Unless the intermediate host necessary for completing a natural zoonotic jump is identified, the dual‐use gain‐of‐function research practice of viral serial passage should be considered a viable route by which the novel coronavirus arose.

The practice of serial passage mimics a natural zoonotic jump, and offers explanations for SARS‐CoV‐2's distinctive spike‐protein region and its unexpectedly high affinity for angiotensin converting enzyme (ACE2), as well as the notable polybasic furin cleavage site within it.

To date, the origins of SARS‐CoV‐2 remain in doubt, and its behavior enigmatic: It has been reported that “the virus acts like no microbe humanity has ever seen.”[1] Although based on sequence analysis many prominent virologists and other eminent scientists have concluded that the novel coronavirus causing the current pandemic was not designed or manipulated in a laboratory and was the result of a natural zoonotic jump,[2] this assertion fails to fully account for all possible origins of two unique genomic characteristics found in SARS‐CoV‐2, and ignores the long history of serial passage as a method to manipulate viral genomes.

The long‐standing practice of serial passage is a form of gain‐of‐function research that forces zoonosis between species, and requires the same molecular adaptations necessary for a natural zoonotic jump to occur within a laboratory, leaving the same genetic signatures behind as a natural jump but occurring in a much shorter period of time.

This furin cleavage site, which is poly or multibasic by definition since its composed of multiple basic amino acids, is an important virulence feature observed to have been acquired by fusion proteins of avian influenza viruses and Newcastle Disease Virus either grown under experimental conditions or isolated from commercial animal farms—settings that mimic the conditions of serial laboratory passage.

In fact, no influenza virus with a furin cleavage site has ever been found in nature,[3] and it is a feature that has been thoroughly investigated in the literature since it appears to allow the influenza viruses that carry it to establish a systemic multiorgan infection using different cell types including nerve cells,[3] is correlated with high pathogenicity, and also plays a key role in overcoming the species barrier.[4] More generally, despite the fact that not all serially passed viruses have demonstrated an increase in pathogenicity, the fact remains that every highly pathogenic avian influenza virus, defined by having a furin cleavage site, has either been found on commercial poultry farms that create the pseudo‐natural conditions necessary for serial passage, or created in laboratories with gain‐of‐function serial passage experiments.[3].

Although they only emerge under artificial conditions in influenza viruses, these furin cleavage sites are found within several branches of the coronavirus family tree.

However SARS‐CoV‐2 is the only lineage B coronavirus found with one, and the only other coronaviruses known to have them are only at most 60% identical to this novel coronavirus.[5] An intriguing clinical correlate is that furin cleavage sites within influenza viruses are associated with lymphopenia in infected mice, and with neurological conditions following replication in the brains of ferrets,[6] both of which are clinical manifestations observed in hospitalized patients infected by SARS‐CoV‐2 and suffering from COVID‐19.[1] This indicates that furin cleavage sites may be an example of the convergent evolution that dominates virus–host interactions, since viral proteins evolve convergently and often accumulate many of the same linear motifs that mediate many functionally diverse biophysical interactions in order to manipulate complex host processes.[7] It is possible that this novel coronavirus gained its furin cleavage site through recombination in an intermediate host species, however there are also two laboratory processes that may have imbued SARS‐CoV‐2 with its furin cleavage site which will be discussed below.

The dual‐use gain‐of‐function research tool of serial passage was first applied to a strain of H1N1 Swine Flu, a variant of the pandemic influenza virus that was genetically modified before it either leaked out of a Soviet lab or was introduced as part of an attenuated vaccine trial in 1977.

In the years that followed, gain‐of‐function serial passage through ferrets was used to increase the virulence of the H7N1 Bird Flu as well as allowing for its aerosol transmission without first introducing any mutations.[14] Additionally, the H1N1 Bird Flu was also found to become airborne and increase in virulence after in vivo passage through swine.[15, 16] And although serial passage in the laboratory does not invariably increase viral pathogenicity, highly pathogenic influenza viruses all contain furin cleavage sites,[16] which only emerge after serial passage in laboratories or pseudo‐naturally on commercial animal farms.

And regarding the second distinctive feature found in the novel coronavirus: If other viruses have been observed to acquire furin cleavage sites by passage under experimental laboratory conditions, then such a mechanism is theoretically possible for SARS‐CoV‐2 as well.[2].

In the case of influenza viruses like those mentioned above, their gain‐of‐function furin cleavage sites are thought to be a result of two different molecular processes.

The first is either nucleotide insertions or substitutions that are able to be rescued and then eventually selected for due to the high multiplicity of infection found in serial passage protocols.[19] And the second is the recombination of multiple viral RNAs inside a host cell,[20] which may also include additional viruses introduced through accidental laboratory co‐infections.

Unlike influenza viruses, serial passage through ferrets has not been recorded in the literature for coronaviruses.

However, since several branches of coronavirus have furin cleavage sites, a molecular pathway for their emergence must exist and may reemerge during serial passage.

Several factors weigh into the probability that coronaviruses can gain furin cleavage sites following serial passage: The frequency of evolutionary motifs meant to deal with virus–host interactions that are often shared between viruses, the observations that when the infectious bronchitis coronavirus (IBV) coronavirus is serially passed through chickens it developed notable mutations along its spike‐protein genes,[21] and the fact that when a lineage A bovine coronavirus was subject to in vitro serial passage through cell lines, a 12‐nucleotide insert found within only a small minority of the pooled viruses spike‐protein region was strongly selected for and quickly emerged as the dominate strain.[22] These findings all point to the possibility that SARS‐CoV‐2 may have gained its furin cleavage site the same way influenza viruses do—through the in vivo serial passage between the live hosts that presents the immune challenges and intense selective pressure necessary for the recombination and mutations that lead to its emergence to occur.

There is no doubt that the acquisition of the furin cleavage site was one of the key adaptations that enable SARS‐CoV‐2 to efficiently spread in the human populations compared to other lineage B coronaviruses, and provides a gain‐of‐function.[23] In addition to the possibility of obtaining a furin cleavage site through natural recombination in a secondary host or through serial passage either in a laboratory or on a commercial farm, one could have been spliced directly into the novel coronavirus's backbone in a laboratory using classic recombinant DNA technology that has been available for nearly 20 years.

Moreover, when a furin cleavage site was introduced to the IBV coronavirus spike‐protein via recombination, just like influenza viruses hosting this feature, it appeared to impart it with increased lethality as well as inflict neurological symptoms that had never previously been reported in studies of the murine IBV coronavirus.[25] The presence of this cleavage site also increased damage to the respiratory and urinary systems, paralleling SARS‐CoV‐2 systemic multiorgan symptoms—especially reports that infection with the novel coronavirus not only targets the lungs where it binds to ACE2 receptors, but also the entire cardiovascular system,[27] the nervous system,[28] and our kidneys as well.[29] It might be more than a coincidence that the Vero cells often used in serial passage are derived from kidney epithelial cells extracted from African green monkeys, which have ACE2 receptors very similar to those found in humans and would be shared by the humanized mice that are also used for serial passage research.

And in subsequent years, EcoHealth Alliance received funding for project proposals outlining gain‐of‐function research to be done in Wuhan, hoping to use cell cultures and humanized mice as well as “[spike]‐protein sequence data, infectious clone technology, in vitro and in vivo infection experiments and analysis of receptor binding”[33] to manipulate bat coronavirus genomes—all of which are consistent with the wet‐work that would be needed to engineer this novel coronavirus in a laboratory.

This relationship was further supported by reports out of the Netherlands that the novel coronavirus had spread among thirteen different mink farms there, and also to at least one farm in Denmark[39] and to another in Spain where 87% of the mink were infected.[40] Minks are a closely related subspecies of ferret that can produce fertile offspring together, and so the fact that not only did the virus spread to fifteen different farms in three countries, but also appears to have spread from minks into farm workers[41] indicates that accidental commercial serial passage through minks could have played a role in its creation, as an alternative to laboratory ferrets?

Further research around SARS‐CoV‐2's affinity to ferrets and minks, as well as other possible intermediate hosts seems warranted, and certainly the examination of all past gain‐of‐function serial passage research by the scientific community at large should occur to determine what other definitive genomic signatures serial passage leaves besides the creation of furin cleavage sites, in case more of those can be found in this novel coronavirus

The release of the H1N1 Swine Flu in 1977 first initiated the discussion about the moral and physical hazards involved with dual‐use gain‐of‐function research, and it was the creation of extraordinarily virulent H5N1 Bird Flu strains—using the same technique of serial passage through an animal host in a lab—that contributed to the NIH imposing a moratorium on dual‐use gain‐of‐function research from 2014 until 2017, after which it was relaxed explicitly to allow influenza strains as well as coronaviruses to be studied

And whether or not gain‐of‐function research is determined to have played a role in SARS‐CoV‐2's emergence, the fact that it creates opportunities for pandemic viruses to leak out of labs calls for a re‐examination of the moratorium against this practice, because the emergence of this novel coronavirus has demonstrated that the international public health community is not prepared to handle the leak of a pandemic virus