Non-Reviewed Preprint Favors Natural Origins Based on Weak Assumptions, Speculation, Logic Flaws, and Overdrawn Conclusions

It's very odd that PMC already has the full version online before any journal has accepted the manuscript. Given the serious flaws, it is unclear how and why the authors reached their conclusion.

A non-peer-reviewed paper concludes that wildlife trade at the Huanan Seafood Wholesale Market in Wuhan, China, is the most likely conduit for the origin of the COVID-19 pandemic. The authors suggest that multiple likely intermediate host species, including raccoon dogs, civets, bamboo rats, and other wildlife sold as food at the market, could have been involved in zoonotic spillover events that transmitted SARS-CoV-2 to humans. They support this conclusion with genetic tracing of animal DNA in SARS-CoV-2 positive environmental samples and phylogenetic evidence that links viral genomes from the market to the global pandemic. However, they acknowledge that while their findings are consistent with zoonotic origins, they do not provide direct proof of which specific species or individual animals transmitted the virus to humans.

The paper suffers from numerous flaws, including weak assumptions, speculation, logic flaws, and overdrawn conclusions. It is worth noting that the credibility and reputation of many of the authors depend on society accepting their conclusions - yet the paper includes no mention of this major conflict of interest.

Weak Assumptions on Zoonotic Origins

The authors assume that the wildlife trade and zoonotic transmission are the most likely origin of the SARS-CoV-2 virus, as seen in their strong association of wildlife stalls with the outbreak (e.g., raccoon dogs, civets). However, this is built on several untested or partially tested assumptions:

Insufficient Testing of Animal Hosts: The paper points out that no samples from raccoon dogs or civets were directly tested for SARS-CoV-2 via qPCR, meaning that there is no direct evidence of SARS-CoV-2 infection in the wildlife thought to have carried the virus.

Unverified Intermediate Hosts: The study leans heavily on genetic materials linked to raccoon dogs and other species being present in environmental samples. However, they do not present clear evidence that these species were infected or capable of shedding the virus at the Huanan market at the necessary time to initiate human infections. The lack of qPCR or serological testing on these animals is a critical gap.

This leads to an overreliance on indirect evidence and thus weakens the assumption that these animals were key intermediaries.

Quote:

"No samples from raccoon dogs, civets, or porcupines on sale in the market were tested by qPCR, and no serology from animals in the market has been described."
— Page 5

Overdrawn Conclusions About Genetic Tracing

The paper asserts that the presence of animal genetic material in SARS-CoV-2-positive samples supports a wildlife-to-human spillover, which is speculative. This conclusion relies on correlational analysis rather than direct evidence of animal infections. The authors acknowledge the possibility that viral shedding by early human cases could explain the positive environmental samples, meaning the zoonotic spillover remains unconfirmed.

Furthermore, the connection between genetic diversity in the market and global pandemic emergence assumes that the genetic similarities are due to market origins. This conclusion might be overdrawn because it doesn't eliminate other possible sites of viral amplification or initial human infections that could also explain these patterns.

Flaw: Correlation between the presence of animal DNA in SARS-CoV-2 positive samples and a zoonotic spillover is speculative.

Quote:

"The presence of genetic material from raccoon dogs and hoary bamboo rats was highly frequent across the wildlife stalls, constituting the two most commonly detected mammalian wildlife species."
— Page 10

Speculative Correlation of Animal and Viral Data

The correlation analysis between mammalian species abundances and SARS-CoV-2 RNA (rho values for raccoon dogs, bamboo rats, etc.) is presented as supportive evidence, but these results suffer from temporal and compositional biases (acknowledged by the authors). The samples collected on different dates and across a decaying viral RNA environment make these correlations speculative rather than conclusive. These temporal gaps challenge the authors' interpretation that wildlife species were shedding SARS-CoV-2, making the conclusions drawn from the correlational analysis less robust.

Flaw: Correlational analysis between species abundance and viral RNA lacks robustness due to temporal and compositional biases.

Quote:

"As previously described (Crits-Christoph et al. 2023), a correlational analysis would be unlikely to provide reliable insights into whether any particular species was or was not infected by SARS-CoV-2 within the market."
— Page 11

The authors themselves acknowledge the limitations of using correlational analysis, highlighting that the conclusions drawn from these results are speculative.

Phylodynamic Inferences

The authors' reliance on phylodynamic inference to support the idea of multiple spillovers from animals in the Huanan market rests on assumptions that the common ancestor of the market-associated strains is equivalent to the common ancestor of the pandemic. However, this inference is contingent upon adequate and representative sampling, which is a limitation acknowledged by the authors. The high variability in the time to the most recent common ancestor (tMRCA) suggests that multiple interpretations of the data are possible. The suggestion that the timing of the market outbreak is "indistinguishable" from the pandemic's origin might be overextended, given the uncertainties in dating early infections.

Flaw: Phylodynamic analysis assumes a common ancestor at the market but depends heavily on uncertain sampling.

Quote:

"The tMRCA of market-associated genomes reflects the global tMRCA of the pandemic [...] This is consistent with previously published results that identify the ancestral MRCA of SARS-CoV-2 as either the A or B haplotypes or an unobserved intermediate between the two."
— Page 6

This quote assumes a single origin at the market but notes the possibility of other unobserved intermediates, which weakens the inference that the market was definitively the origin.

Logical Gaps in Interpretation of Environmental Samples

The study frequently discusses SARS-CoV-2 RNA decay in environmental samples to explain inconsistencies in detection rates across different wildlife stalls. However, this creates a logical gap: if viral RNA is decaying rapidly, then the interpretation of positive samples as proof of viral shedding from wildlife becomes weaker. This self-acknowledged decaying signal means the evidence is inherently incomplete, and conclusions drawn from decayed RNA are speculative.

Flaw: Decay of viral RNA challenges the conclusions drawn from positive environmental samples.

Quote:

"As time of sampling progressed after the market’s closure, there was a noticeable decrease in SARS-CoV-2 viral abundance, indicating environmental viral RNA decay throughout the market over several weeks."
— Page 8

The decay of viral RNA suggests that conclusions drawn from samples collected later (such as 11 days after initial sampling) may not fully represent viral shedding by animals, introducing a logical gap in the argument.

Speculation on Geographic Origin

The authors speculate on the geographic origin of the animals in the market based on mitochondrial DNA haplotypes but acknowledge that genetic data for these species in southern China are sparse. This makes their conclusions about the source of animals (e.g., raccoon dogs from southern vs. northern China) speculative. They cannot definitively link these animals to the viral spillover into humans based on the limited data they have on animal trade networks and wildlife populations.

Flaw: Limited genetic data makes the conclusion about the geographic origin of animals speculative.

Quote:

"It is unknown how far south the wild or farmed range of the subpopulations detected here extends due to a lack of genetic data for N. procyonoides in southern China."
— Page 15

This quote acknowledges that the geographic origin of raccoon dogs cannot be definitively established, undermining the paper's hypothesis about how these animals may have been involved in viral transmission.

These quotes illustrate how the assumptions, overdrawn conclusions, speculative correlations, phylodynamic uncertainties, and logical gaps in the study make the conclusions less robust than they initially appear.

In summary, while the paper provides valuable genetic tracing and evidence for the presence of wildlife in a market setting, several of the key conclusions are speculative 1and rely on weak or incomplete evidence. The main flaws include the lack of direct animal infection testing, overextended phylodynamic inferences, reliance on correlational data, and gaps in linking genetic diversity to a specific origin of the virus. These shortcomings make the paper's conclusions about zoonotic spillover tentative rather than definitive.

Crits-Christoph A, Levy JI, Pekar JE, Goldstein SA, Singh R, Hensel Z, Gangavarapu K, Rogers MB, Moshiri N, Garry RF, Holmes EC, Koopmans MPG, Lemey P, Popescu S, Rambaut A, Robertson DL, Suchard MA, Wertheim JO, Rasmussen AL, Andersen KG, Worobey M, Débarre F. Genetic tracing of market wildlife and viruses at the epicenter of the COVID-19 pandemic. bioRxiv [Preprint]. 2023 Sep 14:2023.09.13.557637. doi: 10.1101/2023.09.13.557637. PMID: 37745602; PMCID: PMC10515900.

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Hao P, Zhong W, Song S, Fan S, Li X. Is SARS-CoV-2 originated from laboratory? A rebuttal to the claim of formation via laboratory recombination. Emerg Microbes Infect. 2020 Mar 8;9(1):545-547. doi: 10.1080/22221751.2020.1738279. PMID: 32148173; PMCID: PMC7144200.