Despite claims from prominent scientists that SARSxe2x80x90CoVxe2x80x902 indubitably emerged naturally, the etiology of this novel coronavirus remains a pressing and open question: Without knowing the true nature of a disease, it is impossible for clinicians to appropriately shape their care, for policyxe2x80x90makers to correctly gauge the nature and extent of the threat, and for the public to appropriately modify their behavior. Unless the intermediate host necessary for completing a natural zoonotic jump is identified, the dualxe2x80x90use gainxe2x80x90ofxe2x80x90function 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 SARSxe2x80x90CoVxe2x80x902’s distinctive spikexe2x80x90protein region and its unexpectedly high affinity for angiotensin converting enzyme (ACE2), as well as the notable polybasic furin cleavage site within it. Additional molecular clues raise further questions, all of which warrant full investigation into the novel coronavirus’s origins and a rexe2x80x90examination of the risks and rewards of dualxe2x80x90use gainxe2x80x90ofxe2x80x90function research.

This article from a fatherxe2x80x93son team explores whether the history and methodology of viral serial passage gainxe2x80x90ofxe2x80x90function research provides a parsimonious explanation for SARSxe2x80x90CoVxe2x80x902, contrasting it with the theory that the novel coronavirus emerged naturally. It also examines the precedents of performing gainxe2x80x90ofxe2x80x90function research on batxe2x80x90borne coronaviruses, and calls for a rexe2x80x90examination of the risks inherent with gainxe2x80x90ofxe2x80x90function research.n

2.1. Serial Passage and Its Molecular Signatures

Although serial passage mimics many of the natural zoonotic processes that occur during a natural zoonotic jump, because serial passage artificially condenses a natural phenomenon into a small temporal window, some subtle differences can be found. In addition to the inexplicable genetic distance from its sister strains, which screams out for an intermediate relative to complete the phylogenetic picture, SARSxe2x80x90CoVxe2x80x902 has a remarkably strong affinity for spikexe2x80x90protein binding to ACE2xe2x80x94some 10xe2x80x9320 times higher than SARSxe2x80x90CoV’s.^[nn18nn^] That affinity may have emerged after mutational events either in an intermediate natural host or after a zoonotic jump into humans that theoretically could have occurred earlier than the first documented infection, which would give it time to increase that significantly. So logically, it could also have emerged via selection after serial passage through laboratory cell cultures or laboratory animals as well. 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 SARSxe2x80x90CoVxe2x80x902 as well.^[nn2nn^]n

In the case of influenza viruses like those mentioned above, their gainxe2x80x90ofxe2x80x90function 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.^[nn19nn^] And the second is the recombination of multiple viral RNAs inside a host cell,^[nn20nn^] which may also include additional viruses introduced through accidental laboratory coxe2x80x90infections.

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 virusxe2x80x93host 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 spikexe2x80x90protein genes,^[nn21nn^] and the fact that when a lineage A bovine coronavirus was subject to in vitro serial passage through cell lines, a 12xe2x80x90nucleotide insert found within only a small minority of the pooled viruses spikexe2x80x90protein region was strongly selected for and quickly emerged as the dominate strain.^[nn22nn^] These findings all point to the possibility that SARSxe2x80x90CoVxe2x80x902 may have gained its furin cleavage site the same way influenza viruses doxe2x80x94through 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. And just like influenza viruses are only able to preserve their furin cleavages in artificial environments since the heightened virulence they impart kills their hosts before they can propagate in a natural setting, based on the known taxonomy lineage B coronaviruses do not appear to be able to support furin cleavages in nature.

There is no doubt that the acquisition of the furin cleavage site was one of the key adaptations that enable SARSxe2x80x90CoVxe2x80x902 to efficiently spread in the human populations compared to other lineage B coronaviruses, and provides a gainxe2x80x90ofxe2x80x90function.^[nn23nn^] 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. This allows for the removal of the restriction site junctions that are the telltale sign of direct genetic manipulation and permits reassembly without introducing nucleotide changesxe2x80x94creating a virus without any evidence of manipulation using the aptly named xe2x80x9cNo See’m technology.xe2x80x9d^[nn24nn^] So although the entire spikexe2x80x90protein RBD was not assembled from scratch, it is certainly plausible that the 12xe2x80x90nucleotidexe2x80x90long furin cleavage site could have been spliced directly into SARSxe2x80x90CoVxe2x80x902. Furin cleavages already have been successfully spliced into other coronaviruses, including the IBV,^[nn25nn^] and even into SARSxe2x80x90CoV, where it increased cellxe2x80x90toxe2x80x90cell fusion in in vitro experiments that only examined only the spikexe2x80x90protein’s function, which would presumably heighten its infectivity in vivo.^[nn26nn^]n

Moreover, when a furin cleavage site was introduced to the IBV coronavirus spikexe2x80x90protein 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.^[nn25nn^] The presence of this cleavage site also increased damage to the respiratory and urinary systems, paralleling SARSxe2x80x90CoVxe2x80x902 systemic multiorgan symptomsxe2x80x94especially reports that infection with the novel coronavirus not only targets the lungs where it binds to ACE2 receptors, but also the entire cardiovascular system,^[nn27nn^] the nervous system,^[nn28nn^] and our kidneys as well.^[nn29nn^] 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.

2.2. Natural Origin, or Gainxe2x80x90ofxe2x80x90Function Lab Escape?

Gainxe2x80x90ofxe2x80x90function research on batxe2x80x90borne coronaviruses has been ongoing for nearly a decade everywhere from the University of North Carolina to the Wuhan’s Institute of Virology, which is supported by related facilities such as Wuhan’s Center for Disease Control and Prevention as well as Wuhan University. A coronavirus that targets the ACE2 receptor like SARSxe2x80x90CoVxe2x80x902 was first isolated from a wild bat in 2013 by a team out of Wuhan. This research was funded in part by EcoHealth Alliance,^[nn30nn^] and set the stage for the manipulation of batxe2x80x90borne coronavirus genomes that target this receptor and can become airborne. Many more viruses have been collected in Wuhan over the years, and one research expedition captured as many as 400 wild viruses,^[nn31nn^] which were added to a private repository that has since grown to over 1500 strains of virus,^[nn32nn^] meaning that the Wuhan Center for Disease Control and Prevention has a massive catalogue of largely undisclosed viruses to draw from for experiments. And in subsequent years, EcoHealth Alliance received funding for project proposals outlining gainxe2x80x90ofxe2x80x90function research to be done in Wuhan, hoping to use cell cultures and humanized mice as well as xe2x80x9c[spike]xe2x80x90protein sequence data, infectious clone technology, in vitro and in vivo infection experiments and analysis of receptor bindingxe2x80x9d^[nn33nn^] to manipulate bat coronavirus genomesxe2x80x94all of which are consistent with the wetxe2x80x90work that would be needed to engineer this novel coronavirus in a laboratory. But for whatever reason, the Wuhan Institute of Virology has refused to release the lab notebooks of its researchers, which are ubiquitous in even the simplest laboratories and are expected to be meticulously detailed given the sensitive and delicate work that takes place in BSLxe2x80x904 research labs intent on documenting their intellectual property, despite the fact that these notebooks would likely be enough to exonerate the lab from having any role in the creation of SARSxe2x80x90CoVxe2x80x902.^[nn34nn^]n

Although it does not prove a laboratory origin, another gainxe2x80x90ofxe2x80x90function experiment demonstrates one possible step along the way to engineering SARSxe2x80x90CoVxe2x80x902: the synthetic reconstruction of the SARS coronavirus to impart this virus with a high affinity for ACE2. This involved isolating a progenitor coronavirus from civets and then serially passing it through mammalian ACE2 receptorxe2x80x90expressing cellsxe2x80x94serial passage through host cell lines instead of entire hosts, which imparted a strong affinity for ACE2,^[nn35nn^] and another novel strain of coronavirus that was also presumably airborne. A few years after this study, more gainxe2x80x90ofxe2x80x90function research was performed that involved the creation of a chimeric batxe2x80x90borne coronavirus by directly manipulating the bat coronavirus spikexe2x80x90protein gene,^[nn36nn^] which created a coronavirus so virulent that it evoked the following dire warning from Simon Wainxe2x80x90Hobson, a virologist with the Pasteur Institute in Paris: xe2x80x9cIf the [new] virus escaped, nobody could predict the trajectory.xe2x80x9d^[nn37nn^]n

Although SARSxe2x80x90CoVxe2x80x902’s efficient solution for ACE2 binding has been accurately described as something that could not be intentionally engineered nucleotidexe2x80x90byxe2x80x90nucleotide,^[nn2nn^] it could well be selected for after serial passage through ferrets or cell cultures in a lab. The only origin for the SARSxe2x80x90CoVxe2x80x902 spikexe2x80x90protein RBD that the sequence data excludes is the deliberate manufacturing and introduction of the entire SARSxe2x80x90CoV spikexe2x80x90protein RBD sequence to create SARSxe2x80x90CoVxe2x80x902. Otherwise, there are no genetic data to distinguish among natural and engineered possibilities at the present time.

2.3. Ferreting Out the Signs of Serial Passage

Curiously, studies examining SARSxe2x80x90CoVxe2x80x902’s infectivity in ferrets found that it spreads readily among them, and also appears airborne in that animal model.^[nn38nn^] This lends support to the idea that ferrets may have been used for serial passage since viruses typically take a significant many months if not years to acclimate enough to spread at all among any new species, nonetheless become airborne, which requires further mutations.

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^[nn39nn^] and to another in Spain where 87% of the mink were infected.^[nn40nn^] 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^[nn41nn^] indicates that accidental commercial serial passage through minks could have played a role in its creation, as an alternative to laboratory ferrets. Nevertheless, regardless of where any possible serial passage occurred, the fact that SARSxe2x80x90CoVxe2x80x902 spreads from humans to minks and then back to humans demonstrates a high affinity for both species, despite neither nominally being a natural reservoir. Further support for the possibility that serial passage through lab ferrets or throughout mink farms played a role in the genesis of this novel coronavirus is provided by a preprint that notes the obvious ease with which it passes through the air between ferrets, since SARSxe2x80x90CoVxe2x80x902 was transmitted through the air to three out of four indirect recipient ferrets monitored for airborne passage of the novel coronavirus.^[nn42nn^] It seems reasonable to think that SARSxe2x80x90Covxe2x80x902’s apparent affinity for ferrets and minks should lead to an investigation of mink farms in the Hubei province were the novel coronavirus was discovered, since a viable pathway for its emergence could be infected bats defecating on commercial mink farms, which would loosely parallel the emergence of MERSxe2x80x90CoV from herds of camels following putative fecal contamination by local bats.^[nn43nn^]n

The prospect that serial passage through lab animals or on commercial farms may have played a role in the creation of SARSxe2x80x90CoVxe2x80x902 is also raised by an April 2020 preprint, which appears to have been retracted after Chinese authorities implemented the censorship of any papers relating to the origins of the novel coronavirus.^[nn44nn^] This paper found that coronaviruses that target the ACE2 receptor bind with ferret cells more tightly than any other species except the tree shrew, which only scored about 2% higher. Tree shrews have also been used for serial viral passage, and have been promoted as a preferable animal host for laboratory experimentation since they are cheaper, smaller, easier to handle, and closer to humans evolutionarily and physiologically than ferrets.^[nn45nn^] However, one does not exclude the other as a possible host, and a recent preprint examining SARSxe2x80x90CoV’s binding affinity in humans raises additional questions about its initial emergence. It found that the novel coronavirus appears to be far more adapted to human ACE2 receptors than those found in bats, which is unexpected given that bats are the virus’s assumed source, and which lead the lead research to observe that SARSxe2x80x90CoVxe2x80x902 was perfectly adapted to infect humans since its first contact with us, and had no apparent need to for any adaptive evolution at all.^[nn46nn^]n

Although the novel coronavirus also appears to have a high affinity for the pangolin ACE2 receptor,^[nn47nn^] phylogenetic analysis of the neutral sites that best determine shared heritage^[nn48nn^] and a distinctive amino acid sequence both indicate that pangolins are unlikely to have served as an intermediate host,^[nn47nn^] so this affinity is likely due to the convergent motifs that often mark viral evolution and not shared heritage. The unexpected immediate affinity for humans was also reflected by another preprint, which observed that SARSxe2x80x90CoVxe2x80x902 appeared just as adapted to humans at the very start of its epidemic as SARSxe2x80x90CoV was in the latest stages of its emergence,^[nn49nn^] an unexpected finding since viruses are expected to mutate substantially as they acclimate to a new species.^[nn50nn^] SARSxe2x80x90CoVxe2x80x902’s muddled origins are made even more Gordian by a study published March 2018 that examined people who live in villages about a kilometer away from bat caves. This study revealed that only 2.7% of those villagers had antibodies indicating any past exposure to bat coronaviruses. The authors also sampled people living in Wuhan, and found no evidence of exposure to SARSxe2x80x90CoVxe2x80x90like coronaviruses at all.^[nn51nn^]n

This means there is very little serological evidence of any exposure to these coronaviruses even in Chinese villagers living in close proximity to bat caves, and at the epicenter of the current outbreakxe2x80x94no previous exposure was found at all. These data do not support the idea that SARSxe2x80x90CoVxe2x80x902 was circulating in humans prior to the outbreak began in Wuhan in the early winter or fall of 2019, making a zoonotic jump even more unlikely since natural jumps leave wide serological footprints in their new host populations as early variants of a prospective virus make limited and unsuccessful jumps into individuals of the new host species, a trialxe2x80x90andxe2x80x90error that must occur before mutations that allow adaptation to a new host species are selected.^[nn50nn^] However these results do not rule out a much earlier jump into humans somewhere outside Hubei province, an alternative that is awaiting empirical support.

Taken together, the available evidence does not point definitively toward a natural origin for SARSxe2x80x90CoVxe2x80x902, rather, much of it is more consistent with what would be found if the novel coronavirus had arisen from serial passage of a xe2x80x9cprecursorxe2x80x9d progenitor virus in a lab, or from bats infecting a commercial mink farm somewhere in China, which would also provide the conditions for serial passage. However, more evidence is required before a conclusive judgement can be made one way or the other.

Further research around SARSxe2x80x90CoVxe2x80x902’s affinity to ferrets and minks, as well as other possible intermediate hosts seems warranted, and certainly the examination of all past gainxe2x80x90ofxe2x80x90function 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. Two additional unique genomic signature are already being researched, as one preprint indicates that SARSxe2x80x90CoVxe2x80x902 possesses a genomic region not found in other coronaviruses that appears to cloak the novel coronavirus from white blood cells, a characteristic also found with HIV.^[nn52nn^] And the second preprint identifies a region on the spikexe2x80x90protein gene found in no other batxe2x80x90borne coronavirus that is nearly identical to superantigenic and neurotoxic motifs found in some bacteria, which may contribute to the immune overreaction that leads to the Kawasakixe2x80x90like multisystem inflammatory syndrome in children, and cytokine storms in adults.^[nn53nn^] Given the unique traits found in SARSxe2x80x90CoVxe2x80x902 and all the open questions there still are around its emergence, until either a natural or laboratory origin is conclusively demonstrated both avenues should be robustly investigated by the scientific community.