve Gamblin’s lab at the Francis Crick Institute in the United Kingdom specializes in understanding how changes in the shapes of proteins on the surface of the influenza virus allow it to jump between different species, Donald Benton, a postdoc in the Gamblin lab, tells The Scientist. Earlier this year, when it became clear that SARS-CoV-2 was gaining steam, they decided to devote their expertise to asking the same type of questions about the coronavirus, focusing on its iconic spike proteins that protrude from the viral surface.

Previous work showed that the SARS-CoV-2 spike protein must be cut between two amino acids at the junction between the portion of the protein that binds a receptor and the domain of the protein responsible for fusing with the host cell membrane. Rather than cutting the protein in two, this cleavage event—performed for SARS-CoV-2 by the human protease furin—is thought to increase flexibility in the protein so that it can enter mammalian cells. To investigate how this cleavage affects the structure of the protein, Benton and colleagues generated a version of the SARS-CoV-2 spike protein with the furin cleavage site intact and then exposed that protein to furin to generate a cleaved version.

See “Scientists Scan for Weaknesses in the SARS-CoV-2 Spike Protein”

In the uncleaved form, the protein was stable, with three components known as receptor-binding domains (RBDs), which are thought to bind ACE2, tightly tucked into the top of the protein. After furin cleavage, one of the RBDs rotated to open a surface at the top of the protein for ACE2 interaction. The findings indicate that furin cleavage appears to make the spike protein more likely to adopt an open shape that allows it bind to the receptor and enter human cells.

According to a study published in February, SARS-CoV-2 and RaTG13 share about 96 percent of their genomes and about 93 percent sequence similarity in their spike protein genes, making RaTG13 the closest SARS-CoV-2 relative found yet. In work published in April, researchers showed that the amino acid sequences of the two proteins were least similar—around 90 percent—in the RBDs and that the furin cleavage site in SARS-CoV-2’s spike protein is absent in RaTG13, findings Benton and colleagues confirmed.

The authors of the new paper also observed that the SARS-CoV-2 spike protein binds ACE2 about 1,000 times more tightly than the RaTG13 spike protein.

“It looks as if this particular bat virus wouldn’t directly be able to infect humans because of its weak ability to bind to the human receptor,” Benton tells The Scientist.

“We probably still haven’t found the correct bat virus that actually did make this leap” to people, he says, though there are some coronaviruses in pangolins—scale-covered mammals found in Asia and Africa—that have similar RBDs in their spike proteins.

In a study published July 1, researchers proposed that recombination events between multiple coronaviruses from different species—potentially including RaTG13—could have led to the emergence of SARS-CoV-2, an idea that’s supported by findings of the current study, according to Benton and his colleagues. “That’s not just plausible, I think it’s also parsimonious,” says Adam Frost, a structural biologist at the University of California, San Francisco, who did not participate in either study. “Where that recombination event took place remains unknown . . . and it may be very hard to be absolutely sure.”

Beyond the evolutionary insight, these new structures may also help researchers generate tools—such as antibodies and synthetic ACE2 domains—that could attach to the RBD and prevent it from engaging the endogenous ACE2, Frost explains. “Big picture, these new structural states will help us both develop and understand those kinds of therapeutic reagents.”


Online Methods

Sampling

Bats were trapped in their natural habitat as described previously 5 . Throat and faecal swab samples were collected in viral transport medium (VTM) composed of Hank’s balanced salt solution, pH 7.4, containing BSA (1%), amphotericin (15 μg ml −1 ), penicillin G (100 U ml −1 ) and streptomycin (50 μg ml −1 ). To collect fresh faecal samples, clean plastic sheets measuring 2.0 by 2.0 m were placed under known bat roosting sites at about 18:00 h each evening. Relatively fresh faecal samples were collected from sheets at approximately 05:30–06:00 the next morning and placed in VTM. Samples were transported to the laboratory and stored at −80 °C until use. All animals trapped for this study were released back to their habitat after sample collection. All sampling processes were performed by veterinarians with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH05210201) and EcoHealth Alliance under an inter-institutional agreement with University of California, Davis (UC Davis protocol no. 16048).

RNA extraction, PCR and sequencing

RNA was extracted from 140 μl of swab or faecal samples with a Viral RNA Mini Kit (Qiagen) following the manufacturer’s instructions. RNA was eluted in 60 μl RNAse-free buffer (buffer AVE, Qiagen), then aliquoted and stored at −80 °C. One-step RT–PCR (Invitrogen) was used to detect coronavirus sequences as described previously 15 . First round PCR was conducted in a 25-μl reaction mix containing 12.5 μl PCR 2× reaction mix buffer, 10 pmol of each primer, 2.5 mM MgSO4, 20 U RNase inhibitor, 1 μl SuperScript III/ Platinum Taq Enzyme Mix and 5 μl RNA. Amplification of the RdRP-gene fragment was performed as follows: 50 °C for 30 min, 94 °C for 2 min, followed by 40 cycles consisting of 94 °C for 15 s, 62 °C for 15 s, 68 °C for 40 s, and a final extension of 68 °C for 5 min. Second round PCR was conducted in a 25-μl reaction mix containing 2.5 μl PCR reaction buffer, 5 pmol of each primer, 50 mM MgCl2, 0.5 mM dNTP, 0.1 μl Platinum Taq Enzyme (Invitrogen) and 1 μl first round PCR product. The amplification of RdRP-gene fragment was performed as follows: 94 °C for 5 min followed by 35 cycles consisting of 94 °C for 30 s, 52 °C for 30 s, 72 °C for 40 s, and a final extension of 72 °C for 5 min.

To amplify the RBD region, one-step RT–PCR was performed with primers designed based on available SARS-CoV or bat SL-CoVs (first round PCR primers F, forward R, reverse: CoVS931F-5′-VWGADGTTGTKAGRTTYCCT-3′ and CoVS1909R-5′-TAARACAVCCWGCYTGWGT-3′ second PCR primers: CoVS951F-5′-TGTKAGRTTYCCTAAYATTAC-3′ and CoVS1805R-5′-ACATCYTGATANARAACAGC-3′). First-round PCR was conducted in a 25-μl reaction mix as described above except primers specific for the S gene were used. The amplification of the RBD region of the S gene was performed as follows: 50 °C for 30 min, 94 °C for 2 min, followed by 35 cycles consisting of 94 °C for 15 s, 43 °C for 15 s, 68 °C for 90 s, and a final extension of 68 °C for 5 min. Second-round PCR was conducted in a 25-μl reaction mix containing 2.5 μl PCR reaction buffer, 5 pmol of each primer, 50 mM MgCl2, 0.5 mM dNTP, 0.1 μl Platinum Taq Enzyme (Invitrogen) and 1 μl first round PCR product. Amplification was performed as follows: 94 °C for 5 min followed by 40 cycles consisting of 94 °C for 30 s, 41 °C for 30 s, 72 °C for 60 s, and a final extension of 72 °C for 5 min.

PCR products were gel purified and cloned into pGEM-T Easy Vector (Promega). At least four independent clones were sequenced to obtain a consensus sequence for each of the amplified regions.

Sequencing full-length genomes

Degenerate coronavirus primers were designed based on all available SARS-CoV and bat SL-CoV sequences in GenBank and specific primers were designed from genome sequences generated from previous rounds of sequencing in this study (primer sequences will be provided upon request). All PCRs were conducted using the One-Step RT–PCR kit (Invitrogen). The 5′ and 3′ genomic ends were determined using the 5′ or 3′ RACE kit (Roche), respectively. PCR products were gel purified and sequenced directly or following cloning into pGEM-T Easy Vector (Promega). At least four independent clones were sequenced to obtain a consensus sequence for each of the amplified regions and each region was sequenced at least twice.

Sequence analysis and databank accession numbers

Routine sequence management and analysis was carried out using DNAStar or Geneious. Sequence alignment and editing was conducted using ClustalW, BioEdit or GeneDoc. Maximum Likelihood phylogenetic trees based on the protein sequences were constructed using a Poisson model with bootstrap values determined by 1,000 replicates in the MEGA5 software package.

Sequences obtained in this study have been deposited in GenBank as follows (accession numbers given in parenthesis): full-length genome sequence of SL-CoV RsSHC014 and Rs3367 (KC881005, KC881006) full-length sequence of WIV1 S (KC881007) RBD (KC880984-KC881003) ACE2 (KC8810040). SARS-CoV sequences used in this study: human SARS-CoV strains Tor2 (AY274119), BJ01 (AY278488), GZ02 (AY390556) and civet SARS-CoV strain SZ3 (AY304486). Bat coronavirus sequences used in this study: Rs672 (FJ588686), Rp3 (DQ071615), Rf1 (DQ412042), Rm1 (DQ412043), HKU3-1 (DQ022305), BM48-31 (NC_014470), HKU9-1 (NC_009021), HKU4 (NC_009019), HKU5 (NC_009020), HKU8 (DQ249228), HKU2 (EF203067), BtCoV512 (NC_009657), 1A (NC_010437). Other coronavirus sequences used in this study: HCoV-229E (AF304460), HCoV-OC43 (AY391777), HCoV-NL63 (AY567487), HKU1 (NC_006577), EMC (JX869059), FIPV (NC_002306), PRCV (DQ811787), BWCoV (NC_010646), MHV (AY700211), IBV (AY851295).

Amplification, cloning and expression of the bat ACE2 gene

Construction of expression clones for human and civet ACE2 in pcDNA3.1 has been described previously 29 . Bat ACE2 was amplified from a R. sinicus (sample no. 3357). In brief, total RNA was extracted from bat rectal tissue using the RNeasy Mini Kit (Qiagen). First-strand complementary DNA was synthesized from total RNA by reverse transcription with random hexamers. Full-length bat ACE2 fragments were amplified using forward primer bAF2 and reverse primer bAR2 (ref. 29). The ACE2 gene was cloned into pCDNA3.1 with KpnI and XhoI, and verified by sequencing. Purified ACE2 plasmids were transfected to HeLa cells. After 24 h, lysates of HeLa cells expressing human, civet, or bat ACE2 were confirmed by western blot or immunofluorescence assay.

Western blot analysis

Lysates of cells or filtered supernatants containing pseudoviruses were separated by SDS–PAGE, followed by transfer to a nitrocellulose membrane (Millipore). For detection of S protein, the membrane was incubated with rabbit anti-Rp3 S fragment (amino acids 561–666) polyantibodies (1:200), and the bound antibodies were detected by alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (1:1,000). For detection of HIV-1 p24 in supernatants, monoclonal antibody against HIV p24 (p24 MAb) was used as the primary antibody at a dilution of 1:1,000, followed by incubation with AP-conjugated goat anti-mouse IgG at the same dilution. To detect the expression of ACE2 in HeLa cells, goat antibody against the human ACE2 ectodomain (1:500) was used as the first antibody, followed by incubation with horseradish peroxidase-conjugated donkey anti-goat IgG (1:1,000).

Virus isolation

Vero E6 cell monolayers were maintained in DMEM supplemented with 10% FCS. PCR-positive samples (in 200 μl buffer) were gradient centrifuged at 3,000–12,000g, and supernatant were diluted 1:10 in DMEM before being added to Vero E6 cells. After incubation at 37 °C for 1 h, inocula were removed and replaced with fresh DMEM with 2% FCS. Cells were incubated at 37 °C for 3 days and checked daily for cytopathic effect. Double-dose triple antibiotics penicillin/streptomycin/amphotericin (Gibco) were included in all tissue culture media (penicillin 200 IU ml −1 , streptomycin 0.2 mg ml −1 , amphotericin 0.5 μg ml −1 ). Three blind passages were carried out for each sample. After each passage, both the culture supernatant and cell pellet were examined for presence of virus by RT–PCR using primers targeting the RdRP or S gene. Virions in supernatant (10 ml) were collected and fixed using 0.1% formaldehyde for 4 h, then concentrated by ultracentrifugation through a 20% sucrose cushion (5 ml) at 80,000g for 90 min using a Ty90 rotor (Beckman). The pelleted viral particles were suspended in 100 μl PBS, stained with 2% phosphotungstic acid (pH 7.0) and examined using a Tecnai transmission electron microscope (FEI) at 200 kV.

Virus infectivity detected by immunofluorescence assay

Cell lines used for this study and their culture conditions are summarized in Extended Data Table 5. Virus titre was determined in Vero E6 cells by cytopathic effect (CPE) counts. Cell lines from different origins and HeLa cells expressing ACE2 from human, civet or Chinese horseshoe bat were grown on coverslips in 24-well plates (Corning) incubated with bat SL-CoV-WIV1 at a multiplicity of infection = 10 for 1 h. The inoculum was removed and washed twice with PBS and supplemented with medium. HeLa cells without ACE2 expression and Vero E6 cells were used as negative and positive controls, respectively. At 24 h after infection, cells were washed with PBS and fixed with 4% formaldehyde in PBS (pH 7.4) for 20 min at 4 °C. ACE2 expression was detected using goat anti-human ACE2 immunoglobulin (R&D Systems) followed by FITC-labelled donkey anti-goat immunoglobulin (PTGLab). Virus replication was detected using rabbit antibody against the SL-CoV Rp3 nucleocapsid protein followed by Cy3-conjugated mouse anti-rabbit IgG. Nuclei were stained with DAPI. Staining patterns were examined using a FV1200 confocal microscope (Olympus).

Virus infectivity detected by real-time RT–PCR

Vero E6, A549, PK15, RSKT and HeLa cells with or without expression of ACE2 of different origins were inoculated with 0.1 TCID50 WIV-1 and incubated for 1 h at 37 °C. After removing the inoculum, the cells were cultured with medium containing 1% FBS. Supernatants were collected at 0, 12, 24 and 48 h. RNA from 140 μl of each supernatant was extracted with the Viral RNA Mini Kit (Qiagen) following manufacturer’s instructions and eluted in 60 μl buffer AVE (Qiagen). RNA was quantified on the ABI StepOne system, with the TaqMan AgPath-ID One-Step RT–PCR Kit (Applied Biosystems) in a 25 μl reaction mix containing 4 μl RNA, 1 × RT–PCR enzyme mix, 1 × RT–PCR buffer, 40 pmol forward primer (5′-GTGGTGGTGACGGCAAAATG-3′), 40 pmol reverse primer (5′-AAGTGAAGCTTCTGGGCCAG-3′) and 12 pmol probe (5′-FAM-AAAGAGCTCAGCCCCAGATG-BHQ1-3′). Amplification parameters were 10 min at 50 °C, 10 min at 95 °C and 50 cycles of 15 s at 95 °C and 20 s at 60 °C. RNA dilutions from purified WIV-1 stock were used as a standard.

Serum neutralization test

SARS patient sera were inactivated at 56 °C for 30 min and then used for virus neutralization testing. Sera were diluted starting with 1:10 and then serially twofold diluted in 96-well cell plates to 1:40. Each 100 μl serum dilution was mixed with 100 μl viral supernatant containing 100 TCID50of WIV1 and incubated at 37 °C for 1 h. The mixture was added in triplicate wells of 96-well cell plates with plated monolayers of Vero E6 cells and further incubated at 37 °C for 2 days. Serum from a healthy blood donor was used as a negative control in each experiment. CPE was observed using an inverted microscope 2 days after inoculation. The neutralizing antibody titre was read as the highest dilution of serum which completely suppressed CPE in infected wells. The neutralization test was repeated twice.

Recombination analysis

Full-length genomic sequences of SL-CoV Rs3367 or RsSHC014 were aligned with those of selected SARS-CoVs and bat SL-CoVs using Clustal X. The aligned sequences were preliminarily scanned for recombination events using Recombination Detection Program (RDP) 4.0 (ref. 19). The potential recombination events suggested by RDP owing to their strong P values (<10–20) were investigated further by similarity plot and bootscan analyses implemented in Simplot 3.5.1. Phylogenetic origin of the major and minor parental regions of Rs3367 or RsSHC014 were constructed from the concatenated sequences of the essential ORFs of the major and minor parental regions of selected SARS-CoV and SL-CoVs. Two genome regions between three estimated breakpoints (20,827–26,553 and 26,554–28,685) were aligned independently using ClustalX and generated two alignments of 5,727 base pairs and 2,133 base pairs. The two alignments were used to construct maximum likelihood trees to better infer the fragment parents. All nucleotide numberings in this study are based on Rs3367 genome position.


Evolution and origins of life Edit

  • Origin of life. Exactly how and when did life on Earth originate? Which, if any, of the many hypotheses is correct? What were the metabolic pathways used by the earliest life forms?
    • Origins of viruses. Exactly how and when did different groups of viruses originate?
    • Extraterrestrial life. Might life which does not originate from planet Earth also have developed on other planets? Might this life be intelligent?
    • What are the chemical origins of life? How did non-living chemical compounds generate self-replicating, complex life forms?
      . What is the cause of homosexuality, especially in the human species?
  • Biochemistry and cell biology Edit

    • What do all the unknown proteins do? Almost two decades since the first eukaryotes were sequenced, the "biological role" of around 20% of proteins are still unknown. [2] Many of these proteins are conserved across most eukaryotic species and some are conserved in bacteria, indicating a role fundamental for life. [3][4][5]
    • Determinants of cell size. How do cells determine what size to grow to before dividing?
    • Golgi apparatus. In cell theory, what is the exact transport mechanism by which proteins travel through the Golgi apparatus?
    • Mechanism of action of drugs. The mechanisms of action of many drugs including paracetamol, lithium, thalidomide and ketamine[6] are not completely understood.
    • Protein folding. What is the folding code? What is the folding mechanism? Can we predict the native structure of a protein from its amino acid sequence? Is it possible to predict the secondary, tertiary and quaternary structure of a polypeptide sequence based solely on the sequence and environmental information? Inverse protein-folding problem: Is it possible to design a polypeptide sequence which will adopt a given structure under certain environmental conditions? [7][8] This was achieved for several small globular proteins in 2008. [9] In 2020, it was announced that Google's AlphaFold, a neural network based on DeepMind artificial intelligence, is capable of predicting a protein's final shape based solely on its amino-acid chain with an accuracy of around 90% on a test sample of proteins used by the team. [10]
    • Enzyme kinetics: Why do some enzymes exhibit faster-than-diffusion kinetics? [11]
    • RNA folding problem: Is it possible to accurately predict the secondary, tertiary and quaternary structure of a polyribonucleic acid sequence based on its sequence and environment?
    • Protein design: Is it possible to design highly active enzymes de novo for any desired reaction? [12]
    • Biosynthesis: Can desired molecules, natural products or otherwise, be produced in high yield through biosynthetic pathway manipulation? [13]
    • What is the mechanism of allosteric transitions of proteins? The concerted and sequential models have been hypothesised but neither has been verified.
    • What are the endogenous ligands of orphan receptors?
    • What substance is endothelium-derived hyperpolarizing factor?

    Other Edit

    • Why does biological aging occur? There are a number of hypotheses why senescence occurs including those that it is programmed by gene expression changes and that it is the accumulative damage of biological processes.
    • Consistency of movement. How can we move so controllably, even though the motor nerve impulses seem haphazard and unpredictable? [14]
    • How do organs grow to the correct shape and size?[15] How are the final shape and size of organs so reliably formed? These processes are in part controlled by the Hippo signaling pathway.
    • Can developing biological systems tell the time?[15] To an extent, this appears to be the case, as shown by the CLOCK gene.
    • Why are babies so rarely born with cancer?[16]
    • Handedness: It is unclear how handedness develops, what purpose it serves, why right-handedness is far more common, and why left-handedness exists.
    • Laughter: While it is generally accepted that laughing evolved as a form of social communication, the exact neurobiological process that leads humans to laugh is not well understood.
    • Yawning: It is yet to be established what the biological or social purpose of yawning is. [17]
    • Why do humans have fingerprints? The function of the epidermal ridges on Human fingers (fingerprints) is not well understood. The theory that fingerprints help maintain grip has been disproven. It is likely that fingerprints play some role in texture perception but this has yet to be proven. [18]
    • Decline in male sperm counts: It is unclear what is causing the steady decline of sperm counts worldwide since the twentieth century. [19]
    • Decline in average human body temperature since the 19th century: Medical data suggests that the average body temperature has declined 0.6 celsius since the 19th century. The cause is unclear although it has been suggested that it has some relation with reduced inflammation from reduced exposure to microorganisms. [20]
    • Why are there blood types? It is unclear what the origin and purpose of having blood types is. It's thought that O blood may be an adaptation to malaria and that different blood types respond to different diseases but this hypothesis has yet to be proven. Why did these antigens develop in the first place? What accounts for the differences in blood type? How ancient are the differences in blood types? What accounts for the large number of rare non ABO blood types? What role do blood types have in fighting disease? [21]
    • Photic sneeze effect: What causes the photic sneeze effect? Why is it so common yet not universal?
    • Human sex pheromones: There is contradictory evidence on the existence of human pheromones. Do they actually exist, and if so, how do they affect behavior? [22]
    • Existence of the Grafenberg spot (G-spot): Does the G-spot actually exist? If so is it present in all women? What exactly is it? [23]

    Neuroscience and cognition Edit

    Neurophysiology Edit

    Sleep What is the biological function of sleep? Why do we dream? What are the underlying brain mechanisms? What is its relation to anesthesia?
    Neuroplasticity How plastic is the mature brain?
    General anesthetic What is the mechanism by which it works?
    Neuropsychiatric diseases What are the neural bases (causes) of mental diseases like psychotic disorders (e.g. mania, schizophrenia), Parkinson's disease, Alzheimer's disease, or addiction? Is it possible to recover loss of sensory or motor function?
    Neural computation What are all the different types of neuron and what do they do in the brain?

    Cognition and psychology Edit

    Cognition and decisions How and where does the brain evaluate reward value and effort (cost) to modulate behavior? How does previous experience alter perception and behavior? What are the genetic and environmental contributions to brain function?
    Computational neuroscience How important is the precise timing of action potentials for information processing in the neocortex? Is there a canonical computation performed by cortical columns? How is information in the brain processed by the collective dynamics of large neuronal circuits? What level of simplification is suitable for a description of information processing in the brain? What is the neural code?
    Computational theory of mind What are the limits of understanding thinking as a form of computing?
    Consciousness What is the brain basis of subjective experience, cognition, wakefulness, alertness, arousal, and attention? Is there a "hard problem of consciousness"? If so, how is it solved? What, if any, is the function of consciousness? [24] [25]
    Free will Particularly the neuroscience of free will
    Language How is it implemented neurally? What is the basis of semantic meaning?
    Learning and memory Where do our memories get stored and how are they retrieved again? How can learning be improved? What is the difference between explicit and implicit memories? What molecule is responsible for synaptic tagging?
    Noogenesis - the emergence and evolution of intelligence What are the laws and mechanisms - of new idea emergence (insight, creativity synthesis, intuition, decision-making, eureka) development (evolution) of an individual mind in the ontogenesis, etc.?
    Perception How does the brain transfer sensory information into coherent, private percepts? What are the rules by which perception is organized? What are the features/objects that constitute our perceptual experience of internal and external events? How are the senses integrated? What is the relationship between subjective experience and the physical world?

    Ecology, evolution, and paleontology Edit

    Unsolved problems relating to the interactions between organisms and their distribution in the environment include:

      . The high diversity of phytoplankton seems to violate the competitive exclusion principle.
      . What is the cause of the apparent rapid diversification of multicellular animal life around the beginning of the Cambrian, resulting in the emergence of almost all modern animal phyla?
      . Why does biodiversity increase when going from the poles towards the equator?
      of botany/plants. What is the exact evolutionary history of flowers and what is the cause of the apparently sudden appearance of nearly modern flowers in the fossil record?
    • Absence of Loricifera fossils. There are at least 100 species of this phylum of marine dwelling animals (many undescribed), but none of them is known to be present in the fossil record.
    • Adult form of Facetotecta. The adult form of this animal has never been encountered in the water, and it remains a mystery what it grows into.
    • Origin of snakes. Did snakes evolve from burrowing lizards or aquatic lizards? There is evidence for both hypotheses.
    • Origin of turtles. Did turtles evolve from anapsids or diapsids? There is evidence for both hypotheses.
      . How should Ediacaran biota be classified? Even what kingdom they belong to is unclear. Why were they so decisively displaced by Cambrian biota?

    Ethology Edit

    Unsolved problems relating to the behaviour of animals include:

      . A satisfactory explanation for the neurobiological mechanisms that allow homing in animals has yet to be found.
      . How flocks of birds and bats coordinate their movements so quickly is not fully understood. Nor is the purpose of large flocks like those of starlings which seem to invite predators rather than protect them. [26]
      . How do the descendants of monarch butterfly all over Canada and the US eventually, after migrating for several generations, manage to return to a few relatively small overwintering spots?
      . There is not much data on the sexuality of the blue whale. [27]
      . It is largely unknown how gall wasps induce gall formation in plants chemical, mechanical, and viral triggers have been discussed.

    Non-human organs and biomolecules Edit

    Unsolved problems relating to the structure and function of non-human organs, processes and biomolecules include:


    Viewpoint: Why the Wuhan lab escape theory explaining the origin of the global pandemic isn’t going away anytime soon

    Wuhan Institute of Virology. Credit: Ureem2805/Wikimedia

    In what follows I will sort through the available scientific facts, which hold many clues as to what happened, and provide readers with the evidence to make their own judgments. I will then try to assess the complex issue of blame, which starts with, but extends far beyond, the government of China.

    By the end of this article, you may have learned a lot about the molecular biology of viruses. I will try to keep this process as painless as possible. But the science cannot be avoided because for now, and probably for a long time hence, it offers the only sure thread through the maze.

    The virus that caused the pandemic is known officially as SARS-CoV-2, but can be called SARS2 for short. As many people know, there are two main theories about its origin. One is that it jumped naturally from wildlife to people. The other is that the virus was under study in a lab, from which it escaped. It matters a great deal which is the case if we hope to prevent a second such occurrence.

    I’ll describe the two theories, explain why each is plausible, and then ask which provides the better explanation of the available facts. It’s important to note that so far there is no direct evidence for either theory. Each depends on a set of reasonable conjectures but so far lacks proof. So I have only clues, not conclusions, to offer. But those clues point in a specific direction. And having inferred that direction, I’m going to delineate some of the strands in this tangled skein of disaster.

    A tale of two theories

    After the pandemic first broke out in December 2019, Chinese authorities reported that many cases had occurred in the wet market — a place selling wild animals for meat — in Wuhan. This reminded experts of the SARS1 epidemic of 2002, in which a bat virus had spread first to civets, an animal sold in wet markets, and from civets to people. A similar bat virus caused a second epidemic, known as MERS, in 2012. This time the intermediary host animal was camels.

    The decoding of the virus’s genome showed it belonged a viral family known as beta-coronaviruses, to which the SARS1 and MERS viruses also belong. The relationship supported the idea that, like them, it was a natural virus that had managed to jump from bats, via another animal host, to people. The wet market connection, the major point of similarity with the SARS1 and MERS epidemics, was soon broken: Chinese researchers found earlier cases in Wuhan with no link to the wet market. But that seemed not to matter when so much further evidence in support of natural emergence was expected shortly.

    From early on, public and media perceptions were shaped in favor of the natural emergence scenario by strong statements from two scientific groups. These statements were not at first examined as critically as they should have been.

    “We stand together to strongly condemn conspiracy theories suggesting that COVID-19 does not have a natural origin,” a group of virologists and others wrote in the Lancet on February 19, 2020, when it was really far too soon for anyone to be sure what had happened. Scientists “overwhelmingly conclude that this coronavirus originated in wildlife,” they said, with a stirring rallying call for readers to stand with Chinese colleagues on the frontline of fighting the disease.

    Contrary to the letter writers’ assertion, the idea that the virus might have escaped from a lab invoked accident, not conspiracy. It surely needed to be explored, not rejected out of hand. A defining mark of good scientists is that they go to great pains to distinguish between what they know and what they don’t know. By this criterion, the signatories of the Lancet letter were behaving as poor scientists: They were assuring the public of facts they could not know for sure were true.

    It later turned out that the Lancet letter had been organized and drafted by Peter Daszak, president of the EcoHealth Alliance of New York. Daszak’s organization funded coronavirus research at the Wuhan Institute of Virology. If the SARS2 virus had indeed escaped from research he funded, Daszak would be potentially culpable. This acute conflict of interest was not declared to the Lancet’s readers. To the contrary, the letter concluded, “We declare no competing interests.”

    Peter Daszak, a member of the World Health Organization (WHO) team investigating the origins of the COVID-19 coronavirus, talks on his cellphone at the Hilton Wuhan Optics Valley in Wuhan. Credit: Hector Retamal/AFP/Getty Images

    Virologists like Daszak had much at stake in the assigning of blame for the pandemic. For 20 years, mostly beneath the public’s attention, they had been playing a dangerous game. In their laboratories they routinely created viruses more dangerous than those that exist in nature. They argued that they could do so safely, and that by getting ahead of nature they could predict and prevent natural “spillovers,” the cross-over of viruses from an animal host to people. If SARS2 had indeed escaped from such a laboratory experiment, a savage blowback could be expected, and the storm of public indignation would affect virologists everywhere, not just in China. “It would shatter the scientific edifice top to bottom,” an MIT Technology Review editor, Antonio Regalado, said in March 2020.

    A second statement that had enormous influence in shaping public attitudes was a letter (in other words an opinion piece, not a scientific article) published on 17 March 2020 in the journal Nature Medicine. Its authors were a group of virologists led by Kristian G. Andersen of the Scripps Research Institute. “Our analyses clearly show that SARS-CoV-2 is not a laboratory construct or a purposefully manipulated virus,” the five virologists declared in the second paragraph of their letter.

    Unfortunately, this was another case of poor science, in the sense defined above. True, some older methods of cutting and pasting viral genomes retain tell-tale signs of manipulation. But newer methods, called “no-see-um” or “seamless” approaches, leave no defining marks. Nor do other methods for manipulating viruses such as serial passage, the repeated transfer of viruses from one culture of cells to another. If a virus has been manipulated, whether with a seamless method or by serial passage, there is no way of knowing that this is the case. Andersen and his colleagues were assuring their readers of something they could not know.

    The discussion part of their letter begins, “It is improbable that SARS-CoV-2 emerged through laboratory manipulation of a related SARS-CoV-like coronavirus.” But wait, didn’t the lead say the virus had clearly not been manipulated? The authors’ degree of certainty seemed to slip several notches when it came to laying out their reasoning.

    The reason for the slippage is clear once the technical language has been penetrated. The two reasons the authors give for supposing manipulation to be improbable are decidedly inconclusive.

    First, they say that the spike protein of SARS2 binds very well to its target, the human ACE2 receptor, but does so in a different way from that which physical calculations suggest would be the best fit. Therefore the virus must have arisen by natural selection, not manipulation.

    If this argument seems hard to grasp, it’s because it’s so strained. The authors’ basic assumption, not spelt out, is that anyone trying to make a bat virus bind to human cells could do so in only one way. First they would calculate the strongest possible fit between the human ACE2 receptor and the spike protein with which the virus latches onto it. They would then design the spike protein accordingly (by selecting the right string of amino acid units that compose it). Since the SARS2 spike protein is not of this calculated best design, the Andersen paper says, therefore it can’t have been manipulated.

    But this ignores the way that virologists do in fact get spike proteins to bind to chosen targets, which is not by calculation but by splicing in spike protein genes from other viruses or by serial passage. With serial passage, each time the virus’s progeny are transferred to new cell cultures or animals, the more successful are selected until one emerges that makes a really tight bind to human cells. Natural selection has done all the heavy lifting. The Andersen paper’s speculation about designing a viral spike protein through calculation has no bearing on whether or not the virus was manipulated by one of the other two methods.

    The authors’ second argument against manipulation is even more contrived. Although most living things use DNA as their hereditary material, a number of viruses use RNA, DNA’s close chemical cousin. But RNA is difficult to manipulate, so researchers working on coronaviruses, which are RNA-based, will first convert the RNA genome to DNA. They manipulate the DNA version, whether by adding or altering genes, and then arrange for the manipulated DNA genome to be converted back into infectious RNA.

    Only a certain number of these DNA backbones have been described in the scientific literature. Anyone manipulating the SARS2 virus “would probably” have used one of these known backbones, the Andersen group writes, and since SARS2 is not derived from any of them, therefore it was not manipulated. But the argument is conspicuously inconclusive. DNA backbones are quite easy to make, so it’s obviously possible that SARS2 was manipulated using an unpublished DNA backbone.

    And that’s it. These are the two arguments made by the Andersen group in support of their declaration that the SARS2 virus was clearly not manipulated. And this conclusion, grounded in nothing but two inconclusive speculations, convinced the world’s press that SARS2 could not have escaped from a lab. A technical critique of the Andersen letter takes it down in harsher words.

    Science is supposedly a self-correcting community of experts who constantly check each other’s work. So why didn’t other virologists point out that the Andersen group’s argument was full of absurdly large holes? Perhaps because in today’s universities speech can be very costly. Careers can be destroyed for stepping out of line. Any virologist who challenges the community’s declared view risks having his next grant application turned down by the panel of fellow virologists that advises the government grant distribution agency.

    The Daszak and Andersen letters were really political, not scientific, statements, yet were amazingly effective. Articles in the mainstream press repeatedly stated that a consensus of experts had ruled lab escape out of the question or extremely unlikely. Their authors relied for the most part on the Daszak and Andersen letters, failing to understand the yawning gaps in their arguments. Mainstream newspapers all have science journalists on their staff, as do the major networks, and these specialist reporters are supposed to be able to question scientists and check their assertions. But the Daszak and Andersen assertions went largely unchallenged.

    Doubts about natural emergence. Natural emergence was the media’s preferred theory until around February 2021 and the visit by a World Health Organization (WHO) commission to China. The commission’s composition and access were heavily controlled by the Chinese authorities. Its members, who included the ubiquitous Daszak, kept asserting before, during, and after their visit that lab escape was extremely unlikely. But this was not quite the propaganda victory the Chinese authorities may have been hoping for. What became clear was that the Chinese had no evidence to offer the commission in support of the natural emergence theory.

    This was surprising because both the SARS1 and MERS viruses had left copious traces in the environment. The intermediary host species of SARS1 was identified within four months of the epidemic’s outbreak, and the host of MERS within nine months. Yet some 15 months after the SARS2 pandemic began, and after a presumably intensive search, Chinese researchers had failed to find either the original bat population, or the intermediate species to which SARS2 might have jumped, or any serological evidence that any Chinese population, including that of Wuhan, had ever been exposed to the virus prior to December 2019. Natural emergence remained a conjecture which, however plausible to begin with, had gained not a shred of supporting evidence in over a year.

    And as long as that remains the case, it’s logical to pay serious attention to the alternative conjecture, that SARS2 escaped from a lab.

    Why would anyone want to create a novel virus capable of causing a pandemic? Ever since virologists gained the tools for manipulating a virus’s genes, they have argued they could get ahead of a potential pandemic by exploring how close a given animal virus might be to making the jump to humans. And that justified lab experiments in enhancing the ability of dangerous animal viruses to infect people, virologists asserted.

    With this rationale, they have recreated the 1918 flu virus, shown how the almost extinct polio virus can be synthesized from its published DNA sequence, and introduced a smallpox gene into a related virus.

    These enhancements of viral capabilities are known blandly as gain-of-function experiments. With coronaviruses, there was particular interest in the spike proteins, which jut out all around the spherical surface of the virus and pretty much determine which species of animal it will target. In 2000 Dutch researchers, for instance, earned the gratitude of rodents everywhere by genetically engineering the spike protein of a mouse coronavirus so that it would attack only cats.

    The spike proteins on the coronavirus’s surface determine which animal it can infect. Credit: CDC

    Virologists started studying bat coronaviruses in earnest after these turned out to be the source of both the SARS1 and MERS epidemics. In particular, researchers wanted to understand what changes needed to occur in a bat virus’s spike proteins before it could infect people.

    Researchers at the Wuhan Institute of Virology, led by China’s leading expert on bat viruses, Shi Zheng-li or “Bat Lady,” mounted frequent expeditions to the bat-infested caves of Yunnan in southern China and collected around a hundred different bat coronaviruses.

    Shi then teamed up with Ralph S. Baric, an eminent coronavirus researcher at the University of North Carolina. Their work focused on enhancing the ability of bat viruses to attack humans so as to “examine the emergence potential (that is, the potential to infect humans) of circulating bat CoVs [coronaviruses].” In pursuit of this aim, in November 2015 they created a novel virus by taking the backbone of the SARS1 virus and replacing its spike protein with one from a bat virus (known as SHC014-CoV). This manufactured virus was able to infect the cells of the human airway, at least when tested against a lab culture of such cells.

    The SHC014-CoV/SARS1 virus is known as a chimera because its genome contains genetic material from two strains of virus. If the SARS2 virus were to have been cooked up in Shi’s lab, then its direct prototype would have been the SHC014-CoV/SARS1 chimera, the potential danger of which concerned many observers and prompted intense discussion.

    “If the virus escaped, nobody could predict the trajectory,” said Simon Wain-Hobson, a virologist at the Pasteur Institute in Paris.

    Baric and Shi referred to the obvious risks in their paper but argued they should be weighed against the benefit of foreshadowing future spillovers. Scientific review panels, they wrote, “may deem similar studies building chimeric viruses based on circulating strains too risky to pursue.” Given various restrictions being placed on gain-of function (GOF) research, matters had arrived in their view at “a crossroads of GOF research concerns the potential to prepare for and mitigate future outbreaks must be weighed against the risk of creating more dangerous pathogens. In developing policies moving forward, it is important to consider the value of the data generated by these studies and whether these types of chimeric virus studies warrant further investigation versus the inherent risks involved.”

    That statement was made in 2015. From the hindsight of 2021, one can say that the value of gain-of-function studies in preventing the SARS2 epidemic was zero. The risk was catastrophic, if indeed the SARS2 virus was generated in a gain-of-function experiment.

    Inside the Wuhan Institute of Virology. Baric had developed, and taught Shi, a general method for engineering bat coronaviruses to attack other species. The specific targets were human cells grown in cultures and humanized mice. These laboratory mice, a cheap and ethical stand-in for human subjects, are genetically engineered to carry the human version of a protein called ACE2 that studs the surface of cells that line the airways.

    Shi returned to her lab at the Wuhan Institute of Virology and resumed the work she had started on genetically engineering coronaviruses to attack human cells. How can we be so sure?

    A May 20, 2020, photo of the Wuhan Institute of Virology in Wuhan, where research on bat coronaviruses was conducted. Credit: Kyodo News/Getty Images

    Because, by a strange twist in the story, her work was funded by the National Institute of Allergy and Infectious Diseases (NIAID), a part of the US National Institutes of Health (NIH). And grant proposals that funded her work, which are a matter of public record, specify exactly what she planned to do with the money.

    The grants were assigned to the prime contractor, Daszak of the EcoHealth Alliance, who subcontracted them to Shi. Here are extracts from the grants for fiscal years 2018 and 2019. (“CoV” stands for coronavirus and “S protein” refers to the virus’s spike protein.)

    “Test predictions of CoV inter-species transmission. Predictive models of host range (i.e. emergence potential) will be tested experimentally using reverse genetics, pseudovirus and receptor binding assays, and virus infection experiments across a range of cell cultures from different species and humanized mice.”

    “We will use S protein sequence data, infectious clone technology, in vitro and in vivo infection experiments and analysis of receptor binding to test the hypothesis that % divergence thresholds in S protein sequences predict spillover potential.”

    What this means, in non-technical language, is that Shi set out to create novel coronaviruses with the highest possible infectivity for human cells. Her plan was to take genes that coded for spike proteins possessing a variety of measured affinities for human cells, ranging from high to low. She would insert these spike genes one by one into the backbone of a number of viral genomes (“reverse genetics” and “infectious clone technology”), creating a series of chimeric viruses. These chimeric viruses would then be tested for their ability to attack human cell cultures (“in vitro”) and humanized mice (“in vivo”). And this information would help predict the likelihood of “spillover,” the jump of a coronavirus from bats to people.

    The methodical approach was designed to find the best combination of coronavirus backbone and spike protein for infecting human cells. The approach could have generated SARS2-like viruses, and indeed may have created the SARS2 virus itself with the right combination of virus backbone and spike protein.

    It cannot yet be stated that Shi did or did not generate SARS2 in her lab because her records have been sealed, but it seems she was certainly on the right track to have done so. “It is clear that the Wuhan Institute of Virology was systematically constructing novel chimeric coronaviruses and was assessing their ability to infect human cells and human-ACE2-expressing mice,” says Richard H. Ebright, a molecular biologist at Rutgers University and leading expert on biosafety.

    “It is also clear,” Ebright said, “that, depending on the constant genomic contexts chosen for analysis, this work could have produced SARS-CoV-2 or a proximal progenitor of SARS-CoV-2.” “Genomic context” refers to the particular viral backbone used as the testbed for the spike protein.

    The lab escape scenario for the origin of the SARS2 virus, as should by now be evident, is not mere hand-waving in the direction of the Wuhan Institute of Virology. It is a detailed proposal, based on the specific project being funded there by the NIAID.

    Even if the grant required the work plan described above, how can we be sure that the plan was in fact carried out? For that we can rely on the word of Daszak, who has been much protesting for the last 15 months that lab escape was a ludicrous conspiracy theory invented by China-bashers.

    On December 9, 2019, before the outbreak of the pandemic became generally known, Daszak gave an interview in which he talked in glowing terms of how researchers at the Wuhan Institute of Virology had been reprogramming the spike protein and generating chimeric coronaviruses capable of infecting humanized mice.

    “And we have now found, you know, after 6 or 7 years of doing this, over 100 new SARS-related coronaviruses, very close to SARS,” Daszak says around minute 28 of the interview. “Some of them get into human cells in the lab, some of them can cause SARS disease in humanized mice models and are untreatable with therapeutic monoclonals and you can’t vaccinate against them with a vaccine. So, these are a clear and present danger:

    Interviewer: You say these are diverse coronaviruses and you can’t vaccinate against them, and no anti-virals — so what do we do?

    Daszak: Well I think…coronaviruses — you can manipulate them in the lab pretty easily. Spike protein drives a lot of what happen with coronavirus, in zoonotic risk. So you can get the sequence, you can build the protein, and we work a lot with Ralph Baric at UNC to do this. Insert into the backbone of another virus and do some work in the lab. So you can get more predictive when you find a sequence. You’ve got this diversity. Now the logical progression for vaccines is, if you are going to develop a vaccine for SARS, people are going to use pandemic SARS, but let’s insert some of these other things and get a better vaccine.

    The insertions he referred to perhaps included an element called the furin cleavage site, discussed below, which greatly increases viral infectivity for human cells.

    In disjointed style, Daszak is referring to the fact that once you have generated a novel coronavirus that can attack human cells, you can take the spike protein and make it the basis for a vaccine.

    One can only imagine Daszak’s reaction when he heard of the outbreak of the epidemic in Wuhan a few days later. He would have known better than anyone the Wuhan Institute’s goal of making bat coronaviruses infectious to humans, as well as the weaknesses in the institute’s defense against their own researchers becoming infected.

    But instead of providing public health authorities with the plentiful information at his disposal, he immediately launched a public relations campaign to persuade the world that the epidemic couldn’t possibly have been caused by one of the institute’s souped-up viruses. “The idea that this virus escaped from a lab is just pure baloney. It’s simply not true,” he declared in an April 2020 interview.

    The safety arrangements at the Wuhan Institute of Virology. Daszak was possibly unaware of, or perhaps he knew all too well, the long history of viruses escaping from even the best run laboratories. The smallpox virus escaped three times from labs in England in the 1960’s and 1970’s, causing 80 cases and 3 deaths. Dangerous viruses have leaked out of labs almost every year since. Coming to more recent times, the SARS1 virus has proved a true escape artist, leaking from laboratories in Singapore, Taiwan, and no less than four times from the Chinese National Institute of Virology in Beijing.

    One reason for SARS1 being so hard to handle is that there were no vaccines available to protect laboratory workers. As Daszak mentioned in the December 19 interview quoted above, the Wuhan researchers too had been unable to develop vaccines against the coronaviruses they had designed to infect human cells. They would have been as defenseless against the SARS2 virus, if it were generated in their lab, as their Beijing colleagues were against SARS1.

    A second reason for the severe danger of novel coronaviruses has to do with the required levels of lab safety. There are four degrees of safety, designated BSL1 to BSL4, with BSL4 being the most restrictive and designed for deadly pathogens like the Ebola virus.

    The Wuhan Institute of Virology had a new BSL4 lab, but its state of readiness considerably alarmed the State Department inspectors who visited it from the Beijing embassy in 2018. “The new lab has a serious shortage of appropriately trained technicians and investigators needed to safely operate this high-containment laboratory,” the inspectors wrote in a cable of January 19, 2018.

    The real problem, however, was not the unsafe state of the Wuhan BSL4 lab but the fact that virologists worldwide don’t like working in BSL4 conditions. You have to wear a space suit, do operations in closed cabinets, and accept that everything will take twice as long. So the rules assigning each kind of virus to a given safety level were laxer than some might think was prudent.

    Before 2020, the rules followed by virologists in China and elsewhere required that experiments with the SARS1 and MERS viruses be conducted in BSL3 conditions. But all other bat coronaviruses could be studied in BSL2, the next level down. BSL2 requires taking fairly minimal safety precautions, such as wearing lab coats and gloves, not sucking up liquids in a pipette, and putting up biohazard warning signs. Yet a gain-of-function experiment conducted in BSL2 might produce an agent more infectious than either SARS1 or MERS. And if it did, then lab workers would stand a high chance of infection, especially if unvaccinated.

    Much of Shi’s work on gain-of-function in coronaviruses was performed at the BSL2 safety level, as is stated in her publications and other documents. She has said in an interview with Science magazine that “[t]he coronavirus research in our laboratory is conducted in BSL-2 or BSL-3 laboratories.”

    Shi Zheng-li.

    “It is clear that some or all of this work was being performed using a biosafety standard — biosafety level 2, the biosafety level of a standard US dentist’s office — that would pose an unacceptably high risk of infection of laboratory staff upon contact with a virus having the transmission properties of SARS-CoV-2,” Ebright says.

    “It also is clear,” he adds, “that this work never should have been funded and never should have been performed.”

    This is a view he holds regardless of whether or not the SARS2 virus ever saw the inside of a lab.

    Concern about safety conditions at the Wuhan lab was not, it seems, misplaced. According to a fact sheet issued by the State Department on January 15, 2021, “The U.S. government has reason to believe that several researchers inside the WIV became sick in autumn 2019, before the first identified case of the outbreak, with symptoms consistent with both COVID-19 and common seasonal illnesses.”

    David Asher, a fellow of the Hudson Institute and former consultant to the State Department, provided more detail about the incident at a seminar. Knowledge of the incident came from a mix of public information and “some high end information collected by our intelligence community,” he said. Three people working at a BSL3 lab at the institute fell sick within a week of each other with severe symptoms that required hospitalization. This was “the first known cluster that we’re aware of, of victims of what we believe to be COVID-19.” Influenza could not completely be ruled out but seemed unlikely in the circumstances, he said.

    Comparing the rival scenarios of SARS2 origin. The evidence above adds up to a serious case that the SARS2 virus could have been created in a lab, from which it then escaped. But the case, however substantial, falls short of proof. Proof would consist of evidence from the Wuhan Institute of Virology, or related labs in Wuhan, that SARS2 or a predecessor virus was under development there. For lack of access to such records, another approach is to take certain salient facts about the SARS2 virus and ask how well each is explained by the two rival scenarios of origin, those of natural emergence and lab escape. Here are four tests of the two hypotheses. A couple have some technical detail, but these are among the most persuasive for those who may care to follow the argument.

    1) The place of origin

    Start with geography. The two closest known relatives of the SARS2 virus were collected from bats living in caves in Yunnan, a province of southern China. If the SARS2 virus had first infected people living around the Yunnan caves, that would strongly support the idea that the virus had spilled over to people naturally. But this isn’t what happened. The pandemic broke out 1,500 kilometers away, in Wuhan.

    Beta-coronaviruses, the family of bat viruses to which SARS2 belongs, infect the horseshoe bat Rhinolophus affinis, which ranges across southern China. The bats’ range is 50 kilometers, so it’s unlikely that any made it to Wuhan. In any case, the first cases of the COVID-19 pandemic probably occurred in September, when temperatures in Hubei province are already cold enough to send bats into hibernation.

    Bats hibernating. Credit: Anita Glover

    What if the bat viruses infected some intermediate host first? You would need a longstanding population of bats in frequent proximity with an intermediate host, which in turn must often cross paths with people. All these exchanges of virus must take place somewhere outside Wuhan, a busy metropolis which so far as is known is not a natural habitat of Rhinolophus bat colonies. The infected person (or animal) carrying this highly transmissible virus must have traveled to Wuhan without infecting anyone else. No one in his or her family got sick. If the person jumped on a train to Wuhan, no fellow passengers fell ill.

    It’s a stretch, in other words, to get the pandemic to break out naturally outside Wuhan and then, without leaving any trace, to make its first appearance there.

    For the lab escape scenario, a Wuhan origin for the virus is a no-brainer. Wuhan is home to China’s leading center of coronavirus research where, as noted above, researchers were genetically engineering bat coronaviruses to attack human cells. They were doing so under the minimal safety conditions of a BSL2 lab. If a virus with the unexpected infectiousness of SARS2 had been generated there, its escape would be no surprise.

    2) Natural history and evolution

    The initial location of the pandemic is a small part of a larger problem, that of its natural history. Viruses don’t just make one time jumps from one species to another. The coronavirus spike protein, adapted to attack bat cells, needs repeated jumps to another species, most of which fail, before it gains a lucky mutation. Mutation — a change in one of its RNA units — causes a different amino acid unit to be incorporated into its spike protein and makes the spike protein better able to attack the cells of some other species.

    Through several more such mutation-driven adjustments, the virus adapts to its new host, say some animal with which bats are in frequent contact. The whole process then resumes as the virus moves from this intermediate host to people.

    In the case of SARS1, researchers have documented the successive changes in its spike protein as the virus evolved step by step into a dangerous pathogen. After it had gotten from bats into civets, there were six further changes in its spike protein before it became a mild pathogen in people. After a further 14 changes, the virus was much better adapted to humans, and with a further four, the epidemic took off.

    But when you look for the fingerprints of a similar transition in SARS2, a strange surprise awaits. The virus has changed hardly at all, at least until recently. From its very first appearance, it was well adapted to human cells. Researchers led by Alina Chan of the Broad Institute compared SARS2 with late stage SARS1, which by then was well adapted to human cells, and found that the two viruses were similarly well adapted. “By the time SARS-CoV-2 was first detected in late 2019, it was already pre-adapted to human transmission to an extent similar to late epidemic SARS-CoV,” they wrote.

    Even those who think lab origin unlikely agree that SARS2 genomes are remarkably uniform. Baric writes that “early strains identified in Wuhan, China, showed limited genetic diversity, which suggests that the virus may have been introduced from a single source.”

    A single source would of course be compatible with lab escape, less so with the massive variation and selection which is evolution’s hallmark way of doing business.

    The uniform structure of SARS2 genomes gives no hint of any passage through an intermediate animal host, and no such host has been identified in nature.

    Proponents of natural emergence suggest that SARS2 incubated in a yet-to-be found human population before gaining its special properties. Or that it jumped to a host animal outside China.

    All these conjectures are possible, but strained. Proponents of a lab leak have a simpler explanation. SARS2 was adapted to human cells from the start because it was grown in humanized mice or in lab cultures of human cells, just as described in Daszak’s grant proposal. Its genome shows little diversity because the hallmark of lab cultures is uniformity.

    Proponents of laboratory escape joke that of course the SARS2 virus infected an intermediary host species before spreading to people, and that they have identified it — a humanized mouse from the Wuhan Institute of Virology.

    3) The furin cleavage site

    The furin cleavage site is a minute part of the virus’s anatomy but one that exerts great influence on its infectivity. It sits in the middle of the SARS2 spike protein. It also lies at the heart of the puzzle of where the virus came from.

    Credit: SciTechDaily

    The spike protein has two sub-units with different roles. The first, called S1, recognizes the virus’s target, a protein called angiotensin converting enzyme-2 (or ACE2) which studs the surface of cells lining the human airways. The second, S2, helps the virus, once anchored to the cell, to fuse with the cell’s membrane. After the virus’s outer membrane has coalesced with that of the stricken cell, the viral genome is injected into the cell, hijacks its protein-making machinery and forces it to generate new viruses.

    But this invasion cannot begin until the S1 and S2 subunits have been cut apart. And there, right at the S1/S2 junction, is the furin cleavage site that ensures the spike protein will be cleaved in exactly the right place.

    The virus, a model of economic design, does not carry its own cleaver. It relies on the cell to do the cleaving for it. Human cells have a protein cutting tool on their surface known as furin. Furin will cut any protein chain that carries its signature target cutting site. This is the sequence of amino acid units proline-arginine-arginine-alanine, or PRRA in the code that refers to each amino acid by a letter of the alphabet. PRRA is the amino acid sequence at the core of SARS2’s furin cleavage site.

    Viruses have all kinds of clever tricks, so why does the furin cleavage site stand out? Because of all known SARS-related beta-coronaviruses, only SARS2 possesses a furin cleavage site. All the other viruses have their S2 unit cleaved at a different site and by a different mechanism.

    How then did SARS2 acquire its furin cleavage site? Either the site evolved naturally, or it was inserted by researchers at the S1/S2 junction in a gain-of-function experiment.

    Consider natural origin first. Two ways viruses evolve are by mutation and by recombination. Mutation is the process of random change in DNA (or RNA for coronaviruses) that usually results in one amino acid in a protein chain being switched for another. Many of these changes harm the virus but natural selection retains the few that do something useful. Mutation is the process by which the SARS1 spike protein gradually switched its preferred target cells from those of bats to civets, and then to humans.

    Mutation seems a less likely way for SARS2’s furin cleavage site to be generated, even though it can’t completely be ruled out. The site’s four amino acid units are all together, and all at just the right place in the S1/S2 junction. Mutation is a random process triggered by copying errors (when new viral genomes are being generated) or by chemical decay of genomic units. So it typically affects single amino acids at different spots in a protein chain. A string of amino acids like that of the furin cleavage site is much more likely to be acquired all together through a quite different process known as recombination.

    Recombination is an inadvertent swapping of genomic material that occurs when two viruses happen to invade the same cell, and their progeny are assembled with bits and pieces of RNA belonging to the other. Beta-coronaviruses will only combine with other beta-coronaviruses but can acquire, by recombination, almost any genetic element present in the collective genomic pool. What they cannot acquire is an element the pool does not possess. And no known SARS-related beta-coronavirus, the class to which SARS2 belongs, possesses a furin cleavage site.

    Proponents of natural emergence say SARS2 could have picked up the site from some as yet unknown beta-coronavirus. But bat SARS-related beta-coronaviruses evidently don’t need a furin cleavage site to infect bat cells, so there’s no great likelihood that any in fact possesses one, and indeed none has been found so far.

    The proponents’ next argument is that SARS2 acquired its furin cleavage site from people. A predecessor of SARS2 could have been circulating in the human population for months or years until at some point it acquired a furin cleavage site from human cells. It would then have been ready to break out as a pandemic.

    If this is what happened, there should be traces in hospital surveillance records of the people infected by the slowly evolving virus. But none has so far come to light. According to the WHO report on the origins of the virus, the sentinel hospitals in Hubei province, home of Wuhan, routinely monitor influenza-like illnesses and “no evidence to suggest substantial SARSCoV-2 transmission in the months preceding the outbreak in December was observed.”

    So it’s hard to explain how the SARS2 virus picked up its furin cleavage site naturally, whether by mutation or recombination.

    That leaves a gain-of-function experiment. For those who think SARS2 may have escaped from a lab, explaining the furin cleavage site is no problem at all. “Since 1992 the virology community has known that the one sure way to make a virus deadlier is to give it a furin cleavage site at the S1/S2 junction in the laboratory,” writes Steven Quay, a biotech entrepreneur interested in the origins of SARS2. “At least 11 gain-of-function experiments, adding a furin site to make a virus more infective, are published in the open literature, including [by] Dr. Zhengli Shi, head of coronavirus research at the Wuhan Institute of Virology.”

    4) A question of codons

    There’s another aspect of the furin cleavage site that narrows the path for a natural emergence origin even further.

    As everyone knows (or may at least recall from high school), the genetic code uses three units of DNA to specify each amino acid unit of a protein chain. When read in groups of 3, the 4 different kinds of DNA unit can specify 4 x 4 x 4 or 64 different triplets, or codons as they are called. Since there are only 20 kinds of amino acid, there are more than enough codons to go around, allowing some amino acids to be specified by more than one codon. The amino acid arginine, for instance, can be designated by any of the six codons CGU, CGC, CGA, CGG, AGA or AGG, where A, U, G and C stand for the four different kinds of unit in RNA.

    Here’s where it gets interesting. Different organisms have different codon preferences. Human cells like to designate arginine with the codons CGT, CGC or CGG. But CGG is coronavirus’s least popular codon for arginine. Keep that in mind when looking at how the amino acids in the furin cleavage site are encoded in the SARS2 genome.

    Now the functional reason why SARS2 has a furin cleavage site, and its cousin viruses don’t, can be seen by lining up (in a computer) the string of nearly 30,000 nucleotides in its genome with those of its cousin coronaviruses, of which the closest so far known is one called RaTG13. Compared with RaTG13, SARS2 has a 12-nucleotide insert right at the S1/S2 junction. The insert is the sequence T-CCT-CGG-CGG-GC. The CCT codes for proline, the two CGG’s for two arginines, and the GC is the beginning of a GCA codon that codes for alanine.

    There are several curious features about this insert but the oddest is that of the two side-by-side CGG codons. Only 5 percent of SARS2’s arginine codons are CGG, and the double codon CGG-CGG has not been found in any other beta-coronavirus. So how did SARS2 acquire a pair of arginine codons that are favored by human cells but not by coronaviruses?

    Proponents of natural emergence have an up-hill task to explain all the features of SARS2’s furin cleavage site. They have to postulate a recombination event at a site on the virus’s genome where recombinations are rare, and the insertion of a 12-nucleotide sequence with a double arginine codon unknown in the beta-coronavirus repertoire, at the only site in the genome that would significantly expand the virus’s infectivity.

    “Yes, but your wording makes this sound unlikely — viruses are specialists at unusual events,” is the riposte of David L. Robertson, a virologist at the University of Glasgow who regards lab escape as a conspiracy theory. “Recombination is naturally very, very frequent in these viruses, there are recombination breakpoints in the spike protein and these codons appear unusual exactly because we’ve not sampled enough.”

    Robertson is correct that evolution is always producing results that may seem unlikely but in fact are not. Viruses can generate untold numbers of variants but we see only the one-in-a-billion that natural selection picks for survival. But this argument could be pushed too far. For instance, any result of a gain-of-function experiment could be explained as one that evolution would have arrived at in time. And the numbers game can be played the other way. For the furin cleavage site to arise naturally in SARS2, a chain of events has to happen, each of which is quite unlikely for the reasons given above. A long chain with several improbable steps is unlikely to ever be completed.

    For the lab escape scenario, the double CGG codon is no surprise. The human-preferred codon is routinely used in labs. So anyone who wanted to insert a furin cleavage site into the virus’s genome would synthesize the PRRA-making sequence in the lab and would be likely to use CGG codons to do so.

    “When I first saw the furin cleavage site in the viral sequence, with its arginine codons, I said to my wife it was the smoking gun for the origin of the virus,” said David Baltimore, an eminent virologist and former president of CalTech. “These features make a powerful challenge to the idea of a natural origin for SARS2,” he said. [1]

    A third scenario of origin

    There’s a variation on the natural emergence scenario that’s worth considering. This is the idea that SARS2 jumped directly from bats to humans, without going through an intermediate host as SARS1 and MERS did. A leading advocate is the virologist David Robertson who notes that SARS2 can attack several other species besides humans. He believes the virus evolved a generalist capability while still in bats. Because the bats it infects are widely distributed in southern and central China, the virus had ample opportunity to jump to people, even though it seems to have done so on only one known occasion. Robertson’s thesis explains why no one has so far found a trace of SARS2 in any intermediate host or in human populations surveilled before December 2019. It would also explain the puzzling fact that SARS2 has not changed since it first appeared in humans — it didn’t need to because it could already attack human cells efficiently.

    One problem with this idea, though, is that if SARS2 jumped from bats to people in a single leap and hasn’t changed much since, it should still be good at infecting bats. And it seems it isn’t.

    “Tested bat species are poorly infected by SARS-CoV-2 and they are therefore unlikely to be the direct source for human infection,” write a scientific group skeptical of natural emergence.

    Still, Robertson may be onto something. The bat coronaviruses of the Yunnan caves can infect people directly. In April 2012 six miners clearing bat guano from the Mojiang mine contracted severe pneumonia with COVID-19-like symptoms and three eventually died. A virus isolated from the Mojiang mine, called RaTG13, is still the closest known relative of SARS2. Much mystery surrounds the origin, reporting and strangely low affinity of RaTG13 for bat cells, as well as the nature of 8 similar viruses that Shi reports she collected at the same time but has not yet published despite their great relevance to the ancestry of SARS2. But all that is a story for another time. The point here is that bat viruses can infect people directly, though only in special conditions.

    So who else, besides miners excavating bat guano, comes into particularly close contact with bat coronaviruses? Well, coronavirus researchers do. Shi says she and her group collected more than 1,300 bat samples during some eight visits to the Mojiang cave between 2012 and 2015, and there were doubtless many expeditions to other Yunnan caves.

    Imagine the researchers making frequent trips from Wuhan to Yunnan and back, stirring up bat guano in dark caves and mines, and now you begin to see a possible missing link between the two places. Researchers could have gotten infected during their collecting trips, or while working with the new viruses at the Wuhan Institute of Virology. The virus that escaped from the lab would have been a natural virus, not one cooked up by gain of function.

    The direct-from-bats thesis is a chimera between the natural emergence and lab escape scenarios. It’s a possibility that can’t be dismissed. But against it are the facts that 1) both SARS2 and RaTG13 seem to have only feeble affinity for bat cells, so one can’t be fully confident that either ever saw the inside of a bat and 2) the theory is no better than the natural emergence scenario at explaining how SARS2 gained its furin cleavage site, or why the furin cleavage site is determined by human-preferred arginine codons instead of by the bat-preferred codons.

    Where we are so far. Neither the natural emergence nor the lab escape hypothesis can yet be ruled out. There is still no direct evidence for either. So no definitive conclusion can be reached.

    That said, the available evidence leans more strongly in one direction than the other. Readers will form their own opinion. But it seems to me that proponents of lab escape can explain all the available facts about SARS2 considerably more easily than can those who favor natural emergence.

    It’s documented that researchers at the Wuhan Institute of Virology were doing gain-of-function experiments designed to make coronaviruses infect human cells and humanized mice. This is exactly the kind of experiment from which a SARS2-like virus could have emerged. The researchers were not vaccinated against the viruses under study, and they were working in the minimal safety conditions of a BSL2 laboratory. So escape of a virus would not be at all surprising. In all of China, the pandemic broke out on the doorstep of the Wuhan institute. The virus was already well adapted to humans, as expected for a virus grown in humanized mice. It possessed an unusual enhancement, a furin cleavage site, which is not possessed by any other known SARS-related beta-coronavirus, and this site included a double arginine codon also unknown among beta-coronaviruses. What more evidence could you want, aside from the presently unobtainable lab records documenting SARS2’s creation?

    Proponents of natural emergence have a rather harder story to tell. The plausibility of their case rests on a single surmise, the expected parallel between the emergence of SARS2 and that of SARS1 and MERS. But none of the evidence expected in support of such a parallel history has yet emerged. No one has found the bat population that was the source of SARS2, if indeed it ever infected bats. No intermediate host has presented itself, despite an intensive search by Chinese authorities that included the testing of 80,000 animals. There is no evidence of the virus making multiple independent jumps from its intermediate host to people, as both the SARS1 and MERS viruses did. There is no evidence from hospital surveillance records of the epidemic gathering strength in the population as the virus evolved. There is no explanation of why a natural epidemic should break out in Wuhan and nowhere else. There is no good explanation of how the virus acquired its furin cleavage site, which no other SARS-related beta-coronavirus possesses, nor why the site is composed of human-preferred codons. The natural emergence theory battles a bristling array of implausibilities.

    The records of the Wuhan Institute of Virology certainly hold much relevant information. But Chinese authorities seem unlikely to release them given the substantial chance that they incriminate the regime in the creation of the pandemic. Absent the efforts of some courageous Chinese whistle-blower, we may already have at hand just about all of the relevant information we are likely to get for a while.

    So it’s worth trying to assess responsibility for the pandemic, at least in a provisional way, because the paramount goal remains to prevent another one. Even those who aren’t persuaded that lab escape is the more likely origin of the SARS2 virus may see reason for concern about the present state of regulation governing gain-of-function research. There are two obvious levels of responsibility: the first, for allowing virologists to perform gain-of-function experiments, offering minimal gain and vast risk the second, if indeed SARS2 was generated in a lab, for allowing the virus to escape and unleash a world-wide pandemic. Here are the players who seem most likely to deserve blame.

    1. Chinese virologists

    First and foremost, Chinese virologists are to blame for performing gain-of-function experiments in mostly BSL2-level safety conditions which were far too lax to contain a virus of unexpected infectiousness like SARS2. If the virus did indeed escape from their lab, they deserve the world’s censure for a foreseeable accident that has already caused the deaths of three million people. True, Shi was trained by French virologists, worked closely with American virologists and was following international rules for the containment of coronaviruses. But she could and should have made her own assessment of the risks she was running. She and her colleagues bear the responsibility for their actions.

    I have been using the Wuhan Institute of Virology as a shorthand for all virological activities in Wuhan. It’s possible that SARS2 was generated in some other Wuhan lab, perhaps in an attempt to make a vaccine that worked against all coronaviruses. But until the role of other Chinese virologists is clarified, Shi is the public face of Chinese work on coronaviruses, and provisionally she and her colleagues will stand first in line for opprobrium.

    2. Chinese authorities

    China’s central authorities did not generate SARS2, but they sure did their utmost to conceal the nature of the tragedy and China’s responsibility for it. They suppressed all records at the Wuhan Institute of Virology and closed down its virus databases. They released a trickle of information, much of which may have been outright false or designed to misdirect and mislead. They did their best to manipulate the WHO’s inquiry into the virus’s origins, and led the commission’s members on a fruitless run-around. So far they have proved far more interested in deflecting blame than in taking the steps necessary to prevent a second pandemic.

    3. The worldwide community of virologists

    Virologists around the world are a loose-knit professional community. They write articles in the same journals. They attend the same conferences. They have common interests in seeking funds from governments and in not being overburdened with safety regulations.

    Virologists knew better than anyone the dangers of gain-of-function research. But the power to create new viruses, and the research funding obtainable by doing so, was too tempting. They pushed ahead with gain-of-function experiments. They lobbied against the moratorium imposed on Federal funding for gain-of-function research in 2014, and it was raised in 2017.

    The benefits of the research in preventing future epidemics have so far been nil, the risks vast. If research on the SARS1 and MERS viruses could only be done at the BSL3 safety level, it was surely illogical to allow any work with novel coronaviruses at the lesser level of BSL2. Whether or not SARS2 escaped from a lab, virologists around the world have been playing with fire.

    Their behavior has long alarmed other biologists. In 2014 scientists calling themselves the Cambridge Working Group urged caution on creating new viruses. In prescient words, they specified the risk of creating a SARS2-like virus. “Accident risks with newly created ‘potential pandemic pathogens’ raise grave new concerns,” they wrote. “Laboratory creation of highly transmissible, novel strains of dangerous viruses, especially but not limited to influenza, poses substantially increased risks. An accidental infection in such a setting could trigger outbreaks that would be difficult or impossible to control.”

    When molecular biologists discovered a technique for moving genes from one organism to another, they held a public conference at Asilomar in 1975 to discuss the possible risks. Despite much internal opposition, they drew up a list of stringent safety measures that could be relaxed in future — and duly were — when the possible hazards had been better assessed.

    When the CRISPR technique for editing genes was invented, biologists convened a joint report by the US, UK and Chinese national academies of science to urge restraint on making heritable changes to the human genome. Biologists who invented gene drives have also been open about the dangers of their work and have sought to involve the public.

    You might think the SARS2 pandemic would spur virologists to re-evaluate the benefits of gain-of-function research, even to engage the public in their deliberations. But no. Many virologists deride lab escape as a conspiracy theory, and others say nothing. They have barricaded themselves behind a Chinese wall of silence which so far is working well to allay, or at least postpone, journalists’ curiosity and the public’s wrath. Professions that cannot regulate themselves deserve to get regulated by others, and this would seem to be the future that virologists are choosing for themselves.

    4. The US role in funding the Wuhan Institute of Virology [2]

    From June 2014 to May 2019, Daszak’s EcoHealth Alliance had a grant from the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, to do gain-of-function research with coronaviruses at the Wuhan Institute of Virology. Whether or not SARS2 is the product of that research, it seems a questionable policy to farm out high-risk research to foreign labs using minimal safety precautions. And if the SARS2 virus did indeed escape from the Wuhan institute, then the NIH will find itself in the terrible position of having funded a disastrous experiment that led to the death of more than 3 million worldwide, including more than half a million of its own citizens.

    The responsibility of the NIAID and NIH is even more acute because for the first three years of the grant to EcoHealth Alliance there was a moratorium on funding gain-of-function research. When the moratorium expired in 2017, it didn’t just vanish but was replaced by a reporting system, the Potential Pandemic Pathogens Control and Oversight (P3CO) Framework, which required agencies to report for review any dangerous gain-of-function work they wished to fund.

    The moratorium, referred to officially as a “pause,” specifically barred funding any gain-of-function research that increased the pathogenicity of the flu, MERS or SARS viruses. It defined gain-of-function very simply and broadly as “research that improves the ability of a pathogen to cause disease.”

    But then a footnote on p.2 of the moratorium document states that “[a]n exception from the research pause may be obtained if the head of the USG funding agency determines that the research is urgently necessary to protect the public health or national security.”

    This seemed to mean that either the director of the NIAID, Anthony Fauci, or the director of the NIH, Francis Collins, or maybe both, would have invoked the exemption in order to keep the money flowing to Shi’s gain-of-function research, and later to avoid notifying the federal reporting system of her research.

    “Unfortunately, the NIAID Director and the NIH Director exploited this loophole to issue exemptions to projects subject to the Pause –preposterously asserting the exempted research was ‘urgently necessary to protect public health or national security’—thereby nullifying the Pause,” Dr. Richard Ebright said in an interview with Independent Science News.

    But it’s not so clear that the NIH thought it necessary to invoke any loopholes. Fauci told a Senate hearing on May 11 that “the NIH and NIAID categorically has not funded gain-of-function research to be conducted in the Wuhan Institute of Virology.”

    This was a surprising statement in view of all the evidence about Shi’s experiments with enhancing coronaviruses and the language of the moratorium statute defining gain-of-function as “any research that improves the ability of a pathogen to cause disease.”

    The explanation may be one of definition. Daszak’s EcoHealth Alliance, for one, believes that the term gain-of-function applies only to enhancements of viruses that infect humans, not to animal viruses. “So gain-of-function research refers specifically to the manipulation of human viruses so as to be either more easily transmissible or to cause worse infection or be easier to spread,” an Alliance official told The Dispatch Fact Check.

    If the NIH shares the EcoHealth Alliance view that “gain of function” applies only to human viruses, that would explain why Fauci could assure the Senate it had never funded such research at the Wuhan Institute of Virology. But the legal basis of such a definition is unclear, and it differs from that of the moratorium language which was presumably applicable.


    The origins of Covid-19: A laboratory-made virus and a massive cover-up by culpable parties? An analysis

    The connection to US-based organisations’ funding to experiments in Wuhan labs, conflict of interest in articles published by researchers denying a lab leak and China’s reluctance towards releasing more information on the experiments in Wuhan lab raise a lot of questions about Covid-19’s origin.

    Daszak had admitted to developing SARS coronaviruses in China that have no vaccines, just before the pandemic started

    Wade writes that in an interview on December 9, just before the pandemic raged, Daszak had himself boasted that they have been conducting research to manipulate coronaviruses and they have developed over 100 new coronaviruses that can get into human cells in a lab, they are untreatable with antibody models and there are no vaccines against them. He also boasted that coronaviruses are pretty easy to manipulate. The relevant part can be seen at the 28-minute mark in the interview below.

    The place of SARS2 origin

    The closest known relatives of SARS2 were found in the caves of Yunnan, but SARS2 was found infecting people 1,500 KM away in Wuhan. The range of bats is around 50 K.M. it is highly unlikely they travelled from Yunnan to Wuhan.

    The theory of an intermediary person or animal travelling to Wuhan does not stand either as no one between Yunnan and Wuhan got infected by the virus. Wuhan is home to China’s top centres of coronavirus research. As noted before, scientists were able to create genetically engineered bat viruses capable of attacking humans.

    Evolution from bats to humans

    One theory suggests that the intermediary is yet to be found. Those who believe in this theory say that it is possible the jump from bat to human took place outside China. Another theory suggests directly jumps from bats to humans. In that case, the virus should not have changed much. If the virus jumped from bat to humans directly, it should have been able to infect bats as well, which is not the case.

    No strong evidence of natural emergence or leak from the lab

    There is no direct evidence of either of the theories. Till no definitive conclusion can be reached out, both natural emergence and the lab escape hypothesis have to be factored in. Notably, possibilities do weigh towards a slab leak. It is well documented that researchers at Wuhan Institute of Virology were doing gain-of-function experiments and were working under minimal safety conditions of the BSL2 lab. Thus escape from the virus would not be a surprise.

    The lab’s records can make things clearer, but the Chinese government is unlikely to release the documents. The connection to US-based organisations’ funding to experiments in Wuhan labs, conflict of interest in articles published by researchers denying a lab leak and China’s reluctance towards releasing more information on the experiments in Wuhan lab raise a lot of questions about Covid-19’s origin.

    The detailed report by Nicholas Wade on the Origin of Covid-19 virus can be read here.


    Pangolin coronavirus could jump to humans, research suggests

    Scientists at the Francis Crick Institute have found important structural similarities between SARS-CoV-2 and a pangolin coronavirus, suggesting that a pangolin coronavirus could infect humans.

    While SARS-CoV-2 is thought to have evolved from a bat coronavirus, its exact evolutionary path is still a mystery. Uncovering its history is challenging as there are likely many undiscovered bat coronaviruses and, due to differences between bat coronaviruses and SARS-CoV-2, it is thought that the virus may have passed to humans via at least one other species.

    In their study, published in Nature Communications, the scientists compared the structures of the spike proteins found on SARS-CoV-2, the most similar currently identified bat coronavirus RaTG13, and a coronavirus isolated from Malayan pangolins which were seized by authorities after being smuggled to China. They found that the pangolin virus was able to bind to receptors from both pangolins and humans. This differs to the bat coronavirus, which could not effectively bind with human or pangolin receptors.

    Antoni Wrobel, co-lead author and postdoctoral training fellow in the Structural Biology of Disease Processes Laboratory at the Crick, says: "By testing if the spike protein of a given virus can bind with cell receptors from different species, we're able to see if, in theory, the virus could infect this species."

    "Importantly here, we've shown two key things. Firstly, that this bat virus would unlikely be able to infect pangolins. And secondly that a pangolin virus could potentially infect humans."

    The team used cryo-electron microscopy to uncover in minute detail the structure of the pangolin coronavirus' spike protein, which is responsible for binding to and infecting cells. While some parts of the pangolin virus' spike were found to be incredibly similar to SARS-CoV-2, other areas differed.

    In terms of understanding the evolutionary path of SARS-CoV-2, this work does not confirm whether or not this pangolin virus is definitely part of the chain of evolution for SARS-CoV-2. But the findings do support various possible scenarios for how the coronavirus jumped from bats to humans. One potential route is that SARS-CoV-2 originated from a different, currently unknown bat coronavirus which could infect pangolins, and from this species it then moved to humans. Or alternatively, RaTG13 or a similar bat coronavirus might have merged with another coronavirus in a different intermediate species, other than a pangolin.

    Donald Benton, co-lead author and postdoctoral training fellow in the Structural Biology of Disease Processes Laboratory at the Crick, says: "We still don't have evidence to confirm the evolutionary path of SARS-CoV-2 or to prove definitively that this virus did pass through pangolins to humans."

    "However, we have shown that a pangolin virus could potentially jump to humans, so we urge caution in any contact with this species and the end of illegal smuggling and trade in pangolins to protect against this risk."

    Steve Gamblin, group leader of the Structural Biology of Disease Processes Laboratory at the Crick says: "A lot is still to be uncovered about the evolution of SARS-CoV-2, but the more we know about its history and which species it passed through, the more we understand about how it works, and how it may continue to evolve."

    This work builds upon previous studies from the Crick team, including research published in July 2020, which found that the bat coronavirus RaTG13 could not effectively bind to human receptors.

    The team are continuing to examine the spikes of SARS-CoV-2 and related coronaviruses, including other bat viruses, to better understand the mechanisms of infection and evolution.


    Understanding viruses and challenges in microbiology

    Virology holds a central position in both microbiology and public perception, never more than now as we face the challenge of a new viral pathogen. This section focuses on viruses, their structure and how we can manipulate viruses to benefit society.

    Virology and viral disease

    Nicola J. Stonehouse and Natalie Kingston

    Viruses infect all forms of life, and while they can be extremely variable, the survival and propagation of all viruses is dependent upon living host cells. At their simplest, they are protein shells, with a nucleic acid centre. The shells (called capsids) protect the nucleic acid and serve to deliver this to new cells in order to spread infection. It is the nucleic acid that initiates disease. Cells can therefore become factories for the production of new viruses that then go on and infect other cells within the same host or infect new hosts.

    © Nicola Stonehouse

    Although it is viruses such as Ebola and Zika that make the headlines, information from studies of a range of viruses is what informs the development of prophylactic vaccines and therapeutic treatments. Indeed, understanding such details of virus structure and lifecycle has revealed parallels in simple viruses that infect bacteria and yeast with those that infect plants and mammals. However, small changes can have big consequences in terms of both severity of infection and susceptibility of the host. Furthermore, new viruses are always emerging. This is mainly due to the speed at which the viral genomes are copied and the errors that can be introduced as a result. This ability to change quickly can mean that viruses can &rsquojump&rsquo to infect new species.

    Ongoing research is essential in order to better understand viruses, and to be in a position to respond rapidly to new and re-emerging viral disease.

    Nicola J. Stonehouse

    Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, West Yorkshire, UK

    Nicola Stonehouse was awarded a PhD in 1992 on dental enamel development. She then moved from studies of inorganic crystals to protein crystals and structural studies on RNA bacteriophages. Postdoctoral studies took her to Uppsala and Leeds and, in 1997, she was awarded an MRC Career Development Fellowship. Her research moved from phage to picornaviruses, maintaining a strong interest in RNA. She was appointed Lecturer in Leeds in 2001, then promoted to Senior Lecturer and to Chair of Molecular Virology in 2013.

    Natalie Kingston

    Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, West Yorkshire, UK

    Natalie Kingston completed a PhD at Monash University, Australia, in 2017. Her research focused on the generation of chimeric virus-like particle vaccines against Plasmodium. She then moved to the University of Leeds where she currently works on the characterisation of enteroviruses and the development of enterovirus vaccines with Nicola Stonehouse and David Rowlands.

    What is the best career decision you&rsquove ever made?

    Nicola: After my PhD, I moved fields to start working on viruses. At the time, this allowed me to translate my skills to a new biological problem, which was bacteriophage structure. But this opened up the world of viruses to me and I&rsquom still fascinated by the virus lifecycle and of finding new antiviral strategies.

    Natalie: Moving to Leeds and changing specialisation to molecular virology. This change has opened up new research areas for me that continue to be both exciting and have the potential to improve vaccine design.

    Why does microbiology matter?

    Nicola: Microbes are everywhere and affect almost all aspects of our lives. Harnessing the power of microbiology can therefore bring health, environmental, social, cultural, industrial and economic benefits to our society.

    Viruses: the good, the bad and the useful

    Hollie French, Elizaveta Elshina, Emmanuelle Pitre and Aartjan te Velthuis

    Viruses are the most abundant and perhaps most diverse biological entities on Earth. They are simple life forms and are entirely dependent on hijacking host cells to replicate their genomes. However, contrary to common belief, not all viruses cause disease, since some are beneficial. By studying viruses, we can learn about the biology of host cells and organisms, develop strategies against viral disease and manipulate viruses for our own purposes.

    Some viruses are only a single self-replicating gene, while others can encode almost a thousand proteins and be the size of a bacterium. Life cycles also vary among viruses, with some lasting millions of years and others less than an hour. Yet, in spite of vast structural and molecular differences, all viruses need to gain entry into a cell, find a site to replicate, and spread.

    Binding and entry

    Virus entry can only occur if a cell expresses surface proteins that a virus can bind to. Because this is not always the case, virus infections are restricted to specific cell types, organs and organisms. Knowledge of a virus&rsquo tropism is important for estimating the potential that a virus emerges from a reservoir population and causes an outbreak in another species. For instance, a recently discovered bat influenza virus can enter human cells via the same receptor that it uses to enter bat cells, suggesting that bat flu might spill over into the human population.

    Once inside a cell, viruses move their genetic material to sites of replication. Researchers can follow that movement by tracking single, fluorescently labelled viruses using a microscope. Studies of the interaction of the virus with the host have revealed how viruses use the cytoskeleton and intracellular membranes for viral translocation and replication, but also the importance of these host components for normal cellular signalling, movement and immunity.

    Some viruses depend on the machinery in the nucleus and thus need to cross the nuclear membrane as well. To do this, influenza viruses and HIV-1 mimick host cell signals and employ cellular importins to carry their genome into the nucleus. In the nucleus, retroviruses express their genes by integrating them into the host genome. Such integration events can lead to cancer, but also shape animal evolution. One striking example is the &lsquodomestication&rsquo of the retrovirus HERV-W envelope protein, now known as syncytin, as a component for placenta formation in mammals.

    Replication and adaptation

    Whether they replicate in the cytoplasm or nucleus, all viral genomes are copied by a polymerase. X-ray crystallography and electron microscopy techniques have enabled researchers to reveal the structure of these enzymes and develop drugs that frustrate viral replication. Some antiviral strategies can also exploit the high mutation rate of some viruses by pushing the error rate even higher, ultimately causing the viral population to collapse.

    To prevent viruses from stealing their resources, cells express sensors that can identify viral genomes and proteins. The activation of these sensors triggers signalling, prevents virus spread and clears the infection. Many viruses encode proteins that can suppress or prevent these innate immune responses, but their function needs to be adapted to a host. Emerging viruses thus often trigger stronger responses than adapted viruses.

    Vectors and spread

    When new copies of the viral genome are ready to leave the host cell, some viruses fuse the infected cell with a neighbouring cell to allow faster spread. Other viruses condense their genome inside a protective protein shell with the right receptors to get the new virus to the next cell. To condense their genome, some herpesviruses and bacteriophages use a powerful molecular motor that can build up a pressure of 50 atmospheres!

    Ultimately, a virus may need to spread between organisms. It can then rely on the natural behaviour of the host or a vector, such as a tick, or manipulate its host&rsquos behaviour. The rabies virus, for instance, uses a snake-venom-like compound that makes animals aggressive and froth at the mouth with virus-laden saliva in order to increase the chance that a bite will spread the virus. Similarly, some baculoviruses can turn caterpillars into &lsquozombies&rsquo that climb up to high leaves and burst, spreading infectious virus particles to healthy caterpillars below.

    Although viruses use their host, they are also incredibly useful. Molecular biology uses viral enzymes to manipulate RNA and DNA. Moreover, we can now alter viral receptors to re-target viruses to specific cells, such as cancerous cells. In addition, we can create attenuated viruses that can only proliferate in cancer cells, which often lack antiviral sensors we can lyse tumours and keep healthy tissues, which are still able to control the infection, unharmed.

    Viruses are masters at infecting cells, utilising life&rsquos diverse abundance of molecules, systems and behaviours for their propagation. By studying them, we are learning from their expertise about ourselves and other organisms. This knowledge, combined with advances in other scientific fields, is enabling us to re-engineer viruses for our own purposes. Viruses may not only be the most abundant and diverse biological entities, but also some of the most useful.

    Hollie French

    Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 0QQ, UK

    Hollie French is a Research Assistant. She holds a BA in Natural Sciences (University of Cambridge) and now works on flu aberrant replication and innate immunity at the University of Cambridge. Her interests are in infectious disease virology and public health. She was previously an intern in the Global Polio Eradication Initiative (WHO, Geneva). Hollie has been a member of the Microbiology Society since 2017.

    Elizaveta Elshina

    Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 0QQ, UK

    After completing a BSc in Infectious Diseases at the University of Edinburgh, Elizaveta Elshina worked in preclinical vaccine development at the University of Oxford. She started to work on influenza virus during her MSc at the University of Zurich and is currently researching erroneous activity of influenza virus polymerase for her PhD. She has been a Microbiology Society member since 2018.

    Emmanuelle Pitre

    Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 0QQ, UK

    Emmanuelle Pitre graduated with a master&rsquos in Fundamental Virology from Sorbonne University and the Pasteur Institute. She is now working towards a PhD at the University of Cambridge, on influenza viruses&rsquo replication mechanisms.

    Aartjan te Velthuis

    Division of Virology, Department of Pathology, University of Cambridge, Cambridge, CB2 0QQ, UK

    Aartjan te Velthuis is a Henry Dale Fellow and Group Leader at the Department of Pathology of the University of Cambridge. He is interested in influenza virus replication and how aberrant viral RNA triggers innate immune responses. His research is funded by the Wellcome Trust, the Royal Society and NIH/NIAID.

    Why does microbiology matter?

    Joint answer: Microbiology has impacted human lives since the dawn of history. For centuries, it has played a key role in how we grow, prepare, flavour and preserve our foods. We used yeast for making beer before we knew how to make clean water and learnt to add salt to foods to prevent microbial growth. We also depend on microbiology to understand how viruses, bacteria and fungi cause disease, and how we can fight pathogens. In particular, the discovery of penicillin, a product of a fungus that can kill bacteria, has saved many lives. But microbiology is equally important for our future. It is helping us find ways to break down oil and plastic, to develop alternatives for other harmful products and to find (and possibly survive on) a habitable planet. Microbiology is without a doubt one of the most important research fields today.

    What is the most rewarding part of your job?

    Joint answer: In our lab, we study how influenza viruses replicate in human cells, how the viral genome is mutated, and how the efficiency of viral replication contributes to disease. It is extremely exciting to study this virus and being one of the first to uncover unknown molecular mechanisms. It feels like being an explorer discovering a new country or navigating a new mountain top. But one of the most rewarding aspects of our work is showing others how interesting microbiology is, either by presenting our work or using games such as our &lsquovirus roulette&rsquo table to teach children (and adults) how infections and antibodies work.

    Understanding viruses at the atomic scale: a history of virus structure research

    David Bhella

    As a PhD student in the crystallography department of Birkbeck University of London, I was struck by the proud heritage of that institution. Among the pioneers of structural biology from that department, J.D. Bernal, Rosalind Franklin and Aaron Klug made extraordinary contributions to structural virology. As a young researcher entering the field, a sense of walking in the footsteps of such towering historical figures was awe-inspiring. Over the intervening 25 years, I have been equally astounded by the technological developments in structural biology that have propelled the field forwards. In particular, the evolution of cryogenic electron microscopy (cryoEM), which has become a powerful tool for high-resolution structure determination, particularly suited to large macromolecular assemblies such as viruses.

    Viruses are fascinating targets for structural biology research, being simultaneously the smallest (and most abundant) life-forms on the planet and the largest of macromolecular assemblies to be understood at the atomic level.

    The shape of viruses

    Our earliest insights into the shapes of viruses came when Helmut Ruska imaged plant and animal viruses for the first time in the transmission electron microscope (TEM). These images were published in 1939. At this time, J.D. Bernal and Isidor Fankuchen were beginning to work on X-ray diffraction of concentrated preparations of plant viruses, including tobacco mosaic virus (TMV) and tomato bushy-stunt virus (TBSV) &ndash showing the former to be a long filamentous structure and the latter to be a spherical one.

    The work started by Bernal was continued at Birkbeck college, where he recruited Rosalind Franklin to study the detailed structure of TMV. She showed it to be a helical assembly and defined the spatial arrangements of protein and RNA. Based on the work of Franklin&rsquos collaborator and friend Don Caspar, Crick and Watson proposed that spherical viruses assemble with icosahedral symmetry. Using delightfully anachronistic language, these viruses were said to be likely to resemble a &lsquorather symmetrical mulberry&rsquo assembling from 60 protein subunits.

    © https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-0056

    Initial theories of icosahedral symmetry in spherical viruses were insufficient, as many viruses were shown to form larger assemblies comprising many hundreds of capsid proteins. Don Caspar and Aaron Klug addressed this by formulating their theory of quasi-equivalent packing in icosahedral viruses. Building on emerging knowledge, they set out how larger capsids might be assembled by the introduction of small variations in bonding relationships between protein subunits.

    Atomic modelling and structure

    The first atomic model of a spherical virus was calculated for TBSV by Steve Harrison et al. in 1978, revealing a shell comprising 180 copies of the major capsid protein. This was followed by structures of two small RNA-containing viruses that infect humans: rhinovirus, solved by Michael Rossmann and colleagues, and poliovirus solved by Jim Hogle et al., both published in 1985. These studies revealed a common fold in the capsid proteins of these plant and animal viruses: an eight-stranded &beta-barrel known as the &beta-jelly roll.

    The potential to use electrons rather than X-rays to determine virus structure was demonstrated in 1968 when David DeRosier and Aaron Klug calculated low-resolution 3D density maps of the contractile tail of phage T4 from TEM images. Exploiting the helical symmetry of the assembly allowed the density to be reconstructed from single images of phage particles stained with a heavy metal salt. A method to determine the structures of icosahedral objects followed, developed by Tony Crowther and colleagues. Many aspects of TEM were severely limiting, however, and the first step towards overcoming these challenges came with the invention of cryogenic methods for imaging biological material in the TEM. In 1985, Marc Adrian and colleagues published methods for the preparation of virus particles suspended in thin layers of vitreous ice. The absence of stain and chemical fixative meant that cryo-EM yielded images of macromolecular assemblies in a close to native state. Several technical advances in cryo-EM were required to move from early density maps at 30&ndash40 angstroms resolution to where we are now &ndash where 3D reconstructions at better than 4 angstroms resolution allow the construction of reliable atomic models.

    Technological advances

    The introduction of the first generation of digital cameras for TEM brought about a technological revolution in cryo-EM, facilitating the development of automated data collection and electron tomography (cryo-ET). Cryo-ET allows structure analysis of morphologically unique entities, by rotating them in the electron microscope and recording a tilt-series of images. These data may then be processed to compute a 3D reconstruction at intermediate resolution. A notable application of this method led to the calculation of an atomic model of the HIV capsid in the laboratory of John Briggs at EMBL Heidelberg.

    For much of the first decade of the 21 st century, virus structure research combined intermediate-resolution cryo-EM maps with X-ray data to yield pseudo-atomic models of, for example, complexes of viruses and host proteins such as antibodies. The first ab initio atomic model built into a single particle cryo-EM map of a virus was that of cytoplasmic polyhedrosis virus published by the laboratory of Z. Hong Zhou in 2008. The cryo-EM resolution revolution has since transformed this method to the point that atomic models of icosahedral virus capsids may be rapidly calculated. At the time of writing there are 175 capsid structures in the protein data bank solved by cryo-EM at better than 4 angstroms resolution. Notable recent achievements include high-resolution structures of two very large viruses: herpes simplex virus and African swine fever virus.

    Recent developments in image reconstruction software have allowed investigators to probe asymmetry in viruses, revealing instances where deviating from symmetry is critical for the viral life cycle. One recent example from my own laboratory is our discovery that the calicivirus minor capsid protein VP2 forms a portal at a unique three-fold vertex following receptor binding. We believe that this is the mechanism by which the virus injects its genome into the cell.

    Looking to the future of virus structure research, both X-rays and cryo-EM offer the tantalising prospect of viewing virus behaviour within the cell. Cryo soft X-ray microscopy is emerging as a powerful tool for imaging whole cells, revealing organelle rearrangements associated with virus infections. Cryo-ET of virus-infected cells is allowing researchers to analyse virus structures in situ, providing valuable biological context to structure data and promising that in the not too distant future it will be possible to solve structures of viruses in their natural habitat.

    Harnessing structural biology in a crisis

    In January 2020, SARS-Coronavirus 2 emerged in the Chinese city of Wuhan and has rapidly spread across the world, causing severe illness and deaths. This has led to the widespread lockdown of cities and whole nations. The scientific community has mobilised to address this crisis, including structural biologists. A testament to the significant advances in both X-ray crystallography and cryo-EM is the speed with which researchers have solved atomic structures for critical viral proteins. At the time of writing (20 March 2020), 29 protein structures for SARS-CoV-2 have been deposited in the Protein Data Bank, including the protease Mpro bound to several inhibitors, the S protein that mediates attachment and entry, and a complex of the S-protein&rsquos receptor binding domain and the virus&rsquo cellular receptor ACE2. These data will inform the development of antivirals and vaccines and are a major contribution to the global effort to defeat COVID-19.

    David Bhella

    MRC-University of Glasgow Centre for Virus Research (CVR), Sir Michael Stoker Building, Garscube Campus, 464 Bearsden Road, Glasgow G61 1QH, UK

    David Bhella is Professor of Structural Virology at the Medical Research Council &ndash University of Glasgow CVR. He is also Associate Director of the CVR and Director of the Scottish Centre for Macromolecular Imaging (SCMI). David started his career working as a diagnostic virologist at the Royal London Hospital before undertaking a PhD with Professor Helen Saibil FRS at Birkbeck College. He then moved to Glasgow&rsquos MRC Virology Unit where he developed his programme of structural virology research.

    Why does microbiology matter?

    Microbiology impacts many key elements of human endeavour. Understanding microbes as primary drivers of the planet&rsquos ecosystems, disease-causing agents, irreplaceable components of our own biological processes and as tools is a vital scientific need. Investigating the biology of micro-organisms has the potential to allow us to prevent and treat infectious and metabolic diseases, as well as feeding ourselves while minimising our ecological impacts.

    What is the most rewarding part of your job?

    For much of my career the primary driver has been the sheer thrill of discovery. That moment when your experiments lead to a new insight into the biology of an important virus is so often unexpected and startling. For a moment you are the only person in the history of humanity to know something important. Recent events have reminded me why I first chose to become a virologist. Understanding viruses at the molecular level is key to preventing serious diseases and saving lives. Microbiology is a worldwide endeavour and I am proud to play my own small part in this.

    Thumbnail: Bacteriophage T4. Eye of Science/Science Photo Library.

    Transmission electron micrograph of human rhinovirus, the main causative agent of the common cold. Nicola Stonehouse.


    Tracing the Virus’s Origins

    Experts from China and the World Health Organization joint team visit Wuhan Tongji Hospital in Wuhan, Hubei Province, China, February 23, 2020. (China Daily via Reuters)

    On the menu today: One scientific research paper makes the case that SARS-CoV-2 — the type of coronavirus that causes COVID-19 — must have spent time percolating in a pangolin, the anteater-like species that is one of the world’s most heavily trafficked animals. But how does that square with other studies suggesting the Huanan Seafood Market wasn’t the origin point for the virus, and other scientists contending that a jump straight from bats to humans was more likely?

    Does COVID-19 All Trace Back to Pangolins?

    Yesterday someone sent me this article in Nature Medicine, arguing that the scenario of an accidental laboratory infection of SARS-CoV-2 — the type of coronavirus that causes COVID-19 — is unlikely, because the virus has certain features that indicate it evolved through a considerable period of natural selection, and those traits are just too similar to coronaviruses from pangolins for the virus to have jumped straight from bats to humans.

    Basic research involving passage of bat SARS-CoV-like coronaviruses in cell culture and/or animal models has been ongoing for many years in biosafety level 2 laboratories across the world27, and there are documented instances of laboratory escapes of SARS-CoV28. We must therefore examine the possibility of an inadvertent laboratory release of SARS-CoV-2.

    In theory, it is possible that SARS-CoV-2 acquired RBD mutations (Fig. 1a) during adaptation to passage in cell culture, as has been observed in studies of SARS-CoV11. The finding of SARS-CoV-like coronaviruses from pangolins with nearly identical RBDs, however, provides a much stronger and more parsimonious explanation of how SARS-CoV-2 acquired these via recombination or mutation.

    The authors of this study are cautious they note that we don’t know what we don’t know: “Although no animal coronavirus has been identified that is sufficiently similar to have served as the direct progenitor of SARS-CoV-2, the diversity of coronaviruses in bats and other species is massively undersampled.” It’s conceivable that somewhere out there is a bat with a strain of the coronavirus that is nearly identical to SARS-CoV-2 found in humans, but that bat hasn’t been found yet.

    While it is possible that one of the two major laboratories in Wuhan, China, researching coronaviruses also had pangolins in their labs, at this point we have no evidence indicating that those labs used those animals. (We do have strong evidence that they both used bats.)

    If the genomes of the SARS-CoV-2 virus point to this virus percolating in pangolins for a while before jumping to humans, they point to a pangolin, and make the lab accident or improperly disposed viral material scenarios less likely. There certainly is enough evidence to suggest that illegal consumption of pangolins was occurring, probably throughout China and almost certainly within Wuhan.

    But this piece of the puzzle is a challenging fit with other research that was pointing away from the origin of COVID-19 being the Huanan Seafood Market.

    First, as that February article in The Lancet noted, “27 (66%) of 41 patients had been exposed to Huanan seafood market.” This meant that a third couldn’t be traced back to the market. A separate study in the New England Journal of Medicine could only tie 30 of the first 47 cases back to the market.

    As noted yesterday, two microbiologists in Australia, John S. Mackenzie and David W. Smith, are more skeptical that COVID-19 jumped to humans through pangolins:

    The closest known wildlife sequence to SARS-CoV-2 remains the sequence from the virus isolated from an Intermediate horseshoe bat, but there were significant differences in the receptor-binding domain between the two viruses. Malayan pangolins (Manis javanica) have been suggested as potential intermediate hosts, and SARS-like viruses have been identified in pangolins seized in anti-smuggling operations in southern China, but they only shared about 85–92% homology with SARS-CoV-2.

    But probably the most glaring not-fitting puzzle piece is the study of the genomic data that suggesting the strain of the virus found in the market evolved from other strains found elsewhere, not the other way around:

    By applying the reported bat coronavirus genome (bat-RaTG13-CoV) as the outgroup, we found that haplotypes H13 and H38 might be considered as ancestral haplotypes, and later H1 (whose descendants included all samples from the Huanan Seafood Wholesale Market) was derived from the intermediate haplotype H3. The population size of the SARS-CoV-2 was estimated to have undergone a recent expansion on 06 January 2020, and an early expansion on 08 December 2019. Phyloepidemiologic analyses suggested that the SARS-CoV-2 source at the Huanan Seafood Wholesale Market was potentially imported from elsewhere. The crowded market then boosted SARS-CoV-2 circulation and spread it to the whole city in early December 2019.

    Did someone encounter or eat a pangolin elsewhere in Wuhan away from the Huanan Seafood Market, contract SARS-CoV-2, travel around the city infecting others, then go to the market and set off the bigger outbreak? And from that, they ended up contracting a strain of the coronavirus that originated in bats was unrelated to the research on coronaviruses in bats going on in two major laboratories in the city? It is possible, but it is a remarkable sequence.

    I feel like the “laboratory accident” scenario is being dismissed out of hand by some people who do not realize that this sort of sequence of events already happened in China 16 years ago:

    SARS has sent the head of another top official in China rolling. Yesterday, director Li Liming of the Center for Disease Control and Prevention (CDC) resigned, along with several lower-ranking officials, after a report by a panel of experts blamed China’s most recent outbreak of severe acute respiratory syndrome on a series of flaws at the CDC’s National Institute of Virology in southern Beijing.

    The outbreak earlier this year, which sickened eight people in Beijing and Anhui Province and killed one (ScienceNOW, 27 April), started when two workers at the CDC lab, independently from each other, developed SARS. The most likely source of their infection, the report concludes, is a batch of supposedly inactivated SARS virus that was brought from a high-containment facility into a low-safety diarrhea research lab where the two were working. Apparently, the inactivation process–adding a mix of detergents to the virus–did not work properly, according to the study, of which only a five-paragraph summary has been released. In a breach of standard safety procedures, the researcher who carried out the inactivation–identified only by a family name, “Ren”–had not tested whether the virus was truly inactive, according to the panel.

    Some scientists hailed the report and Li’s resignation. “This is a clear sign to Chinese scientists and the rest of the world that the Chinese government is taking [biosafety] seriously,” says Guan Yi, a virologist at the University of Hong Kong. But others are disappointed that many details about the incident and the lab’s operating procedures remain hidden. “I was hoping for a full, more open account of what happened,” says Tony Della-Porta, an Australian biosafety consultant who helped investigate earlier SARS escapes in Singapore and Taiwan.

    (If you’ve ever thought your job was difficult, just think of the words “low-safety diarrhea research lab.”)

    For what it’s worth, the “accidental release from a lab” scenario is apparently being contemplated by senior officials in the British government, and David Ignatius’ column in the Washington Post Friday suggested that either U.S. intelligence officials or unspecified “scientists” can’t rule out that scenario.

    CNN recently spoke to some of the country’s top virologists and found there isn’t a clear consensus yet:

    Rutgers University bioweapons expert Dr. Richard Ebright told CNN, “the possibility that the virus entered humans through a laboratory accident cannot and should not be dismissed… It is absolutely clear the market had no connection with the origin of the outbreak virus, and, instead, only was involved in amplification of an outbreak that had started elsewhere in Wuhan almost a full month earlier.”

    “I think people went into the fish market who were already infected,” Vincent Racaniello, a microbiology professor at Columbia University, told CNN. “In bats, these viruses are intestinal viruses, and they are shedding the bat feces, which we call guano,” he said. “And if you go into a bat cave, it is littered with guano. And farmers in many countries harvest the guano to fertilize their fields.”

    Dr. Simon Anthony, a professor at the public health grad school of Columbia University, told CNN, “Early in the outbreak … everyone was talking about the thing having emerged from the wet market. And now I think the data calls into question whether or not that’s really true.”

    Some might ask why we should bother looking into this if clear answers are likely to never be found the Chinese government certainly isn’t going to welcome outside investigators to go poking around the city of Wuhan and its own labs, trying to determine the source of the virus. But the origin of the coronavirus is the biggest and most consequential mystery in the world right now. Until we know how this virus got into humans, we live with the risk of it happening again with another strain — perhaps an even more virulent or contagious one.