The Only Unequivocally Human-Created Gain-of-Function Phenomenon: The Immune-Escape Pandemic Driven by Mass COVID-19 Vaccination

A new study shows how coronaviruses can generate genetic insertions naturally. Does that vindicate the zoonotic-origin hypothesis of SARS-CoV-2? Perhaps it answers the wrong question.

A fascinating new publication from the laboratory of Paul Bieniasz has added another piece to the molecular puzzle of coronavirus (CoV) evolution.

At first sight, it could easily be interpreted as good news for proponents of a conventional zoonotic origin of SARS-CoV-2 (SC-2).

The study, ‘Genetic innovation in coronaviruses driven by a viral nuclease’, reports that the CoV endoribonuclease NSP15 can participate in the generation of novel sequence insertions. Working with both HCoV-OC43 and SC-2, Bianco and colleagues provide evidence for an endogenous viral mechanism capable of generating insertional genetic diversity (https://www.biorxiv.org/content/10.64898/2026.08.15.744938v1).
That is important. One of the most discussed peculiarities of SC-2 since the beginning of the pandemic has been the insertion at the S1/S2 junction of the spike (S) protein that created its polybasic furin-cleavage site (FCS). The 2020 Nature Medicine article ‘The Proximal Origin of SC-2’ specifically identified this as one of the notable genomic features requiring explanation (https://www.nature.com/articles/s41591-020-0820-9).

So has Bieniasz now provided the missing explanation? Has Mother Nature finally produced her receipt?

Not quite.

The SC-2 FCS is anything but a decorative curiosity in the genome.

Experimental evidence indicates that it contributes critically to the phenotype that made SC-2 such an efficient respiratory virus. When Peacock and colleagues removed the S1/S2 cleavage site, the resulting virus showed impaired replication in relevant human airway cells and, strikingly, failed to transmit between ferrets under conditions in which the parental virus transmitted efficiently. The key point is that this experiment tells us that the FCS is functionally important. It does not tell us how SC-2 acquired it (https://www.nature.com/articles/s41564-021-00908-w).

The polybasic FCS of SC-2 was conspicuous because it was absent from the closest known SC-2-related sarbecoviruses. Other members of the wider CoV family do possess furin-sensitive cleavage sites and insertions at or near the S1/S2 junction have also been observed naturally in related bat CoVs. But none of this retrospectively establishes how the specific Pro–Arg–Arg–Ala (PRRA) insertion in SC-2 arose.

Theoretically, one possibility is that it arose spontaneously during natural CoV evolution. Another is that such an insertion arose spontaneously during experimental propagation or selection. A third possibility is that a functionally important cleavage site was deliberately introduced into a progenitor virus during experimental work.

The ferret experiments cannot distinguish among these possibilities. Indeed, their significance for the origin question lies elsewhere: they show that acquisition of the FCS potentially represent a major phenotypic step toward efficient mammalian respiratory transmission.

This makes the history of that site more - not less - interesting.

The new Bieniasz study adds another layer. It provides evidence that CoVs possess an endogenous NSP15-associated mechanism capable of generating insertional genetic novelty. This strengthens the biological plausibility that an insertion could arise spontaneously.

But biological plausibility is not historical reconstruction.

Demonstrating that a CoV can generate an insertion by its own molecular machinery does not demonstrate that the SC-2 PRRA insertion actually arose in a naturally infected animal population. Nor does it exclude the possibility that exactly the same endogenous mechanism operated during experimental propagation. Importantly, demonstrating that the FCS performs a biologically meaningful function cannot distinguish among spontaneous evolution, experimental propagation, or deliberate genetic introduction; each of these routes could produce a functional viral phenotype.

The scientifically relevant question is therefore not merely whether the FCS could arise naturally, but how SC-2 came to contain a feature that contributed materially to its transmission phenotype ─ and in what evolutionary or experimental context that feature emerged or was introduced.

Suppose the PRRA-associated insertion arose through an entirely endogenous CoV mechanism. No scientist designed the nucleotide sequence. No cloning system was used to place it there. The viral replication machinery generated it spontaneously.

Would that prove zoonotic emergence? No.

An endogenous mutation occurring during experimental propagation remains an endogenous mutation. Likewise, selection of a fitter virus during serial passage is still Darwinian evolution. The fact that researchers created the circumstances under which selection occurred does not make the mutation itself unnatural.

This is why the familiar dichotomy - natural evolution versus laboratory manipulation - is biologically inadequate. There is a large grey zone between deliberately constructing a viral genome, for example, and having no human influence whatsoever on the circumstances of its evolution. A naturally occurring CoV could be collected from wildlife and propagated. It could undergo repeated replication in cultured cells or animals. Its population could diversify spontaneously while selection determines which variants prevail. Every mutation emerging during that process could be fully compatible with ordinary CoV biology while the evolutionary environment itself was created by humans. That is how I myself generated variants of FIP CoV (the virus causing Feline Infectious Peritonitis) in the past under experimental laboratory conditions, by repeatedly passaging the virus in Crandell feline kidney-cellen (CrFK/CRFK).

A natural molecular mechanism does not identify the evolutionary environment in which that mechanism operated.

One of the most striking characteristics of is SC-2 what happened shortly after the virus entered the global human population: it evolved not merely by accumulating neutral mutations but through repeated replacement by viral variants with measurable fitness advantages!

The D614G substitution in the S protein appeared remarkably early and rapidly became dominant. Experimental work showed that D614G increased infectivity and transmission fitness. Alpha then emerged with a substantial infectivity and transmission advantage over previously circulating lineages. Beta and Gamma also carried convergent adaptive mutations and Delta later displaced Alpha in many regions.

The timing matters. D614G emerged and dominated in prevalence during the first half of 2020. Alpha appeared in the United Kingdom around September 2020, while Beta and Gamma emerged independently in South Africa and Brazil during the second half of 2020. All of this preceded the launch of the insane C-19 mass vaccination program and, therefore, occurred before suboptimal population-level immunity became the dominant evolutionary pressure on SC-2.

This means that at least an important part of the earliest SC-2 evolution must be understood as adaptation toward greater intrinsic viral infectiousness in the new human host. This is to say that, although the Wuhan virus was clearly capable of sustained human-to-human transmission, it subsequently evolved into more infectious variants that enabled more efficient and prolonged pandemic spread.

The early fitness sweeps therefore raise an important question:
if SC-2 entered the human population already fully adapted for sustained pandemic transmission, why was there so much immediately accessible evolutionary headroom for variants with markedly higher intrinsic fitness?

Importantly, none of this implies that the selective forces acting on SC-2 remained unchanged throughout the pandemic.

The early replacement of the Wuhan lineage by D614G and the subsequent emergence and expansion of the Alpha, Beta and Gamma variants began before the C-19 mass vaccination. Those early events therefore cannot reasonably be attributed to collective vaccine-induced immune pressure on the virus. They are more parsimoniously interpreted as reflecting adaptation toward greater intrinsic viral fitness in a new human host.

However, once large fractions of highly C-19-vaccinated population had acquired substantial antibody responses through severe disease and primarily vaccination, or both, the adaptive viral landscape profoundly changed. Viral fitness was no longer determined only by intrinsic infectiousness. It increasingly depended on the virus’s ability to escape neutralizing antibodies and sustain transmission in immunologically experienced hosts.

This is precisely where the later phase of the pandemic must be distinguished from the early one. The initial fitness-adaptation phase and the subsequent immune-landscape phase as shaped by the large-scale C-19 vaccination program are not contradictory explanations; they are successive evolutionary regimes. In my view, mass C-19 vaccination during ongoing widespread transmission is to be considered the main contributor to that later population-level selective environment, favoring variants able to maintain transmission despite vaccine-induced immune pressure. Repeated vaccine-breakthrough infections and the resulting immune focusing further promoted viral immune escape, thereby selecting increasingly infectious and immune-resistant viral phenotypes while progressively driving population immunity toward inefficiency and, ultimately, dysfunction.

Thus, recognizing strong pre-vaccine selection for increased intrinsic infectiousness does not contradict the later conversion of the pandemic into one increasingly shaped by vaccine-driven immune escape. Rather, it suggests a shift in the dominant selective forces over time: from positive selection for greater intrinsic infectivity to selection for escape from the immune pressure exerted by highly C-19-vaccinated populations.

The comparison with the 1918 influenza pandemic is instructive. Genetic material recovered directly from victims of the 1918-1919 pandemic indicates that its H1N1 virus remained remarkably conserved across geographically distant locations and across successive pandemic sampling periods (https://pmc.ncbi.nlm.nih.gov/articles/PMC2720273/).
Although minor genetic variation and two co-circulating receptor-binding configurations existed, there is no evidence for the serial global replacement of the original pandemic strain by successively more transmissible variants comparable to what occurred with SC-2.

SC-2 behaved strikingly differently.

Beginning with D614G and followed by Alpha and subsequent variants, fitter viral populations repeatedly displaced those that preceded them. Importantly, this process had already begun before mass vaccination and before widespread population immunity could have provided the dominant selective pressure.

This suggests that SC-2 entered humans only partially adapted and subsequently completed a substantial part of its optimization within unusually permissive human transmission environments, eventually becoming fully adapted for sustained pandemic transmission in human populations.

The comparison should not be overstated. The 1918 virus was not literally genetically frozen; sequence diversity existed. However, the relevant observation is that the archival evidence does not show anything resembling the conspicuous succession of globally sweeping, progressively fitter lineages seen with SC-2. That historical contrast deserves attention!

If a newly emerged animal CoV requires several successive steps of enhanced intrinsic infectiousness before achieving the transmission robustness ultimately observed for SC-2, then demonstrating that any individual mutation - such as the FCS insertion - can arise spontaneously does not solve the origin problem. One must also demonstrate the existence of an evolutionary pathway that allowed the intermediate forms to survive, replicate and undergo positive selection.

That is the critical point. Evolution requires variation and selection. Bieniasz may have illuminated an important mechanism generating the former.
A novel insertion is evolutionarily irrelevant unless the virus carrying it survives. A modest improvement in attachment and ACE2 engagement or S proteolytic priming/activation is irrelevant unless the virus gets transmitted. A partially adapted animal CoV crossing into humans may simply disappear after one or a few infections. For an incompletely adapted virus to cross the evolutionary gap toward robust human transmission, it needs repeated replication and transmission opportunities.

But what circumstances enabled SC-2 to cross this evolutionary gap and develop into a genuine global pandemic?

Imagine a virus whose transmissibility in humans is initially only moderate. In a population with limited contact rates, many transmission chains will terminate. But introduce the same virus into an environment characterized by exceptionally intense interpersonal contact and repeated exposure, and the equation may change!

Extremely dense housing, crowded gatherings or indoor workplaces, overcrowded nursing homes for the elderly and people with disabilities, mass transportation, poorly ventilated buildings and other environments producing frequent close contacts can enormously increase the number of successful transmission chains. This is particularly true when such settings are compounded by poor hygienic conditions.

Such circumstances do more than amplify an outbreak. They can provide an evolutionary incubator. More infected people mean more viral replication. More replication means more genetic variation. More variation increases the probability that advantageous mutants arise and dominate in prevalence.

A mutant with even a modest increase in intrinsic infectiousness can progressively displace its ancestor. In that sense, a dense and highly connected human environment could theoretically allow a virus that was only partly adapted to humans to remain in circulation long enough for positive natural selection to improve it. The impact of these environmental conditions should, of course, be clearly distinguished from the population-level immune pressure exerted on the virus in highly C-19-vaccinated populations, most of which are found in industrialized countries.

Partial human adaptation -> ecological amplification -> massive replication opportunity -> selection of higher intrinsic fitness -> increasingly robust transmission.

Nothing about this scenario strictly requires laboratory involvement. Dense human populations, possibly combined with poor hygienic standards, could theoretically provide an evolutionary bridge to a functionally different phenotype.

This is where the expression ‘GoF’ often creates more confusion than enlightenment. Public debate commonly presents two opposing scenarios: Nature made the virus or scientists made the virus.

Biology permits many possibilities in between. A wild CoV can spill directly into humans. It can pass through an intermediate host. A naturally occurring virus collected during field work could infect a researcher. An isolated virus could be propagated repeatedly without deliberate sequence alteration. It could be serially passaged through cells or animals. But researchers could as well deliberately introduce specific changes. Several of these processes could occur sequentially.

Although these scenarios are scientifically distinct, their outcome may be the same while only some necessarily leave recognizable signs of deliberate engineering. Extensive documentation of deliberately targeted GoF research, for example, strengthens the suspicion that SC-2 may have been artificially generated and, therefore, may not have had a purely natural origin. However, “naturally generated” does not imply “naturally originated.”

The 2020 Proximal Origin paper made a genomic argument that SC-2 did not resemble a virus straightforwardly assembled from an obvious previously described backbone and the authors argued against purposeful manipulation. That is a genomic inference. It is not logically identical to demonstrating that the virus emerged through an entirely natural zoonotic chain.

The Bieniasz findings illustrate why. If CoV enzymes can spontaneously generate insertional diversity, and ordinary selection can amplify advantageous variants, then an experimentally propagated virus could acquire a genome that remains entirely compatible with natural molecular evolution.

The experiment would have altered the evolutionary environment, not necessarily the mutational machinery.

Bieniasz may make the molecular origin of an insertion less mysterious. Peacock made the functional importance of the SC-2 FCS less mysterious. However, neither study tells us how the critical mutation arose, nor can either study determine whether it occurred naturally or was generated artificially.

Any serious discussion of SC-2 origins must acknowledge another part of the historical record: sophisticated CoV reverse genetics and recombinant-virus technology did not suddenly appear in 2020.

Years before the C-19 outbreak, researchers had developed infectious CoV cDNA systems, recombinant CoV vectors, chimeric S constructs, targeted mutagenesis strategies and experimental approaches capable of altering CoV genomes and phenotypes. Patent records and the scientific literature document that such capabilities existed well before 2019.

David Martin has repeatedly drawn attention to this patent landscape and to pre-pandemic CoV constructs. The underlying factual point deserves to be taken seriously: pre-pandemic CoV engineering was real, technically sophisticated and well established. Any origin argument that implicitly assumes otherwise is historically untenable.

But one must distinguish capability from provenance. A patent describing recombinant CoV technology proves that the technology existed. A patent or publication describing chimeric CoV spikes proves that such constructs could be - and in some cases were - made. Those facts do not, by themselves, establish that the specific SC-2 genome first detected in 2019, or its immediate progenitor, was produced by one of those methods.

To establish that connection unambiguously would require a much more specific evidentiary chain: for example an authenticated pre-outbreak viral sequence closely ancestral to SC-2, archived biological material confirming the constructed sequence or epidemiological evidence linking that material to the first infections.

This does not make the patent evidence irrelevant. Quite the opposite: it establishes that human-created environments existed in which CoV genomes could be deliberately altered, recombined, propagated and selected before the pandemic. That fact belongs in any serious assessment of plausibility.

At the same time, the Bieniasz findings remind us that experimental propagation need not involve deliberate insertion of every consequential mutation, such as the insertion of a FCS, for example. A CoV placed in an experimental evolutionary environment can generate mutations through its own endogenous machinery and can then undergo Darwinian selection. The resulting genome may look entirely compatible with natural molecular evolution even though some of the evolutionary circumstances were human-created. Although this now seems entirely possible, it does not imply that deliberate genetic manipulation leading to a similar outcome can be ruled out ─ especially since the molecular techniques capable of generating comparable CoV mutations and constructs were already known and documented before the outbreak of the pandemic.

Bieniasz shows that the virus itself can generate insertional novelty. The pre-2019 patent and experimental construct record shows that researchers already possessed the technical capacity to engineer CoV with comparable genetic alterations. Neither fact, on its own, tells us whether SC-2 arose through natural evolution or was generated, modified, propagated, or selected in a laboratory environment.

Suppose the immediate progenitor of SC-2 was a bat sarbecoviruses capable of engaging human ACE2. Suppose the FCS insertion arose spontaneously in bats or intermediate susceptible mammals, or was artificially created by human intervention. Suppose other necessary genetic changes also arose through ordinary CoV biology accompanying abundant replication and propagation in animal species. Even then, we must still explain how a virus successfully jumped from an animal-adapted state to one capable of sustained human transmission. Receptor binding and FCS insertion are only individual components of human transmissibility. Sustained human-to-human transmission requires many additional functionalities. The evolutionary gap should therefore neither be exaggerated nor reduced to ACE2 binding and FCS insertion alone. The relevant origin question is how the complete combination of traits was assembled, rather than merely how the FCS insertion was acquired.

If intermediate forms carried only partial improvements in human fitness, where could they remain in circulation long enough for subsequent adaptive steps to arise? Did high-density human environments allow a partially adapted virus to replicate and propagate exceptionally well and thereby progressively evolve to improve after spillover?

No study has elucidated how SC-2 shifted so rapidly toward increasingly infectious variants in populations that were initially largely immunologically naïve. However, it was quite clear that the original Wuhan-Hu strain was not sufficiently infectious to spread rapidly enough across the globe. It is therefore reasonable to assume that only by chance ─ through a fortuitous convergence of favorable circumstances ─ the virus eventually acquired enough transmissibility to cause a true pandemic. This occurred around the time that mass C-19 vaccination began. The timing of mass vaccination could hardly have been worse!

Suppose crucial pre-pandemic evolution involved not conspicuous synthetic engineering but selection during repeated replication.

A progenitor virus is propagated. An insertion arises spontaneously. Another mutation improves replication. Another enhances receptor interaction. Another subverts innate immunity. Selection retains the fitter population.

At every stage the mutations could be ordinary viral mutations. The polymerase would be viral. NSP15 would be viral. The selective advantage would be biological.

The resulting genome, however, might contain no forensic marker revealing whether the decisive replication cycles occurred inside an animal in the wild, a human population, or an experimental system.

The genome records which sequence survived. It does not necessarily record where the competition took place.

This may represent a fundamental limit on what sequence analysis alone can ever establish about the origin of SC-2. Given authenticated pre-2019 laboratory records documenting a virus extremely close to SC-2, archived sequence files showing its similarity to existing bat sarbecoviruses, patents confirming the sequence and epidemiological evidence linking that virus to the onset of the C-19 pandemic, proponents of the laboratory-origin hypothesis argue that SC-2 directly resulted from targeted GoF research.

A zoonotic origin has never become overwhelmingly persuasive owing to the absence of archived pre-outbreak animal material containing an immediate progenitor, failure to reconstruct evolutionary intermediates or to establish direct links between infected animals and Wuhan through a documented supply chain, or missing epidemiological connections between the earliest human cases and those animals. The further we move from 2019, the more difficult an unambiguous reconstruction becomes. More troubling still, some of the records required to resolve the question conclusively may never have existed.

Perhaps we have spent years asking the wrong question.

Was SC-2 natural? In the sense that its molecular machinery obeyed normal biological rules, almost certainly. But that is close to trivial.

The deeper question is: what environment or intervention enabled the succession of replication and selection events that transformed its progenitor into a virus capable of sustained human transmission?

At least from a theoretical viewpoint, the answer could involve an animal population, an intermediate host, highly permissive transmission settings in dense populations, an experimental GoF system, or some combination of these. The subsequent pandemic itself then appears to have passed through at least two distinct evolutionary regimes: an early phase dominated largely by gains in intrinsic fitness, followed by an increasingly immune-shaped phase as adaptive, population-level immunity accumulated in highly C-19-vaccinated populations.

These scenarios are not equally supported by current evidence. But they cannot be distinguished merely by demonstrating that the FCS mutation was capable of arising spontaneously.

Despite all the compelling evidence on targeted GoF research, we cannot rule out that possibility. Mother Nature has a surprisingly broad job description. She operates in bats. She operates in intermediate animals. She operates in humans. She operates in crowded cities. This would make a natural origin highly likely, were it not for the existence of a large database documenting the design of a genetically engineered SC-2-like virus derived from bat sarbecovirus sequences.

That is why showing that NSP15 can spontaneously generate CoV insertions, including an FCS insertion, cannot settle the SC-2 origin debate. If anything, it shows why the debate is more nuanced than a simple black-and-white dichotomy between laboratory and natural origin.

A natural molecular mechanism does not identify the way in which that mechanism was generated.

If a newly emerged animal CoV requires several successive steps of enhanced intrinsic infectiousness before achieving the transmission robustness ultimately observed for SC-2, then demonstrating that any individual mutation - such as the FCS insertion - can arise spontaneously does not prove a natural origin of SC-2.
Regardless of its origin, one must also explain the evolutionary pathway that ultimately enabled the intermediate forms to survive, replicate, and undergo positive selection for enhanced infectiousness.

That is the missing part of the story.

· Bianco et al. (2026). Genetic innovation in coronaviruses driven by a viral nuclease. bioRxiv.

· Andersen et al. (2020). The proximal origin of SARS-CoV-2. Nature Medicine.

· Peacock et al. (2021). The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets. Nature Microbiology.

· Reid/Taubenberger-related archival 1918 influenza sequence analysis.

· Selected pre-2019 CoV patent literature: recombinant CoV/ nidovirus reverse-genetics platforms and SARS CoV infectious cDNA/recombinant vectors.

· Pre-pandemic literature on engineered/chimeric CoVs and S constructs: evidence of technical capability and experimental precedent, but not standalone proof of SC-2 provenance.