Treacherous ancestry : simplebooklet.com

AboutProtagonist ScienceI believe that science is the hidden protagonist in anystory that truly matters. Currently, Protagonist Sciencefound its role as an outlet for independent sciencejournalism and debunking of SARS-CoV-2 conspiracymyths. In the end, this science communication endeavor is along and eternally incomplete love letter about findingthe human element in our technological future.Sometimes written, sometimes spoken, sometimesvisualized. Never, ever not worth your timeThe scientific method is one of the most important processes to understandin today’s technological age.3WELCOME MESSAGE 2024About the authorPhilipp Markolin is a Switzerland-based sciencecommunicator and writer. He holds a Bachelor degreein Chemistry and a Master degree in biochemistry fromthe Technical University of Graz, Austria and adoctorate degree in translational biomedicine from theSwiss Federal Institute of Technology. After apostdoctoral position as bioinformatic analyst in amachine learning lab, he decided to apply his hybridbackground to understand viral phenomena in ourmodern information ecosystems, and how they shapediscourse and public perceptions of science andsociety.

Page 4

Prelude: The ongoingConfusionsWhy are US scientists dragged in front of Congress inBenghazi-style show trials? How come highly ratedresearch programs get quietly shut down? Is it justifiedto treat virologists with suspicion, consider them guiltyof recklessness or dishonesty, even leak their subpoenaed private conversations to professionalsmear artists for cherry-picking?The origin controversy is political and polarized.Myths that COVID-19 was somehow a manmadepandemic are impactful, whether they are true or not.Polls have shown that 2 out of 3 US citizens believethat SARS-CoV-2, the virus that started the COVID-19pandemic, came out of a laboratory rather than nature.Scientists worldwide vehemently disagree. Theemerging scientific consensus among domain expertsis that SARS-CoV-2 is a natural virus that enteredhumanity via zoonotic spillover (more importantly, thereis a consensus stemming from the body of evidencethat is entirely unequivocal).Yet fewer and fewer citizens seem to hear or care fortheir evidence-based voices. Domain experts aredistrusted, but how much of that is self-inflicted, andhow much is sown by anti-science actors? We canobserve scientists are increasingly bullied out of publicdiscourse by information combatants or targeted byharassers on social media. Most simply have theirknowledge drowned out by an information ecosystemthat favors emotional falsehoods over complicatedfacts. It is fair to say that science has come underpressure.On top of that, various conspiracy myths about a “lableak” continue to garner attention in the media,international forums, and the halls of US Congress, allwhile elected representatives, senators, anti-scienceactivists, and propagandists seek to gain popularity,profit, or power by instigating a “gain-of-functionresearch” moral panic.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/4ProtagonistS C I E N C EOn moral panics: “As a distinct species of collectivebehavior, moral panics representcontentious and intensely affectivecampaigns to police the parameters ofpublic knowledge and morality. [...]seeking to actuate alarm by influencingthe imagery and representations of themainstream press”.-Walsh JP, International Journal ofCultural Studies, 2020Dr. Kristian Andersen and Dr. Robert Garry called to testify on the originsof COVID-19 before the House Oversight Select Subcommittee on theCoronavirus Pandemic. [Image credit: C-SPAN]I) Virology under suspicion"We keep changing our mindsabout things based on data - thatis science, not fraud." - R. Garry

Page 5

How many concerns about GoF research are justified,and how many just make for a good story? Mediamanipulators are skilled at fabricating false uncertaintyand inventing sensationalist narratives out of wholecloth.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/So where is the real birthplace of SARS-CoV-2? Howexactly did this chimeric virus come about? Whichtrajectory did it take before the first outbreak emergedat the Huanan market in Wuhan? Will we ever find out?Given current geopolitics, deliberate sabotage ofscientific processes, and Beijing’s obfuscation of thematter, many say the true origins of COVID-19 willnever be solved. Some governments explicitly root forthat outcome, preferring to be stuck in perpetualuncertainty forever. I would not take that bet againstscientific inquiry — and after reading this deep dive onthe origin controversy — I believe neither will you.5ProtagonistS C I E N C EII) A scientific question stillneeds a scientific answerAll scientists can hope for is that eventually, citizens willget tired enough of the spectacle and make-belief totake a closer look at what we already know about thetopic.When it comes to supportingevidence, the ‘gain-of-function”emperor is embarrassingly naked.But it makes for a fantastic story.Scientific literacy is a superpower in the(dis)information age. It enables citizens to cut throughthe noise, intelligently engage with a topic of globalcontroversy, and come out wiser. Scientifically literatecitizens however need relevant evidence and thereasoning of scientists to be made accessible to formtheir opinion. On top of that, they will probably needmonths to familiarize themselves with the complexity ofthe origin discussion, and honestly, who has time forthat? Having done the (sometimes painful) legwork,here is where I believe I can offer a helping hand. If yougift me your curiosity and time, this article will be aguide to understand some of the most compellingevidence for a natural origin of this virus.The Wuhan Institute of Virology is often center of speculations about“gain-of-function” research that supposedly created SARS-CoV-2. Noevidence of any lab involvement has yet emerged.Clues that might not only hold the definitive answerto whether SARS-CoV-2 came from nature or thelab, but also how to best anticipate and mitigate therisk of another SARS-related coronavirus pandemic.Outside the heated spotlight, multiple teams ofinternational scientists have quietly honed in on a set ofnew clues to the origin of COVID-19.An asymmetric controversy “It takes more effort to debunk aconspiracy theory than to create one. Ittook only one podcast to make me thinkthe lab leak theory was possible, but ittook months of research to understandwhy it’s not” — Peter Miller, who recently won$100.000 in an 18h origin debate against a lab leak trutherIt is time to put the false “gain-of-function research”myth to bed once and for all, and learn about thecutting-edge science on the hunt for the trueorigins of SARS-CoV-2.Buckle up, or fetch a large cup of tea, this is the originstory told through the lens of viral recombination.Tune out the politics, the brokenmedia, and the bad actors. Let’s returnto the process of scientific inquiry.

Page 6

Chapter 1: Unraveling amysterious chimeraWhen SARS-CoV-2 emerged, it was a confusing virusfor a lot of reasons. First, it was very infectious tohumans for a novel virus, spreading effectivelybetween them via the respiratory route. Second, it didnot cause severe disease in many patients, wassometimes asymptomatic, and subsequently hard totrack. Third, side-by-side comparisons to known SARS-related viruses seemed to show that the novel viruswas a chimera. It had parts of very high geneticsimilarity, and other parts of low genetic similarity, ontop of a few other oddities that gave researchersinitially a hard time to wrap their heads around. Evenseasoned virologists were quoted wondering how “thisgets accomplished in nature” in early February 2020.As the pandemic turned into full swing, multiple “man-made” theories of varied quality were advanced onhow the SARS-COV-2 — and its odd genome — came tobe. From bioweapon development to gain-of-functionresearch, from de-novo genetic engineering to thealleged introduction of HIV sequences, from serialpassage through human cells or humanized mice toarcane vaccine experiments, many asserted that sometype of human manipulation was necessary to explainhow this dangerous patchwork virus of high and lowsequence similarities to other coronaviruses cameabout.Only coronavirologists with decades of experience withthat particular viral family would disagree, theycertainly saw nothing unheard of in the genome. Butthe biggest problem in debunking the plethora of falsenotions was the lack of reference points; namely viralcousins of SARS-CoV-2. Those only gradually cametrickling in once the severity of the outbreak turned intoa global pandemic and jolted more and more scientistsinto urgent action. Rather than speculate oninconclusive data, many researchers set out to findrelated coronaviruses, either by discovering neglectedgenomes in large biomedical databases or by directsampling of bats in nature.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/I) An uncanny genomeThey quickly realized that SARS-CoV-2-related virusesall looked a bit weird and stitched together; forexample, a pangolin virus had an ACE2 receptorbinding domain very closely related and able to bindand infect human cells. Another bat virus discovered inMengla country in China had an insertion reminiscentof the furin cleavage site at the S1/S2 boundary, as wellas what looked like an ancestral genome to SC2, atleast for about the first 2/3rds of its full span.How could these diverse animal viruses seem soclosely related to SARS-CoV-2 in one part, and sodistant in another?6“Everything that seemscounterintuitive withCoVs is actually logicalwhen you work with thema few times”,Jasnah Kholin(pseudonym)a coronavirus reseacherfrom Hong Kong ProtagonistS C I E N C E

Page 7

Almost from day one, coronavirus veterans had beenquick to educate their collaborators that CoV genomesmight just seem unintuitive because they areshaped by a process called recombination.Recombination is a mechanism for genetic exchangebetween two different parental viruses that creates anew viral genome sharing genetic information of both.It requires two (usually distinct) viruses to be present inthe same host cell.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/II) On recombination versusartificial assemblyWhat this means is that the RNA production machinery(RdRp) can jump between instruction templates atany point if more than one is available, producing ahybrid sequence (chimeric genome). I sketched out the mechanistic interplay betweenreplication and viral gene transcription (see next page)to illustrate how template switching is not a freak eventbut an essential step in the viral life cycle.7“Replication of the coronavirusgenome requires continuous RNAsynthesis, whereas transcription isa discontinuous process uniqueamong RNA viruses. Transcriptionincludes a template switch duringthe synthesis of subgenomicnegative-strand RNAs to add acopy of the leader sequence.” — Sola I. et al., Annu Rev Virol., 2015Three proposed molecular mechanisms for copy-choice recombination in CoVs utilizing RdRp template switching. (Wells H. et al., Cell Host Microbe, 2023)ProtagonistS C I E N C EAs a useful but impreciseabstraction, one might think ofrecombination as a form of “virussex” that produces uniqueoffspring sharing a mix ofparental genomic regionsOffspring that came about by recombination is in manycases unproductive, meaning it can either not fulfill allessential functions necessary for the virus to replicateand infect new hosts; or it can do so but worse than theparental lineages, thus getting quickly outcompeted bythem and vanish into nothingness. A virus needs toconstantly spread and adapt to persist, after all.However, sometimes this “virus sex” brings forthrecombinant offspring that is in some aspect better — meaning more fit in its current or new environment — than the parental lineages, ergo it will spread and gainground in the host population or particularenvironmental niche, possibly establishing itself foryears to come.Recombination frequencies vary dramaticallybetween viral families, from the promiscuous tothe prudent. For example, recombination does notseem to play an important role in some Filoviridae(Ebola, Zika) and Paramyxoviridae (Hendra, Nipah). Atthe same time, segmented viruses such as influenzause a different mechanism of genetic exchangealtogether called “re-assortment”.The coronavirus family of negative-strand RNA virusesreportedly recombines frequently. Recombination inCoVs is thought to be facilitated by a molecularmechanism called RNA-dependent RNA polymerase(RdRp) template switching leading to copy-choicerecombination (see figure below).

Page 8

An RdRp that needs to jump back and forth betweengenetic elements constantly might easily jump toanother different genome when thrown into the mix,thus providing ample mechanistic opportunity forrecombination. (Where CoVs get physical opportunityto have so much virus sex and what we can learn fromit we will look at in Chapter 2)This is of course not a secret to experts in the field.That coronaviruses fuck around — sorry recombineconstantly — has been scientifically established formany years before COVID-19. For now, what is important is that the promiscuoussarbecovirus (SARS-related beta-coronavirus)sub-genus gets a lot of recombinant progeny; andover time, established offspring lineages canthemselves engage in more virus sex (= undergorecombination) with other circulating viruses. Fromlong-estranged cousins and viral strangers toincestuous siblings and even their own offspring,anything seemingly goes (hey, not our place to judge!).PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/8Illustration of recombination-prone genome replication in negative-stranded coronaviruses. When two RNA viruses find themselves in the same host cell,recombination is not an unusual event but common. (Individual figures from Sola I. et al., Annu Rev Virol., 2015 and Wells H. et al., Cell Host Microbe, 2023)Again, thinking about this promiscuous mingling ismerely a useful abstraction, because in reality, eachindividual virus genome is just one of many millionscopies in a single infected person. Out of thiscomplexity, recombination can result in ever newchimeric offspring that — if successful — will carryparental genetic segments forth from the generationsthat came before it. However, in the early days withless information, SARS-CoV-2 did not seem veryrecombinant. But maybe something changed withmore bat cousins being found since?ProtagonistS C I E N C EIf SARS-CoV-2 and relatedsarbecoviruses indeed came aboutby frequent recombination, we wouldexpect a colorful mix (a mosaic) ofgenome segments shared betweenthem and their closest cousins. Let’s take a look (next page).CoV’s versatile RdRp usage facilitates template-switching and recombination susceptibility1) Genome replication is a continuous linear process,but transcription is discontinuous (making theRdRp jump back-and-forth between elements)3) Replication and transcription happen simultaneouslyand a single complex 3D protein/RNA machineryguides the RdRp meticulously between continuoussynthesis and jumpy transcription4) RdRp jumping can get messy (template switchingduring replication) when similar RNA sequencesfrom different viruses get mixed together, creatinga new recombinant CoV genome2) RNA is not a straight line. Precise RdRpjumping (template switching) in transcription isinfluenced by higher-order RNA structure

Page 9

Recombination is the only scientific explanation thatcan account for the observation of these naturalchimeras. (There are many more technical details as towhy scientists know that SARS-CoV-2’s mosaicgenome was not assembled and stitched together in alab from a sequence, but you get the main point)On top of that, the observed recombination patternsleft behind by past “sexual” encounters betweenparental lineages can not be reproduced,simulated, or faked in a laboratory. Pause for asecond and read that again.The relevant “sexual history” that played out over thelast five decades or so needed to be lived in the wild, itcould only have happened in nature where all theseviruses meet.All scientific evidence and prudence suggest thereforethat a naturally evolved, immediate bat ancestorto SARS-CoV-2 must have existed at one point. Itdid not spring from a computer sequence, was notdreamt up by a mad scientist, or derived and recklesslyassembled from disparate parts. SARS-CoV-2’s batancestor came about as naturally as all the otherrecombinant children of parental sarbecoviruses thatwe have since discovered in bats. PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/On the larger genetic makeup, this ancestral batversion of SARS-CoV-2 already looked very much likethe one that first surfaced in Wuhan. But this alone isnot sufficient to exclude that such a recombinant batancestor was found by researchers and then “tinkeredwith” in the lab. Recombination has a low resolution,it can not track targeted single mutations. So genetictinkering remains possible and plausible, right?How else can we explain that a bat virus seems sodamn good at infecting human cells?9ProtagonistS C I E N C EHow come every random viralcousin found in the wild looksequally “stitched together”? For SARS-CoV-2, the above figure shows that it sharesregions of high sequence similarity with a bunch ofdifferent bat viruses, namely RmYN02, Banal-103,RpYN06, Banal-52, and RaTG13. Close cousins.However, for each SARS-CoV-2 to bat viruscomparison individually, there might be some regionsof very low sequence similarity (usually around thespike) where they are not similar at all.The observation of this mosaic genome makes clearthat SARS-CoV-2's overall genome could not havebeen designed or derived from any known singleprogenitor virus (for example RaTG13 or a BANAL-likevirus, as lab leak activists like to argue), but rather thatit is a genetic chimera containing bits and piecesfrom multiple related viruses. We know this technicallybecause no engineering or culturing approachcould have magically created the “ancestral”sequence in hundreds of positions any time acousin diverged from SARS-COV-2.Given this reality slowly breaking through, some lableak activists (not prone to hold any coherenthypotheses on this issue anyway) have since moved onto fantasize that SARS-CoV-2 must then have been“stitched together” in a lab from a set of (multipleknown and unknown) progenitor viruses secretlysampled by the Wuhan Institute of Virology. (“What canbe asserted without evidence can also be discardedwithout evidence”, I would say about the never-endingepicycles of conspiratorial ideation)In reality, all of the proposed “stitched together”speculations are of course contradicted byevidence and epistemically risky at face value.Representation of the 15 recombinant fragments of relevantSarbecovirus genomes compared to the SARS-CoV-2 human prototypestrain. Where possible, the closest viral sequence is indicated for eachfragment. In other cases, MULT indicates a group of multiple sequences.(Figure from: Temmam S. et al., Nature, 2022)This is what scientists mean whenthey say the “backbone” of SARS-CoV-2 is natural.These ideas also fail to account for the fact that allother SARS-CoV-2 relatives found in the wild lookequally stitched together from multiple viruses.

Page 10

Another common line of erroneous argumentation bylab leak proponents concerns the idea that becauseSARS-CoV-2 was capable of infecting humans verywell, it must have been somehow pre-adapted oroptimized to do so via serial passaging in human cells,or been given this remarkable ability by thoughtfulengineers. How else could a bat virus circulating in batsdevelop this human affinity just by chance?For me, this argumentation from supposed“optimization” always has a bit of a fallacious character — “How can something marvelous such as the eyehave come about by chance alone?“ — seen manytimes in creationist arguments. Evolution is not thesame as chance. We humans tend to underestimatethe diversity of nature and the power of evolutionaryselection at our peril.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/III) Recombination patterns arenot randomI am glad you asked. CoV virologists have studied overthe years how the S gene — encoding the spike protein — seems to be one of the biggest factors influencingcellular and species tropism (the ability to successfullyinfect different hosts). When scientists plot thefrequency of recombination events along theSarbecovirus genome, they found that there was notan even distribution, but rather “hot spots” and “coldspots” for genetic exchanges, with the spike regionshining bright red. 10[…] non-random and mostlyconserved recombination patternsthat we and others have detectedin various coronavirus subgeneraare likely shaped both byevolutionarily conserved variationsin the mechanistic predispositionsof different genome regions torecombination and by sharedselective processes disfavouringthe survival of recombinants thatexpress improperly foldedproteins.” — Klerk A. et al, Virus Evol., 2022ProtagonistS C I E N C ESo how do we know that naturehas optimized the human affinityof SARS-CoV-2 all by itself?The figure to the left basically shows that within andsurrounding the spike and some accessory proteingenes (orange), observing new productiverecombinant segments (and with it, a highersequence diversity) is much more likely thananywhere else along Sarbecoviruses’ genomes.So finding that SARS-CoV-2 has an “unusual” spikegene sequence compared to many of its relatives isactually not unusual at all. It is the norm amongmembers sarbecovirus family, they all have unusuallydiverse sequences there. The question is why?What does this mean? Well, let us remember thatrecombination almost exclusively causes fucked upoffspring genomes that are unable to procreate orestablish themselves. All scientists ever get toobserve are the survivors, those one-in-a-billion viralchildren who came out fitter than their parents.Recombination region count matrices indicating genome regions thatare most and least commonly transferred during detectable coronavirusrecombination events (Klerk A. et al, Virus Evol., 2022)A recombination event that addsto niche fitness gets to stay, andwhatever impairs fitness getsselected out.

Page 11

In general, high sequence diversity enables viruses tostumble upon new functionalities, from evadingimmune systems to changing their tropism (what typeof cells they can infect), facilitating more efficientcellular entry, or infecting new host organisms.However, developing and maintaining sequencediversity over a viral population is not always easy tocome by.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Gain-of-function experiments,but different?When we think of general genetic diversity as a“potential for new functionalities” and recombination asa way to “shuffle varied genome segments” around,some alarm bells should be ringing.With chimeric sarbecoviruses genomes, we are inessence observing direct evidence of countless potential “gain-of-function” experimentsconducted on a scale hardly imaginable that must havehappened in the past. (More on that in Chapter 2)11[…] recombination also has thepotential to act as an evolutionary“fast-forward” by quickly shufflinggenetic material between vastlydifferent viruses. For the same result to be producedby mutation alone, long spans oftime would be needed for selectiveforces to shape such extensivenucleotide changes, especiallyconsidering the high proofreadingcapacity of coronaviruses.— Wells H. et al., Cell Host Microbe,2023ProtagonistS C I E N C EThe existence of untold numbers ofsuch recombinant cousins in thewild implies that chimericsarbecoviruses must have beenbirthed in some vast, natural “gain-of-function” laboratory.A laboratory where gain-of-function experiments withpromiscuous parental lineages and distinct geneticfunctionalities relentlessly produce chimeric offspringthat might have tricks from both parents. Over time, the constant combinatoric mixing ofvaried elements and functionalities will on occasionproduce some very elaborate traits, such as theability to infect multiple different hosts (broad speciestropism), including us humans.I think it is worth looking at that in detail.In coronaviruses, recombination is also assumed as anefficient mechanism for how these long, proofreadingRNA viruses can rapidly create genetic diversity from an existing pool of sequences; presumably tocope with quickly changing niche environmentalconditions. (More on that in Chapter 2)Single point mutations arising from replicationerrors rarely get the job done, and face obstaclessuch as purifying selection.On top of that, RNA viruses with large genomesrequire a strong proofreading ability to correctreplication errors that give rise to deleteriousmutations.That is why for some RNA viruses, such as HIV, it isthought that sequence diversity more frequentlyarises through recombination than point mutation.

Page 12

Broken down to a structural level, the majordeterminant of CoVs to infect various host species hasto do with the makeup of the receptor-binding domain(RBD), a part of the 3D organization of the larger viralspike protein that has to fit the 3D scaffold of hostreceptor (ACE2 in humans) exposed on host cells. Thisis a bit of the lock-and-key principle.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/IV) Of locks, keys, and the doorto human infectionViral recombination in CoV spike genes is considered amajor evolutionary mechanism that drives newadaptation processes, such as viral host switching. (Ifyou already have a key factory within the family, andpromiscuous virus sex where successful keys getpassed around like a hot potato, all new chimeras needis the physical opportunity to try them on previouslylocked doors). Here is an interesting little sidebar:12Sarbecoviruses exhibit extensivegenetic diversity in RBM, likelyarising from frequentrecombination and the highselective pressure associated withinter-species host jumping. — Si J. et al., biorxiv, 2024ProtagonistS C I E N C EA very recent illuminating preprint from Chinesescientists — including Zhengli Shi, the unjustly blamed“Batwoman” herself— took a deep dive to understandhow exactly RBM motifs (key shapes) relate to broadACE2 tropism.Experimentally, they produced 56 individual cell linesexpressing ACE2 orthologues (locks) from bats andselected mammals and then tried 14 different RBDs(keys) from Sarbecoviruses.Since 2021, researchers have not only found morecousins of SARS-CoV-2, but they also discovered thatsome of these cousins had keys very much identical toit, proving that at least for now, nature is and remainsthe ultimate key master for this particular set of keys,so to speak.What they discovered was thatsome keys could open almost alllocks, whereas others couldopen only one, or even none ofthe locks presented.The higher the tolerated genetic diversity ofthe RBM (the genetic sequences coding for theRBD in the spike protein), the bigger the repertoireof potential keys available to CoVs.In any case, we do not need to speculate aboutwhether it is possible or what potential ambitionsgenome engineers might have had or not. Much of this is not unique, but based on specificdeletions in specific positions of the RBM (seenext page).

Page 13

On top of this, unique recombination patterns in thespike gene and the discovery of identical RBMs innature make it unequivocally clear that SARS-CoV-2’sRBD was not “designed”, “created” or “swapped in froman unknown virus” by researchers in any kind of “gain-of-function” setup.It acquired this receptor binding domain in nature.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/13Sarbecovirus RBMs exhibit a non-random pattern for narrowing or widening ACE2 affinity and species tropism. (Figures from Si J. et al., biorxiv, 2024)“We revealed that mostsarbecoviruses with longer RBMs(type-I), present broad ACE2tropism, whereas viruses withsingle deletions in Region 1 (type-II) or Region 2 (type-III) generallyexhibit narrow ACE2 tropism,typically favoring their hosts’ACE2. Sarbecoviruses withdouble region deletions (type-IV)exhibit a complete loss of ACE2usage” — Si J. et al., biorxiv, 2024. The RBD in SARS-CoV-2 has had a very broad bindingaffinity from the start, which is why it is not only great atinfecting humans, but minks, deer, house- and zooanimals as well.ProtagonistS C I E N C EThe supposedly “uncanny” ability ofSARS-CoV-2 to infect human cellsis neither uncanny nor especiallyunique. Some bat CoVs can justinfect humans from the get-go. Nopre-adaption, no magic humanhand, no designer, no arcane labexperiments needed.Many Sarbecoviruses show broad species tropism andcan use hACE2 as a host receptor from the get go1) Binding assay of 14 sarbecovirus RBD against cell cultures expressing ACE2 receptor protein of 56 speciesshow broad binding affinity (incl. multiple bat species, but also pangolin, mouse, civet, camel and human)2) The broader the binding affinity,the more likely that an RBD will also beable to infect human cells via hACE23) Sarbecoviruses RBM motives are highly diverse, with the full-length RBMs typically conferring broad species tropism anddouble region deletions resulting in loss of ACE2 entirely

Page 14

PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/V) Defusing the myth of theFurin cleavage siteYou might hear lab leak proponents lament.Recombination does not have the granularity torule out that this small sequence motif wasartificially introduced. The idea gained traction aftera rejected research proposal from 2018 was played upby credulous amplifiers in the press.In the “man-made” mythology, the furin cleavage site(FCS) — a polybasic cleavage site recognized by furin-like proteases — is a dramatic functional element andallegedly the secret sauce and trigger whoseartificial insertion turned an ordinary bat virus into thepandemic blight pathogen we have today.For lab leak believers, the DEFUSE proposal mentioningthe possibility of introducing FCS coupled with the“suspicious” occurrence of an FCS in SARS-CoV-2is all but proof that the virus was engineered ortinkered with. In other words, researchers wanted tocreate a pandemic virus, or had reckless disregard iftheir work would do so.Another quick sidebar:14ProtagonistS C I E N C ESo let us first talk about why the Spike protein needs tobe cleaved, where it needs to be cleaved, and how theFCS might help. (The next points are going to be highlytechnical, so I made a visual summary to help on thenext page)Despite the DEFUSE proposal being irrelevant, I think itis worth discussing the two associated insinuationsbased on scientific ignorance a bit deeper.First, there is the common confusion that theintroduction of a single genetic element has the powerto make a pandemic pathogen. Second, there is deliberate deception about howlikely it is that nature or engineering came up with theFCS insertion in SARS-CoV-2.Furin pre-cleavage aids TMPRSS2-mediated cellfusionBut what about the furincleavage site? Project DEFUSE?The FCS is probably one of themost misunderstood elements inthe history of the SARS-CoV-2origin controversy.The Spike protein in SARS-COV-2 needs to be cutduring viral processing, first in the S1/S2 region,and second at the S2' siteTMPRSS2 extracellular cleavage facilitates entryat or near the cell surface (cell fusion), as opposedto viral entry through the endosome pathway andlate cleavage by cathepsinsExtracellular cleavage allows the virus to avoid thepotent endosomal/ endolysosomal restrictionfactors — the IFITM proteins — which inhibit viralmembrane fusion and can stall virus replicationTMPRSS2-expression is particularly high for cellsin the upper respiratory tract, so viruses that canuse this route more efficiently will have a selectiveadvantage in respiratory transmissionThe FCS motif allows spike proteins to be pre-cleaved before they leave their host cell; but alsomakes them less stableSpike protein egress that was pre-cleaved by thefurin-like proteases in S1/S2 seem to take awaysome of the processing work of TMPRSS2, bindbetter to ACE2, and thus make viral entry via thismembrane-fusion route more efficient for the viruscontinued on next page

Page 15

PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/15Cleavage at S1/S2 and S2 viral processivity studies highlight the function of the FCS and role in egress priming, respiratory tropism as well as transmissionand infectivity. (Figures from: Lavie M. et al., J Virol., 2022, Jackson JB. et al., Nat Rev Mol Cell Biol., 2022, Peacock T. et al., Nature Microbiology, 2022,Steiner S. et al., Nature Reviews Microbiology, 2024) That an FCS acquisition can aid a respiratory virus isnot exactly unobserved in nature either; for example,the transition from low pathogenic influenza strainstowards high pathogenic versions often happens bythe acquisition of a polybasic cleavage site which canbe recognized by furin-like proteases.However, context is critical:ProtagonistS C I E N C EStudies have shown that TMPRSS2-mediatedentry was particularly potent for virus particlesthat had FCS-containing spike compared with thenon-furin-cleaved mutantsIn ferrets it was shown that virions with FCS-containing SC2 spike protein could spread to newhosts, but did not observe this for virions withFCS-mutated spikesIn circulating lineages, the FCS cleavage is highlyconservedConservation of the FCS since pandemic startargues for its selective advantage in human-to-human transmission as wellTherefore, we know that the FCS in SARS-CoV-2is critically involved in transmission andpathogenicity. It was shown that even furin-deficient cells canstill cleave SARS-CoV-2 spike protein at S1/S2The furin-cleavage site in SARS-COV-2 is quicklylost by normal cell culture techniques because ofslower kinetics, so it impedes the virus in thislaboratory “serial passage” contextThe requirement of FCS and basic residues at S2′for S-mediated cell fusion is entirely cell typedependentan FCS does not make (or necessarily break) anpandemic virus, the whole viral-host contextmatterscontinued from previous page:

Page 16

16ProtagonistS C I E N C EFor a subset of highly pathogenic viruses infectinghumans, an FCS might be a necessary, but notsufficient element to efficiently infect humans orsustain respiratory transmissions.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Necessary, but not sufficientFor example, MERS-CoV and SARS-CoV-1 bothcaused epidemics, one with and one without anFCS. In the case of SARS, some experiments haveshown that the artificial addition of an FCS does notincrease infectivity or pathogenicity. Additionally,SARS-CoV-1 without an FCS is no less infectious inferrets compared to FCS-containing SARS-CoV-2.Some of our endemic human CoVs have an FCS(hCoV-HKU1) but do not use ACE2 as an entryreceptor, others have no FCS but still use ACE2(hCoV-NL63)I think we have to make peacewith the idea that viruses arecomplex, highly optimizedbiological machines working incomplex environments.Their molecular details, hostcontext and environmentalcircumstances matter way morethan single genetic elements.In nature, no genetic elementacts alone However, merely adding an FCScan not magically turn any batsarbecoviruses into a pandemicpathogenFor a pandemic virus, the right genetic elements, hostand environmental conditions have to fall together tocreate a perfect storm. We also would expect for anynatural virus capable to start a pandemic to have someunusual genetic tricks up its sleeve. Otherwise, everyordinary virus would cause a pandemic.So why are many suspiciousabout SARS-CoV-2 having anFCS?The main reason why the short FCS sequence inSARS-CoV-2 (12 nucleotide insertion) has been hotlydiscussed is because it stood out like a sore thumbin side-by-side sequence comparisons to knowncoronaviruses in early 2020. (Since then, scientistshave found plenty of naturally occurring FCS in thewider CoV family, but not in the sarbecovirus cladespecifically)Just because something is rare or even uniqueamong currently known sarbecovirus family membersis however not good evidence for artificialintroduction given the high sequence diversity atS1/S2 (see below). On top of that, there is someevidence to suggest the FCS region itself underwentrecombination with a related sarbecovirus. Again, weunderestimate the diversity of nature at our own peril.Polybasic cleavage sites recognized by furin-like proteases are spreadall over the wider beta-CoV family tree; including human CoVs HKU1and OCT43. S1/S2 boundary region is highly sequence-diverse andsusceptible to nucleotide insertions. These are the ingredientsnecessary to produce an FCS, irrespective of whether it is common orwell-maintained in the bat sarbecovirus family specifically. (Figuresfrom: Andersen KG., Nature Medicine, 2020, Wu Y. et al, Stem CellResearch, 2021, Sander AL. et al., Communications Biology, 2022)

Page 17

Let’s discard for a second our experience with vast andcrafty nature — and the naïve presumption that an FCScan turn any virus into a pandemic threat — and take theidea of a “man-made” FCS introduction as a nullhypothesis.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/17Looking at the specifics of the observed FCS sequence motif, as well as how and where it is placed within the S1/S2 site, makes the hypothesis of anartificial insertion via genetic engineering extremely unlikely. (Figure based on Andersen KG., Nature Medicine, 2020, Garry R., PNAS, 2022)From an engineering perspective, none of these odd,suboptimal, and self-defeating FCS designchoices make any sense and certainly contradict theassumption of “rational design” as a whole. They alsoserve to rule out engineering or mimicry of existingfurin cleavage sites.But that alone is not proof. Who knows what weird andarcane experiments those Chinese researchers wouldcook up in a lab, right? (Some lab leak truthers with nomolecular biology experience would say. For actualgenetic engineers, the situation is already clear as day) Fortunately, we do not need to rely on experience orspeculations about any motivations or experimentalprocedures Chinese scientists might have followed. Because nature always has an ace up it’s sleeve...ProtagonistS C I E N C EIs the FCS engineered?Is there some evidence thatcould help us confirm or rejectthe engineering hypothesis?For an engineered FCS, the observed sequence motifin SARS-CoV-2 is odd, to say the least (see figurebelow for a visual summary)

Page 18

After intensive mechanistic studies of the FCS inSARS-CoV-2, researchers discovered a hithertounknown synergistic interplay between the odd,suboptimal FCS and the genetic backbone of the virus(which we by now know is natural because ofrecombination).PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Hidden synergy Researchers showed beautifully (see figure top left)that there is an intricate functional interplay betweenthe FCS, the loop length, and glycosylation: Theseseparate but co-dependent elements work togethersynergistically and all elements are ultimately requiredfor efficient FCS pre-cleavage (and with it, impact viralreplication and pathogenesis).Co-dependency and synergy between geneticelements are hallmarks of evolutionary historyand selection..18ProtagonistS C I E N C EThese type of complex interactions are extremely hardto design for even with perfect knowledge of allstructural components, which nobody had in 2019. Inthe case of SARS-CoV-2’s FCS, there is no conceivableway how engineers could have designed the observed — but hitherto undiscovered — mechanistic synergypurposefully. Some might still contest that engineers could havestumbled upon this interaction by sheer “luckyaccident”, like winning the lottery. I’d say this:We have learned that pre-cleavage of SARS-CoV-2 S1/S2 by furin is what boost TMPRSS2-mediated cell fusion and thus impacts cellulartropism and transmission dynamics (ergoinfectivity)Even a special pleading for a “miracle accident”evaporates in comparison to what we know aboutnature’s relentless efforts and trillionic opportunities tobring forth such elements. But it turns out, that was not thefull mechanistic story of the FCS.In nature, no genetic elementacts alone.The viral QTQTN amino acid motif is anuncommon natural sequence in severalsarbecovirus spike proteins directly upstream ofthe FCS sequenceIn cell culture experiments, QTQTN is not stableand repeatedly lost, similar to the FCS. Lossresults in impaired viral replication. QTQTN determines how tightly the loop harboringthe FCS is bound to the spike protein, thus itregulates spike protein stability and shapes howwell the Golgi-bound furin-like proteases haveaccess to recognize the FCS motif and pre-cleaveat S1/S2The QTQTN motif is also glycosylated and loss ofthat glycosylation impairs viral replication as well.Synergistic interaction between the FCS, proximal sequence motif(QTQTN) and glycosylation (Figure: Vu MN. et al., PNAS, 2022)To just give you the odds of a “lucky engineering accident” scenario,scientists would have to have tried an unholy combination of FCSsequence/random spacer motifs against the backdrop of total viraldiversity with the right proximal sequence elements available from rareviruses nobody previously described but that are capable of causing apandemic in the first place.

Page 19

19ProtagonistS C I E N C EMechanistic details and probabilistic observations arekey to wrapping our heads around the FCS in SARS-COV-2. If we understand that:PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/We underestimate the virusesnature can come up with“I always say that making arecombinant virus is easy. Butto make a virus like SARS-CoV-2, before nature came upwith it? Impossible.“Prof. Linfa Wang, Duke-NUSSingaporean FCS alone can not turna random bat virus into aweapon of mass disruptionthe FCS has manyhallmarks speaking againstdesign or engineering butstrongly arguing in favor ofevolutionary mechanismsa previous unknownsynergistic interactioncould not have comeabout by engineering orstumbled upon by chancewith any known laboratoryexperimental setupBonus: Zoonoses - not researchhow to stop them - is whatcreates biosecurity threatsThe roles of virus discovery or gain-of-functioninvestigations for pandemic prevention are oftendeliberately misrepresented as cautionary tales againstdual-use research for bioweapons. The contextual andmechanistic intricacies of the FCS should expose thenaivite of such shallow arguments.In nature, no genetic element acts alone andviruses are freaking complex molecular machines thatwe humans have no idea how to bend to our will. Whilenaive suppositions about the FCS get all the unduespotlight, there are other single mutations how nature“weaponized” SARS-CoV-2 in ways no engineercould have ever figured out, Take the role of aminoacid 37 in the nsp6 protein. In bats, the “ancestral”version has a valine at this position (V37), whereasSARS-CoV-2 has a mixture of L37 and F37 that seemsto play a critical role in asymptomatic spread. We donot even have a concept of how one would ever figurethis out starting with a bat virus, nor experimentally testfor it in any research setting.Weaponization is generally much harder thanpeople realize. Even extremely well funded programsin the past were not successful for multiple complexreasons beyond technical capabilities. For example,receptor binding is not the sole determinant of speciestropism, and for a lot of viruses, it has nothing to dowith pathogenicity. Additionally, viral fitness in apermissive host cell or model says nothing about in vivofitness or transmissibility. Weaponizing transmissibilityor pathogenicity would need to be tested in humancohorts, not animal models.In my opinion, it is not virus discovery or gain-of-function research that create an “informationhazard” by “increasing the stockpile of select agentsand threat of biowarfare”. Naive nonsense.It is letting zoonotic spillovers happen wherenature figures out all the tough parts of viralengineering. This collective neglect forces the worldto deal with ever-new and possibly effective organismsa dded to the select agents list. A novel pathogen thatbad actors can then attempt to deploy by simplymodifying key residues to avoid prior immunity.…only then can we fully appreciate the scope andintricacies of gain-of-function experiments nature runsevery day.They are of a sophistication and breadth humanengineers yet can not hope to match, nor fully fathom.Activists, spooks and politicians concerned withbioweapons, biosecurity and national security shouldprobably worry about nature’s bioweapon R&Dprogram a lot more than they currently do.

Page 20

Speculations that the “odd and unusual” geneticfeatures of SARS-CoV-2 came about by gain-of-function research, genetic engineering, design, or anyother type of human tinkering — while hard to disprovein 2020 — are today contradicted by available evidenceand published scientific literature. Science did notstand still in the last four years, researchers learned anincredible amount about the intricacies of the virus thatdisrupted the world.Taking together various recombination analyses,sequence diversity in nature, and hallmarks of evolutionin the recombinant backbone, RBM and FCS leave onlyone parsimonious conclusion:PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/20There is a large and detailed body of evidence that any scientific hypothesis needs to be able to explain. The zoonotic spillover hypothesis isnot only consistent with but powerfully explains every piece of evidence we have. No such consistent hypothesis currently exists for thespeculations surrounding a research-related accident. (Figure source: here)Note: A natural virus by itself does not disprove alab leak, nor all conceivable research-related originscenarios. But it certainly disproves all notions of “gain-of-function” research causing the pandemic. Science works with evidence, so it is oftenimpossible to prove a negative — that something did nothappen — scientifically, as there would be no evidenceof something “not occurring”. Conspiracy myths areadaptive and built to sustain the belief that theevidence-based “mainstream” explanation is wrong.This often makes their scientific refutations difficultespecially when the goalposts of how an eventsupposedly happened are constantly shifting, includingcentral elements such as which lab is supposed to bethe culprit can shift at a moment’s notice.All I am cautioning about is that such cover-upconspiracy myths filled with extraordinary andmagical assumptions are epistemologicallyirresponsible, contradicted by multiple other stronglines of scientific evidence, and can not even explainmost of the body of evidence we observe. (see below)ProtagonistS C I E N C ESARS-CoV-2 is a fully naturalvirus, its specific genomic featurescould not have come about byany known or unknown laboratoryexperiment.VI) Putting the gain-of-functionnarrative to bed

Page 21

“Who even cares about the true origin of this virus?Stopping gain-of-function research causing pandemicsis still a worthy cause, and if it took a little mythmakingto get the ball rolling, fine with me”You might have seen versions of this “greater good”justification for keeping the false “gain-of-function labaccident” narrative alive. I certainly have.There is some logic behind it. Even if this pandemicvirus was not created by researchers, future onesconceivably might be. The reality is that gain-of-function research has potential risks and thus needs tobe regulated, possibly more tightly than it currently is.Biosafety, biosecurity, and dual-use research in labsalso need societal oversight (& should never have beenself-regulated by reckless scientific panels or agencies,some gleefully suggest). For many biosafety expertsand existential risk think tankers, the story that SARS-CoV-2 was created by gain-of-function research finallyshocked the world into action on a long-overduepublicly neglected topic, so overall it served a greatergood irrespective of veracity. Fair enough.Just two little caveats: First, the topic was notneglected and regulations were in place. Second,nobody of the dozens of virologists I talked to is or hasever been opposed to regulation and oversight.Without exception, virologists welcome and aresupportive of efforts aimed at constantly improvingexisting frameworks. This is because — in case you havenot thought this through— their bodies are first-in-linefor deadly lab escapes. Clear, actionable, and effectiveregulations are welcome and desired from all sideshere, and are subjected to ongoing discussions aboutscope, practicality, enforceability, financing, and amillion other nitty-gritty details between differentdomains, stakeholders, and the public.So where is the problem? The false “gain-of-functionlab accident” myth has created a destructiveasymmetry in these discussions, with existential risktypes and biosafety fearmongers getting to have theircake and eat it, subverting the need for any scientificdiscussion, negotiation, or compromise betweendifferent possible implementations and future visions.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Isn’t stopping gain-of-functionresearch causing pandemics aworthy cause?Currently, biosafety advocates (and activists whopretend to be) use the false narrative to paint allvirologists as hopelessly plagued by conflicts ofinterest; that they can not be trusted to have any say orinput in regulations shaping their everyday work. Someare true believers, others do it for self-serving or pettyreasons. Some hope to gain political sway or directmore financial resources to their research departmentsand agendas. Some even demand a total ban on gain-of-function research on viruses. However, thesedramatic societal decisions should not happenunder the exclusion of virologists and domainexperts who know the most about the topic. That isnot just bad policy, but recipe for disaster.For example, knee-jerk regulation currently beingconsidered has already cast a chill over virologicalresearch even before being implemented. Sweepingbans on gain-of-function research come withunbearable risks, or more provocatively stated, apredictable death toll. Lives not saved because medicalinterventions (e.g oncolytic virotherapy, vaccines, genetherapy) were slowed down, research was abandoned,or made inaccessible are still unnecessary deaths; andthat is not even considering the much contested utilityof GoF for pandemic prevention.21ProtagonistS C I E N C E“Even without implementation, theNSABB recommendations arealready having a chilling effect onvirology research. The uncertaintygenerated by these proposedrules is causing scientists and theirinstitutions to avoid research thatis likely to be affected, includingthe development of vaccines andtherapeutics.”Rasmussen AL. et al., Journal ofVirology, 2024

Page 22

No matter where you stand on the gain-of-function discussion in general, I believe it is fair tosay that the continued push for a “gain-of-function labaccident” narrative in politics and media is not drivenby evidence, not justified by remaining scientificuncertainties, and can be safely discarded.We have seen again and again that no amount of hardevidence for any scientific theory has the power toprevent certain activists, politicians, influencers, andother manipulators from trying to sell citizens adifferent — and usually emotionally more satisfying orengaging — story. Climate change denial, anti-vaccineactivism, homeopathy, various alleged alternativemedicines or wonderdrugs; none have any scientificleg to stand on, but are profitable narratives to usefor popularity, persuasion, or power. Those whohave something to gain from pushing false myths willcontinue to mislead no matter the evidence. We just donot have to buy into their debunked falsehoods.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Why false myths are dangerousMy larger point is that biosafety is an important,complex, and omnipresent topic that virologists takeseriously. But do its most fervent advocates in politics,media, and other halls of power? If you observe these activists, politicians, contrarians, orinfluencers who claim gain-of-function research — orany type of human genetic engineering technology — created the virus, you’d be wise to question notonly their motifs but their leadership. (top right)Unfortunately, getting the origin of SARS-COV-2 rightis not just an academic exercise, or avoiding beingfooled. It is about the actions we collectively fail to takewhen falsehoods dominate the discourse.22ProtagonistS C I E N C EAny biosafety advocacy that magically stops atthe lab door — that does not consider nor care for thequantifiably orders-of-magnitude larger natural risks —is not a good basis for collective decision-makingin an interconnected world. The threat we neglect willbe the one we will keep facing.So let me help focus our attention and understandingof where the true origins of this chimeric gain-of-function virus — and its future unwelcome pandemiccousins — actually are to be found. The constant performative pearl-clutching and fearmongeringabout gain-of-function researchor lab biosafety in the mediashould not distract citizens fromthe factual reality that SARS-CoV-2 is a natural virus.A dangerous blindspotThe relentless mythmaking about a“gain-of-function” virus and alleged labbiosafety breaches have exposed adangerous blind spot in its mostfervent purveyors:The inability to appreciate the vastnessof viral diversity and the “gain-of-function” ingenuity of nature, both ofwhich continue to pose a predictableand outsized threat to human healthand prosperity.False narratives and beliefs in a “gain-of-functionlab accident” are not without consequences forsociety, and neither is scientific illiteracy or dishonestyin elected representatives and social elites. Are wetruly okay with too many leaders chasing false mythswhile sleepwalking into the next natural pandemic theycould not be bothered to understand? Whatever yourrisk-to-benefit perception about gain-of-functionresearch in labs, ignorance about the scope andpredictable danger of gain-of-function dynamics in thewild is a surefire recipe for disaster.I hope citizens can at least agreethat if you care about “gain-of-function viruses”, you shouldcare about them anywhere in theworld.

Page 23

Rationally, I knew that the cave housed around two anda half million horseshoe bats, but observing aseemingly never-ending flood of hectic creatures — the fly-out of 2,5 million bats took more than forty-fiveminutes — I realized that I never truly appreciated howmany bats share the world with us.There are around 1500 described bat species that haveemerged from their last common ancestor over 60million years ago. Because they are the only flyingmammals, we conceptualize them all together underan umbrella term called “bats” like we do with “fish” inthe sea. But based on genetic diversity, thatsimplification is rarely adequate. It feels equivalentto lumping together giraffes and cows with dolphinsand whales, all of which diverged from a sharedcommon Artiodactyla — even-toed ungulate — aboutfifty million years ago. Hardly justified we think of themas one and the same, so why do we do it for bats?It is no exaggeration to claim that bats come in almostall sizes, shapes, and forms; from the thumb-sizedCraseonycteris thonglongyai — also known asbumblebee bat weighing just 1,5 grams and holding thetitle of smallest mammal on earth — to various majesticflying foxes with wing spans of over 6 feet, close to twometers. Some bats look almost like dog puppies you’dwant to cuddle and take home, others might appearlike they’ve escaped from a horror-movie production.Maybe we humans just tend to be afraid of what we donot understand. Prelude: A river in the skyPROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Horseshoe bats belong to a group of bats thatecholocate — sending out and receiving sonar waves — primarily through their nostrils. Other bats mostly usetheir mouths. These varied shapes and forms in themiddle of their faces however have intricatefunctionality for shaping their calls, impacting not onlyorientation but for their feeding and social lives too.Given their enormous diversity, bats exist in almost allvariations of social structures, from eremites that don’twant to bother with others, to small family groups, tovillages, to multicultural megacities. Some like tomingle with other bat species, others are territorial andof the “get off my lawn” persuasion, with threateninggrunts and fletching teeth and all. Some horseshoe bat species are not only consideredhypersocial cosmopolitans, they are also the mostprominent host reservoir to SARS-related batcoronaviruses; close viral cousins of both SARS-COV-1 and SARS-CoV-2 that have since caused havoc in ourhuman world.I believe to truly understand where thesedangerous viruses come from, we have to firsttake a look into the lively homes of their hosts.ProtagonistS C I E N C EThe horseshoe bats flying here weresmall insectivores — insect-eating bats — who got their name from the weirdhorseshoe-shaped disfigurement where theirnose should be. Intuitively, we humans findthem rather ugly — I was no exception — atleast at first. I think this is partly because weassume their faces are weirdly deformed, likea fully cleft palate, rather than what theyactually are: optimized.A river of black drew its linein the darkening sky. Abovebat ornaments on crimson roofsfrom the pagodas at Wat KhaoChong Pran, the river flowturned Southeast tonight,towards the crop fields.

Page 24

Chapter 2: Chasing theghosts of SARS-CoV-2Physiologically, bats are extraordinary. They canspeed up their metabolism 16 times, creating immenseheat that would denature our proteins and fry our cells.A bat’s heart can beat up to 1,000 beats per minuteand its body temperature can rise to 42 degreesCelsius [107.6 F] during flight at night. Even if we couldwithstand the initial stress of flight, our inflammatoryresponse after would render us sick. That is not true forbats. Many species have unique immune systems thatdo not overreact to stressors; including viral infections.There are also no reliable markers of aging after theyreach adulthood, bats seem young until they are dead.Some species tend to live up to 40 years in vast, dense,and diverse colonies. All of the above seem to makebats uniquely suited as reservoirs to almost all viralfamilies that befall mammals. But for sarbecovirusesspecifically, that is not the whole story.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/I) The cave(s) where it happensWhile not fully understood today, researchers believethat bat echolocation is not only for orientation andhunting but also for communication and vocal learning.Some bat’s extensive vocal repertoire is needed tocreate specialized social calls used either for parent–offspring reunions, territorial defense, or maintaininggroup integration. Some speculate that the evolutionand differentiation of vocal frequencies within aspecies leads to mating preferences and exclusions,driving niche formation and ultimate species separationinto species complexes.24ProtagonistS C I E N C EVirus outbreaks are always socialphenomena as well, and thatrarely is limited to humans.There is emergent evidence that the lives of theirprimary hosts, the Rhinolophids — horseshoe bats withodd noses — are not only a lot more crowded andcosmopolitan than the average bat, but also a lot moresocially intricate. Horseshoe bats vary in nose shapesbecause that allows them to specialize in echolocationat very wide ranges of frequencies. “No one else was in the room where it happened.The room where it happened. The room where ithappened. No one really knows how the game isplayed, the art of the trade, how the sausage getsmade. We just assume that it happens, but no oneelse is in the room where it happens” - Hamilton (Musical), the Room Where It Happens.A species complex is interesting from the perspectiveof viruses. The genetic stratification of their hostreservoir means that some viruses will specialize on asmall niche or subgroup of the species complex,whereas others might try to develop or maintain broadaffinity to multiple species that might still inhabit thesame spaces, but not mate with each other.Let’s recall an observation from Chapter 1: the RBDsof sarbecoviruses can be very broad (even opening thedoor to non-bat ACE2 receptors), or very niche.Sarbecovirus RBMs and ACE2 tropism are likely a reflection of thepopulation structure and intricate social lives of their host species(Figures from Si J. et al., biorxiv, 2024)

Page 25

While various horseshoe bat species were identified asthe most prominent hosts and natural reservoirs ofsarbecoviruses in the wild, they are certainly not theonly bat family involved in creating overall sarbecoviraldiversity.Viral recombination needs physical opportunityfor other viruses to infect the same cell; parentalviruses can not create offspring if they never meet, buttheir offspring might also not be as diverse if they nevermeet very distinct others. So how do our hypersocialrhinolophids do on that “meeting new people” front?It is reasonable to assume that the intricate populationstructures of Rhinolophids create evolutionarypressures for viruses to constantly adapt to new niches.Recombination as an “evolutionary fast-forward” isthe mechanism of how sarbecoviruses survive in suchan environment, which is why this is such a prominentand critical feature in their evolution.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/25ProtagonistS C I E N C EBeyond mere cuddling with strangers,rhinolophids are known to switch roostsconstantly. There are maternity-only roosts, summerand winter locations, and roosts that are calm or havespecial geographic features. Some bats like to stay aspermanent residents in a single cave, others cycleoutside but come back periodically over the year — maybe kids going to college would be a social analogy,if only we could tell bat age, that is — and many bats dofood tourism with the seasons. All of this leads toconstant turnover, and various bat species mixing andmingling with each other. Researchers observe complexsocial, seasonal, and geographicmixing of various bat species evenwhen just studying single locations,like a specific cave or a forest.Rhinolophus bat species are very diverse and their nostril shapes have been optimized for echolocation at a large range of frequencies. Shown: R. rex, R. stheno, R. I. yunanensis, R. sinicus, R. malayanus (from left to right) Image credit: Prof. Alice Hughes, University of Hong Kong“Recombinant viruses that are mostlikely to succeed under competitivecircumstances with smallpopulation sizes are those for whichthe recombination event hasconferred a strong selectiveadvantage over both parentalviruses.” — Wells H. et al., Cell Host Microbe,2023Some bat species have been observed to regularly co-mingle with otherspecies, especially in cooler caves or during torpor (short-termhibernation). Image credit: Prof. Alice C Hughed, University of Hong Kong.Rhinolophids are gregariousneighbors

Page 26

Some of this mingling and movement was always anatural part of bat lives. More recently, however,human encroachment on bat territory — urbanization,deforestation, hunting, tourism, mining, etc — isconsidered the biggest factor in displacing bats fromtheir traditional roost sites and forcing species togetherthat might not have ordinarily met.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Virus-sharing networks stir thegenetic cauldronBy using a meta-transcriptomics approach — takinga sample and sequencing every single piece of geneticinformation in it — they discovered that many of the batsthey sampled carried more than one virus. On top ofthat, the study found that some of the discoveredviruses were shared between different bat species,suggesting frequent spillover events distributed thevirome over multiple hosts. Dramatic opportunity forvirus sex (recombination) and diversifying the viralgene pool with new genetic elements.26ProtagonistS C I E N C EIf some bat species that diverged often millions of yearsago tend to huddle together — and we already learnedthat the ACE2 tropism of various sarbecovirus RBDscan be very broad — do the diverse viruses they carryinfect each other and influence recombinationpatterns as well?“Some of those different species willroost together, especially when it iscooler or during hibernation. So whenwe have done work in various coolercaves […], we will see clusters.Rhinolophus [horseshoe bats] cuddledup to Miniopterus [long-winged bats],and the next one is Myotis [mouse-eared bats]. Now these are lineagesthat diverged 50 million years ago.” — Bat ecologist Prof. Alice HughesThis is one question researcherswere able to answer recentlywith a resounding yes.Co-infection with multiple viruses is a prerequisite for virus recombination. Diverse virus-sharing network reveals connectivity among viromes of differentbat taxa. Viruses of concern and putative cross-species transmissions are shown in different colors. (Wang J. et al, Nature communications, 2023)

Page 27

PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Nature’s gain-of-functionlaboratory27ProtagonistS C I E N C E“The frequent virus spillover amongphylogenetically related or spatially co-located bats provides an opportunityfor viromes of different bat species toexchange, further expanding geneticdiversity of circulating viruses” — Wang J. et al, Naturecommunications, 2023I think these findings are important to consider.Whatever innate mechanistic ability for recombinationsarbecovirus genomes might have, it is largely thesocial lives of their host reservoir that create theopportunity and evolutionary pressures forrecombination to shape sarbecovirus genomes.“The phylogeographic structure in bathosts (and their diverse immunestrategies) […] creates a landscape ofselective pressure; the trajectory ofviruses’ coevolutionary response is, inturn, constrained by their opportunitiesfor either specialization ordiversification through host jumps andrecombination” — Forero-Muñoz NR. et al., Virusevolution, 2024In other words, we should not be surprised to find acomplex mosaic within the genomes of sarbecoviruses,because it reflects the complex mosaic of bat and virusco-evolution. A constant fight for niche survival againstthe merciless competition of ever-changingcircumstances, one where only the most opportunegenetic elements and fittest chimeric viruses willpersist.These caves are nature’s vast“gain-of-function” laboratory, allwhile our human encroachmenton bat territory is stirring thegenetic cauldron ever faster.So now that we know what “gain-of-function labs” weare looking for, do we know where to look? Whereexactly is the birthplace of SARS-CoV-2?We humans tend to underestimate the diversity and vastness of nature. Even over geographically confined regions, vast cave networks can house millionsof bats, dozens of different bat species and sub-species not yet described or sequenced. Image credit: Prof. Alice Hughes, University of Hong Kong

Page 28

PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/II) Undersampled Karst in theWild Wild EastKarst is a topography formed by the erosion ofsoluble carbonate rocks, such as limestone ordolomite. With a total area of 500.000 squarekilometers in China and 400.000 square kilometersspread over various nations in Southeast Asia, thesecountries house some of the largest and mostbiodiverse Karst regions in the world. But it is truly thepower of water over eons that has shaped thelandscape to include spiky spires, enormous sinkholes,underground rivers, and intricate cave systems belowthe old-growth subtropical forest and rainforests.This is bat country, and each valley and limestoneformation can not only house millions of bats but is amicrocosm in itself.28ProtagonistS C I E N C EThe majority of viruses that spill over might just notreplicate well in the host cell, or get shut down by ourinnate human host defense, such as the interferonpathway, without humans ever being the wiser. Evenmost replicating viruses might be unable to transmitbetween humans, or unable to spread effectivelyenough. Outbreaks are also always social phenomena,even a perfectly capable pandemic virus like SARS-CoV-2 would have died out with a 99,5% likelihood if itspilled over in a remote village, rather than a megacity.continued on next page A guano collector in a massive limestone cave housing millions of bats.Bat guano is sold as potent fertilizer and can be harvested periodically.“Each isolated limestone hill can hostmore than 12 unique species foundnowhere else on earth, with up to 100microsnails, endemic begonias,orchids, and geckos, and yet anestimated 90% of cave-dependentspecies are undescribed” — Prof. Alice Hughes, University ofHong KongThe Karst region spanning Southern China andSoutheast Asia forms a unique and biodiverseecosystem that in considerable parts is still relativelyuntouched by humans, albeit that has been changingespecially with deforestation, slash-and-burn, and cashcrop agriculture leading to the degradation of the land.Its importance to the global climate and conservation isprobably second behind the Amazon rainforest, withprojections suggesting around 40% loss until the end ofthe century if we do not dramatically change course.The shifting landscape use and humanencroachment are in turn some of the major drivers ofzoonotic spillover. Some studies suggest that around65.000 spillovers of CoVs happen each year, most ofthem from viruses that are yet undiscovered and thatdo not cause an outbreak for a variety of reasons.Karst rock formations in Cat Ba, Northern Vietnam. Image credit: Prof. Alice Hughes, University of Hong Kong

Page 29

Given most of these sarbecoviruses die out and willnever be known, narrowing down where exactly SARS-CoV-2 came from within an often inaccessible, remoteKarst region 2,5 times bigger than the size of Germanyis a daunting challenge.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/A granularity problem29ProtagonistS C I E N C ESome have summarized the possible origin place atvery low resolution (below) based on the samplinglocation and overall genetic similarity of the variousSC2-related viruses.Most direct evidence comes from sequenced batviruses in China (before sampling in Yunnanbecame forbidden), where researchers found apool of SC2-related viruses in Yunnan province.(China has been more sampled than othernations)Another trove of informative viruses was found byFrench and Laotian researchers in Vientianeprovince in Laos. Here the closest match to SARS-CoV-2 broad-affinity RBD was found, provingnature unlocked that particular door to humaninfection; not human engineering.More distant SC2-related viruses have also beenfound in Cambodia and Thailand; which inthemselves can house informative geneticelements such as insertions at the S1/S2 sitereminiscent of the polybasic cleavage motif in SC2“The risk for zoonotic viral diseasepresence and emergence in humansalso increases in geographic areas withhigher mammal diversity, wherepreviously pristine forests have beenrecently deforested. This makes theareas of Southeast Asia, which supportlarge, intact natural habitats and haveongoing ecosystem fragmentation, athigh risk for disease emergence” — Evans TS. et al., Int J. Infect Dis, 2023Yet this background of viral emergence and constantspillovers gives researchers three lines of attack toat least narrow down where the bat ancestor ofSARS-CoV-2 likely originated.discover the immunological footprint of relatedviruses through serology in humans and farmanimalsdiscover the immunological footprint of relatedviruses through serology in wild batsdiscover related viruses through taking andsequencing bat samplesEvidence for SARS-CoV-2-related sarbecoviruses hasbeen found in China, Laos, Vietnam, Cambodia,Thailand, Myanmar, and Malaysia.Low-resolution likely geographic origin based on overall genetic and RBDsimilarity of a set of closely related SC2-like bat viruses and their originalsampling site (red triangle) Zhao S. et al., J Genet Genomics, 2022Yet such efforts are bound to be highly dependent onsampling biases and offer a low granularity that isalmost meaningless.continued on next page Bat researchers catch bats with nets, transfer them to cotton bags, takemeasurements and various samples (sliver, excrement, tissue) inimpromptu field labs, then release the bats unharmed after

Page 30

The previous map has also not considered some otherrelevant findings such as:PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/All we know is that we are flyingblind30ProtagonistS C I E N C ESC2-related viruses have been found in pangolinssmuggled over the Chinese and Thai bordersEspecially the karst regions bordering Laos,Northern Vietnam, and Myanmar seemed very richin these viruses, with Myanmar lacking any batsampling efforts there so farHowever, serological evidence from Myanmarfurther South found a very high prevalence ofSC2-related viruses in humans that had contactwith wildlife. More than 1 out of 5 had antibodiesand cross-reactivity to a panel of these viruses(most prominently RaTG13), arguing for highprevalence and spillover potential in this region, butthe concrete viruses remain undiscoveredGeographic location of SARS-CoV-2 relatives sampled in Southern China and Southeast Asia. Large parts of the Karst region, especially outside ofChina, remain hopelessly undersampled. Technical names of virus relatives usually include the bat species as first letters. Rm ... Rhinolophus malayanus, Ra... Rhinolophus affinis, Rsh... rhinolophus stheno, Rp ... Rhinolophus pussilis, Rac ... Rhinolophus acuminatus. The displayed color coding roughly indicatesgenetic similarity to SARS-CoV-2 (yellow — medium, orange — high, red — very high)Bat sampling in Japan, Southern Vietnam, andMalaysia shows more ancestral and distantlyrelated sarbecovirusesSampling in Northern Thailand has been politicallydifficult to publish but might be insightful as wellBelow, I have plotted a rough summary of scientist’sefforts so far:Honestly, while a lot of valuableinformation has been gathered inthe last few years, it servesmerely as a snapshot of thescope of our ongoing ignorance.

Page 31

31ProtagonistS C I E N C EThe reality is that there is still much we do notunderstand about our diverse world.According to Prof. Alice Hughes, it is estimated that90% of caves in Southeast Asia alone remainscientifically undescribed and uninventoried, whilesome estimate around 60% of bat species remain to bediscovered, and a staggering many of the bat specieswe know about have never been sequenced. PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Some governments fear morescientific discoveries will leadto them being blamed by aworld that has not madepeace with natural pandemicrisks.Why governments are not keenon virus discovery researchThere is a need to study these complex interactionsbetween viruses, bats, and humans. However, manySoutheast Asian nations, as well as China, have littleinterest in allowing researchers to sample more bats,discover new viruses, or even publish ones they havealready found (personal communication from multiplebat hunters).Especially sarbecoviruses are a sensitive subjectalmost everywhere. Governments are currently notinterested in discovering where these dangerouschimeras are circulating in the wild or what drives theiremergence.We still need to learn much more about viral and batgeography, how human drivers from deforestation tourbanization to tourism interface with bat immunity,viral-host interactions, bat-human contacts, andpropensity for viral shedding.A world that asks for reparations, culprits, or impossibleinsurances, rather than offering a helping hand on acollective problem. A world where leaders seek powerwith geopolitical grandstanding, or worse, gainpopularity by exerting vengeance on inconvenientscientists rather than heeding their warnings.I believe collectively, we need to do better on that front.Scientists are doing their part, despite these artificiallimitations and political constraints. Some of them havebeen tinkering with new methods and ingenious ideasto learn more about the origin history of SARS-CoV-2,and there are some exciting findings to report.We are working with a mapwhere many spots are and willlikely remain blank becauseany research on bats has beenso highly politicized by thedistorted origins debate.While bats remain undersampled, cave tourism in the karst region isbooming. Image: Tham Lot Cave, Northern ThailandHow long can this dangerous mix of ignorance andpower continue?

Page 32

PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/III) Ghost hunting withphylogenetic inferenceGaining knowledge from sparse data is difficult, but notimpossible.To date, only ~200 sarbecoviruses have been found.139 of them are closely related to SARS-CoV-1, and 26are SARS-CoV-2-like. Not much to work with topinpoint a geographic location, and individual genomescan also not be used to reconstruct a faithfulevolutionary history because all of these are chimeras,so molecular clock estimates based on mutationaldivergence are being misled by the “evolutionary fast-forwards” of recombination.32ProtagonistS C I E N C EThe number of sarbecovirus relatives discovered todate is however just good enough to identifyrecombination breakpoints, places where geneticsimilarity from one parental strain stops and similarity toa different parental strain starts. A way to think aboutrecombination breakpoints is as a minimum number ofsexual acts that must have happened in the past thatcan explain the segmented shape of the chimericgenome today.The genomic segments in between recombinationbreakpoints are called non-recombinant regions(NRRs), basically intact genomic segments from aparental strain. On these segments, molecular clockestimates work normally and can thus be informative.Virologists will just have to individually work with asmany clocks and evolutionary histories as there areNRRs, which is 27 in the case of SARS-CoV-2, and 31for SARS-CoV-1. (see figure below)“If you align all viruses in the sameplace, and then chop up thegenome into segments — what wecall non-recombinant regions(NRRs) — then each NRR will havetheir evolutionary history” — Dr. Spyros Lytras, an evolutionaryvirologist at the University of TokyoNon-recombinant regions are informative and need to be analyzed individually to be able to present — to the extent possible — a single evolutionaryhistory of chimeric viruses. (Pekar et al., biorxiv, 2023)

Page 33

PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Separating the histories of NRRs allows researchers togain some critical information. For example, while someNRRs might be very old and just serve as reminders ofsexual conduct decades ago, others might be veryrecent additions; the final sexual (recombination)events that gave rise to the chimera.Indeed, researchers found that the youngestrecombinant additions for SARS-CoV-1 and SARS-CoV-2 to their closest-inferred bat virus ancestorhappened not decades ago, but were in 2001 (SARS-CoV-1 spilled over in 2002) and in 2014 (SARS-CoV-2spilled over in 2019), respectively. This is of coursenot the final word, as the researchers noticed the morecousins are discovered, the closer the time estimatemoves up towards emergence date.33ProtagonistS C I E N C EHaving identified the 27 disparate recombinant piecesconstituting SARS-CoV-2 also allows researchers to dosomething very interesting, which is constructing theclosest common recombinant ancestor (recCA)genome, basically looking at which viral cousin of SC2has the highest similarity to SARS-CoV-2 for each NRR.Making science accessible is a balance act betweenaccuracy, complexity reduction and useful butinaccurate abstractions (i.e “virus sex” forrecombination).Some scientific topics lend themselves to complexityreduction, others not so much. Sometimes acommunicator has a very good understanding orcreativity to explain a technical topic brilliantly easy,but nobody can do or know everything at the requireddepths.The viral phylogenetic inference covered here is adeeply technical topic based on statistical models,probabilistic assumptions, and experienced modellersusing real data for inference.The research is currently under peer-review as this isthe cutting edge of origins research.For more in depths information about this work, adetailed interview with the two first authors can befound here:“The virus pieces that were most similarcirculated in bats very recently” — Dr. Jonathan Pekar, US San Diego.The recCA genome is anaggregate, built from the ghostsof closest-inferred ancestors foreach NRR that must haveexisted at one point.Tellingly, researchers have found that the more batvirus relatives got discovered in nature, the closer thegenome sequence of the recCA resembled the human-infecting SARS-CoV-2 that emerged.In other words, we have evidence to infer that a98.8% identical to SC2 bat virus ancestorcirculated just a few years before emergence; withtemporal and genetic granularity still increasing asmore viral cousins are discovered.But what can we do with that knowledge?On the limits of sciencecommunicationI highly recommend listening to primary sources puttheir work into context, on top of that, it was a really funconversation with two remarkable young scientists.You can also access the video here: https://www.youtube.com/watch?v=ROUdGeCmVYk

Page 34

PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/IV) Reconstructing thephylogeographic historiesAs we have observed in the karst region,sarbecoviruses, just like their bat hosts, show a degreeof geographic structuring; with the border region ofLaos, Myanmar, Northern Vietnam, and Southern Chinaseemingly quite rich in sarbecoviruses closely relatedto SARS-CoV-2.But does that mean SARS-CoV-2 came from thatregion, or is it just a mixture of undersampling andcoincidence?By using these phylogeographic data, a phylogeny(family tree) for each NRR can be constructed that isspatially mapped to sampling locations.34ProtagonistS C I E N C ECombining geographic information from samplinglocations of close ancestors with phylogeny scaledwith units of time of their closest recombinantancestors creates a geo-temporal record for thevarious genetic elements constituting the SARS-CoV-2viral genome.“When we build those family trees, wewant to calibrate them with time” — Jonathan Pekar, US San DiegoPhylogeographic origin dispersion map of SARS-CoV-1 (left) and SARS-CoV-2 (right). Green density map tracks evolutionary history through time,red density (right bottom corners) are the most likely birthplaces of the direct bat ancestors to SARS-COV-1 and SARS-CoV-2 (Pekar et al., biorxiv, 2023)SARS-CoV-1 SARS-CoV-2These geospatial and temporal efforts also allow us tonarrow down a dispersion zone for these “floatinggenetic elements”, and with it, the viruses that arose bytheir (re)combination. In other words, the most likely geographic birthplaces of direct bat ancestors toSARS-CoV-1 and SARS-CoV-2 (see below).This last result gained from studying sarbecovirusancestry unearthed one more essential clue to howSARS-CoV-1 and SARS-CoV-2 emerged.And it starts with a suspicious conclusion:“Direct ancestors of the SARS-CoVslikely could not have reached sites ofemergence via the bat reservoir alone”— Pekar et al., biorxiv, 2023SARS-COV-1 emerged in Guangzhou, around 1000kmaway from its suspected home; and SARS-COV-2emerged in Wuhan, around 1500 km away from wherethe last direct bat ancestor existed.So how exactly did SARS-CoV-2 findits long way from the sarbecovirus“gain-of-function” heart of the Karstregion to a wet market in Wuhan?

Page 35

An outbreak rarely divulges all of its mysteries. How thevirus made it to Wuhan still leaves room for uncertaintyand speculation. Lab leak advocates might be quick tosmell an opportunity to allege that maybe a researcherfrom Wuhan has gotten infected and brought the virusto the city. Weren’t Zhengli’s group and some otherteams sampling in Yunnan after all?PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/V) Smoking guns pointing at thewildlife industryThese insinuations are unfortunately quite shallow,scientifically naive, and ultimately false because theleftover uncertainties are much smaller than one mightappreciate.But one step at a time. I could spill much more digitalink on arguing how a priori unlikely it is for batresearchers and virus hunters to ever stumble upon apandemic-ready chimeric virus from the small batsampling efforts researchers ever get to conduct. Onemight find genetic puzzle pieces, but how nature putthem together only comes to light after selectioncreated those million-to-one lottery winners that spillover into other mammals. If Chinese researchers hadcollected and stored hundreds of those spilloverviruses, maybe suspicions had a ground to stand on.The reality is that Zhengli Shi’s team only ever foundone SARS-CoV-2-related virus, even non-pandemicviruses are hard to find. One could also contend thereis no evidence for any of these speculations. One mightalso list the eerie circumstances surrounding SARS-CoV-2 that look an awful lot like SARS-CoV-1; from thefact that both started in November (suggestingseasonality) to the finding that sampled wildlife farmsaround Wuhan had SARS-CoV-1 lineages potentiallyancestral to the outbreak that happened inGuangzhou... but okay, I am spilling digital ink again, so Iwill stop. See note (left)Again, it is 2024 and scientists have uncovered a lotmore evidence that completely exoneratesChinese and other bat researchers who ever sampledin Yunnan or various places in South East Asia.35ProtagonistS C I E N C EOn popular and contested topics, media manipulatersutilize an arsenal of misleading communication tacticsto sow doubt, imply ambiguity or create confusionabout inconvenient scientific studies. They know thatcitizens can be misled into discarding nuancedfindings and even whole papers by pointing outlimitations, alternative explanations or simpleunintuitive findings that need further investigations.Any scientific study or experiment has at least somearea of messy or incomplete data, unknown orundefined variables, unexplored alternatives or otherlimitation. That is just the nature of science, if we hadperfect data and perfect knowledge, nobody wouldneed to write a paper anymore. So is science doomedby the never-ending contestations of conspiratorialcommunities and motivated manipulators?Of course not. The existance of limitations anduncertainty (artificially inflated or not) with individualstudies does not need to strand us in eternal ignoranceon the origin topic, nor should it entice us to throw thebaby out with the bathwater.This is because of scientific triangulation orsometimes called information convergence; the useof multiple methods or data sources in qualitativeresearch to develop a comprehensive understanding ofphenomena.The more methodologically diverse, complementaryand independent the research approaches and teamsbehind scientific papers on the topic are, the more wecan trust them if they all converge towards the sameconclusions.The body of evidence behind a non-research relatedorigin of SARS-CoV-2 is entirely one-sided, convergingand can explain all the available evidence. Individualuncertainties and alternative speculations do nothave the power to move that mountain ofevidence any more. Nor can any alternativeexplanation be formulated that can explain all theevidence plausibly.In the last section, let us focuson just two puzzle pieces thatI think are illuminating becauseno type of research-relatedaccident can explain them.Note on: Uncertainty inflationversus scientific triangulation

Page 36

The first one is — what I feel — one of the biggestmedia oversights in the discussion, and it has to do withthe damned furin cleavage site again.In Chapter 1, we spend considerable time explainingwhy we know that the FCS does not have anartificial origin. We also know from looking atsarbecoviruses that while these FCS motifs can easilybe created, they do not seem to be maintained in batviral lineages. They are even unstable in many cellculture systems. However — within certain real-lifetransmission contexts — the FCS is strongly preservedas a respiratory adaption.That extends beyond humans; minks, deers, and otheranimals that got infected with SARS-CoV-2 via reversespillover and the respiratory route from humans havebeen shown to preserve the FCS as well. Bats do notappear to do so, for reasons one can speculate (e.g batsarbecoviruses are gut-adapted, not respiratory) butare certainly host-context dependent. No geneticelement acts alone.This observation however means that one has toassume that the closest bat ancestor of SARS-COV-2 almost certainly did not have (or maintain)an FCS.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/The first piece36ProtagonistS C I E N C EIf neither bats nor humanscreated the FCS, where does itcome from?By just following these two evidence-basedassessments to their logical conclusion, it becomesclear that an intermediate host had to be involved.Somewhen between as early as 2014 (likely later)when a recombination event allowed the bat ancestorto reach its final chimeric form, but before it emergedin 2019 at the Huanan wildlife market, the virus musthave acquired and/or sustained it’s FCS in a non-bat host (complimentary evidence like D614Gstabilization also argue for a very recent FCS addition).“The Furin cleavage site gave me thestrongest hint that the progenitor virusof SARS-CoV-2 is not in bats”, — Prof. Linfa Wang, Duke-NUSSingaporeThere is a recent precedent. Evidence for such FCSacquisition in intermediate host species has beenfound in 2023 with trafficked pangolins. A bat HKU4-related merbecovirus (bat ancestors to MERS also donot contain an FCS) has acquired a minimal polybasiccleavage motif experimentally shown to be cleaved byfurin. It seems that for CoV spillovers, intermediateanimal populations tend to bring these polybasiccleavage sites forth and can maintain them.An identical argument for another such respiratoryadaptation that gets maintained could also be madefor the T372A mutation that alters the 3D confirmationof the trimeric S glycoprotein to a more open andrespiratory infectious form. Bat sarbecoviruses do nothave this, humans could not have come up with it. Sowhere did it come from? Same story really.A priori, it is very unlikely that bats directly infect batresearchers, and even extremely unlikely to ever find apandemic pathogen from the pithy sampling efforts batresearchers ever get to conduct. But add to this thelikely requirement of a host-context switch andrespiratory adaptation in an intermediate animal tomaintain an FCS/T372A? Pretty much impossible. I trust this should finally dispel the myth that bat-sampling researchers brought SARS-CoV-2 to Wuhan.There has been a lot of hysteria and myth-makingsurrounding the furin-cleavage site in the media overthe last four years.To understand that the one genetic element manyhave falsely called the “smoking gun for engineering”contributes much more to bat researchers’exoneration than to their incrimination is certainly afitting and long-overdue twist of fate.Talking about non-bat intermediate hosts though...“With the US Intelligence agencies, theyall focus on the Wuhan Institute ofVirology because they have the mostbat samples…... I said, yes, but science says that thisprogenitor comes from a non-bat smallmammal”-Prof. Linfa Wang, Duke-NUS Singapore

Page 37

Something no research-related accident can explain isthe fact that the multiple independent lines of evidencepoint to the Huanan market as the epicenter of thepandemic. Researchers had already established in 2022 thatSARS-CoV-2 susceptible wildlife had been sold atthe market — despite denials and obfuscations fromChinese authorities continuing to this day — and thatthe spreading pattern of two lineages centering fromthe market is best explained by a multi-spilloverscenario from an infected pool of animals.Much more could be said here, but let’s go to thecutting edge right away:In 2023, sequencing data of environmental samplesfrom the market were finally released by Chinesescientists which provided a trove of genetic informationto sift through for new possible clues.And new clues were found.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/The second piece37ProtagonistS C I E N C EGenetic diversity of found SARS-CoV-2 samples isconsistent with viral emergence at the market; alsocontradicts the market as a mere “amplifier” or“superspreading” eventEnvironmental samples that tested positive for SARS-CoV-2 contained lots of DNA/RNA from variousSARS-CoV-2 susceptible wildlife speciesAnimal DNA/RNA correlated spatially with stallsthat housed these animals, but not other places in themarketEnvironmental samples from these wildlife stallscontained sequencing reads for wildlife-specificanimal viruses and SARS-CoV-2, but not otherhuman viruses (arguing for sick animals and againsthuman contamination of these samples)single-nucleotide variation (SNV) analysis if raccoondog reads found that the raccoon dogs at themarket did not belong to a commonly breedspecies used for fur production in large operations inthe north, but rather a wild-caught varietyMetatranscriptomic analysis of environmental samples taken from SC2-susceptible animals at the Huanan market provide evidence for a link tothe wildlife trade, possibly in Southern China. (Figures from Crits-Christoph A. et al., biorxiv, 2023)Metatranscriptomics provides a direct link from animals atthe market to wildlife industry1) Environmental samples positive for SARS-CoV-2are full of wildlife DNA/RNA3) Animal viruses associated with wildlife trade,but not human viruses, are discovered in animalenvironmental samples4) Raccoon dog DNA shows indications of wild-caught raccoon dogs species, rather than morecommonly farmed species for fur production2) Wildlife mtDNA spatially co-localized with a toa small set of stalls reported to house wildlife

Page 38

These market data make it clear that no infected labworker just walked into Huanan to start the pandemic,nor sneezed on the animals to make it look like theywere involved.On top of that, the metatranscriptomics data providemultiple direct links to the wildlife trade andindustry; from the species that were identified to thetypes of viruses that plagued those animals; and evento the geographic region where they might have comefrom. These findings are consistent with the WHOreport that also suggested the involvement of thewildlife industry in Yunnan and beyond.From illegal trafficking of rare animals over the border,hunting and trapping them in the wild for later sale orsustenance, or supplementing breeding in wildlifefarms, the industry has many facets. Some activities areillegal but lack enforcement, some are traditional andculturally valued, while other activities such as wildlifefarms were explicitly promoted before the pandemic.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Remaining uncertainties aresmall 38ProtagonistS C I E N C E“Local officials trumpeted the wildlifetrade as a way to close the rural-urbandivide and to meet ambitious nationaltargets to alleviate poverty.” — Emily Fang, reporting for NPR So that is it? This is how the virus made it toWuhan?Outbreaks rarely divulge all their secrets, and insightshave to be pried from nature and history through theoften painstaking work of scientists, journalists, andother truthseekers.Science is a process of approaching ever more likelyexplanations of reality by rooting out false hypothesesand narrowing down existing uncertainties.The remaining scientific uncertainties of theorigin puzzle (see below) surround politicallysensitive topics such as a rampant regional wildlifeindustry — estimated to be over USD 70 Billion in Chinaand around USD 10 Billion in South East Asia just fortrafficking — as well as more complex patterns of howsocioeconomic behavior and ecosystemicdisruption influences spillover risk factors andpandemic prevention policies at variousvirus/animal/human interfaces. That is why this type of“pandemic origin” research must continue, despiteknowing this virus did not come from any lab. There isstill much more to learn on how to prevent SARS-CoV-3. But that is a topic for a different time.For now, I think we finally reached a (hopefullysatisfying) end of this epic journey towards the cuttingedge of origins research. So what is my personaltakeaway?Accumulating evidence for a bat origin and directancestor of SARS-CoV-2 circulating in the wild Bat ancestor could nothave reached Wuhanwithout intermediateanimalsMetatranscriptomics pointto the wildlife industry fromSouthern ChinaWildlife trade linkwas not allowed tobe investigatedRemaining uncertaintiesAccumulating evidence for the Huanan market beingthe epicenter of the outbreak that started COVID-19FCS occurrencesuggests a non-batintermediate host202420212022202320222023 2021???The remaining uncertainties are narrow but surround the regional wildlife industry’s role in circulating the virus and facilitating its ultimate emergence

Page 39

Conclusion: Don’t betagainst scientific inquiryFinding the origin of a new virus is a lot moredifficult than finding the needle in a haystack. Virusesevolve, adapt, branch out, and almost always die outwithout us ever being the wiser. It’s like finding aparticular grain of sand on a beach, always in danger ofbeing washed away by the next wave. The search forany “bat patient zero” almost four years later would bea hopeless effort even before the deliberate sabotageof scientific processes by governments and maliciousactors.Yet in some twisted sense, we were lucky that thegenome and evolution of sarbecoviruses are sointricate and reliant on recombination; it means thatSARS-CoV-2's evolutionary history contains manyoverlapping stories. Stories that can provide a richerpicture of what, when, where, why, and how ithappened, akin to a set of contemporary witnesseswith limited knowledge, or a set of partial fingerprintsall over a crime scene.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/Yet our understandable fears and suspicions of a “man-made” pandemic — albeit misplaced into too simplenarratives or wrongfully projected onto convenientscapegoats in suspicious white coats — are also notentirely baseless.As often with popular myths and sentiments, there is agrain of larger truths embedded in them. Today, there is plenty of scientific evidence to make thecase that collectively, we humans are not entirelyinnocent. Neither when it comes to fueling the forcesthat create these dangerous viruses, nor the reasonsthat make them spill over into us. In nature, no elementacts alone. That includes us and the circumstances wecreate. As a now publicly smeared and unjustlycharacter-assassinated zoologist once told me:39ProtagonistS C I E N C EPuzzling these genetic andphylogeographic fingerprintstogether, documenting what placesthey touched at what time, andlistening to what story they have totell allows researchers to draw amuch larger picture from the mosaicknowledge of our ignorance.The reality is that the observed viral ancestry of SARS-CoV-2 betrays any conceited notions we might haveheld about virologists — or gain-of-function research — bringing about the deadly chimeric pathogen.Scientists have the time and place of its birth andemergence narrowed down to a sufficient granularityto exclude any lab-related incidents. On top of that, welearned about some viral features that only naturalselection, not human engineering, could bring forth.It is what we humans currently do to the planet andevery other species on it that largely sets theconditions for zoonotic spillover events, and we drivethem at an ever-accelerating pace.What we can and should do about this sobering realityneeds urgent societal discussion.The unscientific speculations and fantasies ofmotivated manipulators do not. Citizens deserve better.They are owed clarity, not spectacle.“Ecosystem disruption, climatechange, deforestation, wildlife tradeand consumption... we see thehuman hand behind almost everyoutbreak”” — Dr. Peter Daszak, EcoHealthAlliance continued on next page

Page 40

I believe that the era where false “gain-of-function”origin myths are wielded as weapons forpersuasion, profit, or power has to end.Not only is this deeply unethical and scientificallyirresponsible in 2024, but it has thus far provencounterproductive for scientific research, pandemicprevention efforts, and the public good.In this article, I focused our attention on aninterconnected set of scientific knowledgerelated to viral recombination and its implications.It is important to mention that this - while verycomprehensive - is just a mere facette of the largerorigin picture. There are other comprehensive lines ofinvestigation that this article could not go into, likeepidemiology, which also points to viral emergence atthe Huanan market and also contradicts any research-related origin scenario. No single article can cover it all. Understanding the origin story through the lens ofviral recombination is incredibly powerful becauseit exposes the shallow ideas and trivial falsehoodsabout this virus thrown into our faces every day. It alsoshows us that nature is much more complex, nuanced,and vast than we commonly give it credit for. It wouldbe wise to exert more caution and compassion beforetrying to shape every last ecosystem to our humanwhims.PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/False myths arecounterproductive to solutions40ProtagonistS C I E N C EA comprehensive body of scientificevidence has shown that theimmediate bat ancestor to SARS-CoV-2 came from one of thecountless natural “gain-of-functionlabs” spanning the vast biodiverseKarst region from Yunnan in SouthernChina towards Myanmar, Laos,Thailand, Cambodia, Vietnam, andmaybe even Malaysia in SE Asia.The lingering and promiscuousendemic viral elements in thatenormous geographic regionconstantly mix and bring forth newchimeric combinations within theirsocially intricate reservoir hosts; whilehuman activities and encroachmenton bat territories stir the geneticcauldron ever faster.Once a particularly combustible set ofgenetic elements produced apotential pandemic pathogen withbroad host tropism, the legal andillegal mammalian wildlife industrylikely became the maturing vesselsthrough which the virus we nowknow as SARS-CoV-2 reached itsfinal explosive form. From there, itwas dragged in front of hundreds ofimmune-naïve future hosts visitingthe largest wet market of oneparticular Chinese megacity wellconnected with the entire world.The rest is history, one we are aboutto repeatMaybe after four years of political myth-making andsocietal inaction, it is time to face scientific reality. Icertainly believe we’d be better off fighting forsolutions rather than for who is to blame.Because no matter what we want to believe, thesenatural chimeras will keep haunting us if science andsociety don’t come together to stop them.The endThe endThe end

Page 41

A word of caution aboutscience communication versuspeer-reviewed research andscientific reviewsIn this article, I explained how multiple lines ofscientific inquiry strongly exclude the possibility ofgain-of-function research leading to COVID-19.Furthermore, the article highlights a body ofliterature warning about the clear and persistentdanger of another zoonotic sarbecovirus spillingover into the human population. Please consider that no single article can summarizeall the valuable scientific contributions that scientistshave made in support of investigating the originquestion. It is a large and still-growing body ofevidence spanning hundreds of papers that aremutually consistent with each other, the extantevidence, and the reality we live in.I want deeply thank Prof. Alice Hughes, Prof.David Robertson, Prof. Linfa Wang, Dr. AlexCrits-Christoph and Dr. Jonathan Pekar forreviewing relevant sections of this article.My humble contribution to this topic was to focuson explaining recent key scientific argumentsthrough the lens of viral recombination for non-experts and also accurately present the conclusionsscientists reached in their work by considering alarge, detailed body of evidence.While I did go to great lengths to avoidmisrepresentations — such as having hours of expertinterviews and article reviews by domain expertsbefore I post — I cannot always control how mywords will be interpreted. If there are uncertaintiesarising from my simplifications, omissions of brevity,or bad analogies, I advise you to first consult theprimary literature for clarification rather thanpresume there is an obvious mistake in the scienceor reasoning of scientists.At this point, the scientific consensus arising from alarge body of evidence is pretty unequivocal on keyissues surrounding the origins of SC2. However, thisdoes not mean that there are no more questions tobe asked or no more lessons to be learned on how toprevent SARS-CoV-3. That is why origin researchcontinues, despite the “lab leak” question beingmostly settled in the scientific literature.Whenever there is a (fake or real) controversy about ascientific topic, I strongly believe that only the fruits ofscientific inquiry — not a toxic combination of politics, media,and activists — will produce reliable knowledge and factualinsights to act upon.All that is missing currently is for some citizen defenders ofan evidence-based worldview to do the legwork of closingthe rift between science and society, for example bybuilding bridges of accessibility where there are none.There is no infrastructure and no money in sciencecommunication, and science desks were the first to go in acrumbling newspaper economy. That is why I have to dothis work without any financial aid or compensation (seemotivation statement); and against a lot of headwinds fromemotionally engaged conspiratorial communities. To be completely honest, the lack of accessible informationand resources on this topic is not only a monetary issue, nora coincidence in a broken information ecosystem.Many virologists have been generous and greatcommunicators trying to compensate for public knowledgegaps before they were publicly smeared and characterassassinated into disengagement (or even defamed) bymotivated actors. Anti-science pressure organizations likeUS-right-to-know (USRTK) target outspoken virologistsexplicitly for that reason.About scientific “controversies”I believe nobody in society should condone anti-science activism and abusive behavior. It would surelyhelp if citizens stood up for scientists unfairly targeted bypoliticians once in a while. That virologists are bitterlydisappointed in institutions, media, and politics thatfacilitate such attacks is an understatement. In the end,science and democracy can not function without a societythat supports them.So I encourage you and other defenders of a “weight-of-evidence”-based worldview to talk about and speak up forthe scientific method, use it to dispel popular myths, orsimply enjoy having understood something importantabout a dramatic world event so you won’t be a suckerfor the next manipulative “gain-of-function” charlatan thatcomes along.41“They extract a high cost for free speech,they coerce the informed into silence” — Nature Magazine editorial about theanti-science pressure group USRTK PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/ProtagonistS C I E N C E

Page 42

As always, my hope and goals are to educate andequip citizens with conceptual tools and newperspectives to make sense of the world weinhabit.This article took a lot of time and effort toconceptualize, research, and produce, actuallyalmost irresponsibly so given that I do notmonetize my scicomm here; and neither do themany scientists that so graciously gifted their timeand expertise to help me and others understandtheir field of expertise.Motivation statement CopyrightYou are also invited to deepen this work or justderive satisfaction from understanding ourchaotic modern world a bit better.So feel free to use, share, or build on top of thiswork, I just ask you to properly attribute (CreativeCommons CC-BY-NC 4.0).Cite this work:Markolin P., “Treacherous ancestry. Aphylogeographic hunt for the ghosts ofSARS-CoV-2”, April 12, 2024. Free direct access link:www.protagonist-science.com/p/treacherousancestryAlso, since this topic is close to my heart, I’d behappy to hear your thoughts42I see this work as a public goodthat I send out into the void of theinternet in hopes others will getinspired to actPROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/ProtagonistS C I E N C E

Page 43

Below I listed some of the key publications referenced in this article, with a short comment on what they are aboutfor easy navigation and further reading.On recombination in CoVs:Sola I. et al., Annu Rev Virol., 2015 (recombination in CoVs is discontinuous with RdRp jumping around; deepmechanism of CoV transcription)Patino-Galindo JA. et al., Molecular Biology and Evolution, 2021 (recombination patterns and frequency betweenviral families)Boni MF. et al., Nature Microbiology, 2020 (recombination event between bats and pangolins not likely to have ledto emergence of SARS-CoV-2, genetic elements of lineage have been circulating in bats for decades)Klerk A. et al, Virus Evol., 2022 (Conserved recombination patterns across and within coronavirus subgenera)Wells H. et al., Cell Host Microbe, 2023 (review article about CoV recombination, requirements and likelymechanisms)Wang J. et al, Nature communications, 2023 (virus-sharing networks between bats highlight ample opportunity forCoV recombination in nature)*recombination as “virus sex” is not accurate mechanistically: The exchange of genetic material betweenviruses is usually non-reciprocal, meaning the recipient of a genome portion does not act as donor of the replacedportion in the original source. In this respect, the term recombination does not have the same meaning in virusesthat it does in diploid, sexually reproducing organisms wherein the exchange of genetic material betweenchromatids in the first meiosis division is reciprocal. (Perez-Losada M., Infect Genet Evol., 2015)On the Furin-cleavage siteAndersen KG., Nature Medicine, 2020 (proximal origin noticing unusual FCS and structural predictions of changedglycosylation patterns)Wu Y. et al, Stem Cell Research, 2021 (highlights FCS sites over wider CoV family)Lavie M. et al., J Virol., 2022 (cleavage at S1/S2 and S2 viral processivity studies highlight the function of the FCS)Jackson JB. et al., Nat Rev Mol Cell Biol., 2022 (FCS cleavage makes S1/S2 unstable, stabilizing mutation D614G.“This perplexing observation suggests that the acquisition of a furin-cleavage site by SARS-CoV-2 may have been arecent event.”)Peacock T. et al., Nature Microbiology, 2022 (FCS cleavage of S1/S2 is essential in ferrets, pre-cleavage of the spikeduring viral egress enhances entry of progeny virions into TMPRSS2-expressing cells such as those abundant inrespiratory tissue; and avoid endosomal IFITM proteins)Fraser BJ. et al., Nature, 2022 (Spike processing by TMPRSS2; cuts extracellular spike in 3 places including S1/S2 ifnot pre-cleaved by furin to enable membrane fusion)Garry R., PNAS, 2022 (FCS was not engineered and certainly not modeled after human EnAC)Chaudhry MZ. et al., Virology, 2022 (FCS gets quickly lost in cell culture because of kinetics)Vu MN. et al., PNAS, 2022 (synergistic interaction with the QTQTN motif proximal to the FCS plays a key role ininfection and pathogenesis)Sander AL. et al., Communications Biology, 2022 (high sequence diversity in S1/S2 and a proto-FCS site found inEuropean SARS-related bat CoVs)Alwine JC. et al., mSphere, 2023 (The FCS not lab adapted — initial SARS-CoV-2 isolates replicate poorly intraditional laboratory models — and not engineered — cleavage site loop length — best we can tell)Neil SD. Cell, 2023 (Furin cleavage sites in intermediate host animals, but not bat reservoirs)Steiner S. et al., Nature Reviews Microbiology, 2024 (recent review on viral entry and what is known about differentmechanisms)References43PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/ProtagonistS C I E N C E

Page 44

On the social lives of batsHughes AC. et al., Acta Chiropterologica, 2011 (Bat calls are destinct; rhinolophids use distinctive constantfrequencies that can facilitate identification)Vernes SC. et al., Phil. Trans. R. Soc. B., 2019 (vocalizations vary between species and include echolocation calls aswell as social calls used either for parent–offspring reunions, territorial defence or maintaining group integration)Irving A. et al., Nature, 2021 (bats are uniquely suited hosts and viral reservoirs)Respicio JM. et al., Journal of Animal Ecology, 2024 (bat aggregation leads more negative and aggressive behavior)On Karst and cavesDavid Gillieson, Oxford Academic, 2005 (With a total area of about 400 000 km2, Southeast Asia contains some ofthe more extensive karst regions in the world. Many of these karst areas are of high relief with spectacular arrays oftower and cone karst. Many have now been inscribed on the World Heritage list in recognition of their uniquegeomorphology and biology) (500.000 km2)On pre-pandemic Sarbeco seroprevalence in South-East AsiaWang N. et al., Virol Sin, 2018 (October 2015 serum sampling from 218 residents in four villages in Jinning County,Yunnan province, China, 6 (2.7%) positive SARSr serology)Li H. et al., Biosafety and Health, 2019 (pre-pandemic: 9 cases (0.6%) of SARS-CoV-1 positive serology from asnowball-sampled cohort of 1600 people in 3 provinces in Southern China between 2015–2017) Sanchez CA et al., Nature Communications, 2022 (65000 CoVs spillovers per year estimated)Manning J. et al., Emerg. Inf Dis, 2022 (SC2 Elisa assays of pre-pandemic blood samples taken in Cambodia between2005–2011 show 4–14% reactivity)Evans TS. et al., Int J. Infect Dis, 2023 (neutralizing antibodies against various SC2r CoVs in Myanmar locals, highseroprevalence for rural (~20%) populations in 4 areas, 0% in city people, sampled pre-pandemic July 2017 toFebruary 2020)Meyer M. et al., Nature Communications, 2024 (bat diversity and disease relationship in response to humanencroachment and habitat change)On sampling location, geographical distribution of SC2r genomes: Li LL. et al., Emerg. Microb Infect. 2021 (RpPrC31, a viral recombinant ancestor, retrospectively discovered from batsamples in Yunnan)Wacharaplusadee S. et al., Nature communications, 2021 (RacSC203, a cousin of SARS-2 discovered in Thailandcontaining S1/S2 insertion; also pangolin CoVs at wildlife checkpoint)Delaune D. et al., Nature communications, 2021 (bat samples from 2010 cambodia find a close relative 92,6% ofSARS-CoV-2 (RSHTT200), one bat was simultaneously co-infected with 4 viruses, 2 of them sarbecoviruses)Lytras S. et al., Gen Evo., 2022 (recombination analysis and geographic distribution of Sarbecoviruses)Temmam S. et al., Nature, 2022 (discovery of human-infectious SC2 relatives in Laos, “Banal” viruses and genomesequences)Zhao S. et al., J Genet Genomics, 2022 (geogenomic distribution patterns)Han Y. et al., Nature communications, 2023 (CoV sampling all over China)Muylaert RL. et al., Nature communication, 2023 (landscape predictions and drivers of SC2 spillover)Gilbert M. et al., Tropical Biomedicine, 2023 (SC2 related coronaviruses in Malaysia, no full genome sequencing yetbut 99% similarity for a fragment)Forero-Muñoz NR. et al., Virus evolution, 2024 (phylogeographic structure in bat hosts creates a landscape ofselective pressure)References44PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/ProtagonistS C I E N C E

Page 45

On moral panics*moral panic: As a distinct species of collective behavior, moral panics represent contentious and intensely affectivecampaigns to police the parameters of public knowledge and morality. As such, they are necessarily dependentupon and constituted by claims-making, with interested parties historically seeking to actuate alarm by influencingthe imagery and representations of the mainstream press. (Walsh JP, International Journal of Cultural Studies, 2020)Recent preprints:Hassanin A. et al., biorxiv, 2023 (phylogenetic divergence of close SC2 relatives between sub-tropical NorthernVietnam and tropical Southern Vietnam)Crits-Christoph A. et al., biorxiv, 2023 (metatranscriptomics data analysis from Huanan market identifies wildlifespecies)Pekar et al., biorxiv, 2023 (geospatial history and origins of SARS-CoV-1 and SARS-CoV-2, video interview here)Si J. et al., biorxiv, 2024 (multi-species ACE2 adaptiveness, Sarbecos can infect humans from the get-go)Other video interviews with origin reseachersVideo: Long-form discussion with bat ecologist Dr. Alice C. Hughes and the risk of SARS-CoV-3Video: Long-form discussion with Dr. Michael Worobey and Dr. Kristian Anderson about their research establishingthe Huanan market as the origin of the SARS-CoV-2 pandemicVideo: Long-form discussion with Dr. Angela Rasmussen and Dr Stephen Goldstein debunking common bad lableakargumentVideo: Long-form discussion with SAGO member Dr. Carlos Morel (SAGO is the WHO’s scientific advisory group onthe origins of pandemics)BonusWow, I am surprised you scrolled all the end to the references. This curiosity (or due diligence) is of courserewarded with a little bonus information:I have been working on an origins book with a twist that I believe will make some waves. It raises questions nobodyelse has yet dared to tackle: Where exactly does the “lab leak” myth come from, how did it move through society,and why were so many citizens susceptible to it? From the remote villages of the Lahu mountain tribes via chaoticconspiracy theorists and needy journalists to the halls of power and even the US presidency, THE LAB-LEAK MYTHwill invite readers into my most challenging project yet; making sense of where the current rift between science andsociety really comes from.You can subscribe for free to keep following my science writing and receive further updates and announcementsper email.https://www.protagonist-science.com/ (Substack newsletter)You can also follow/engage on socials, preferably Bluesky @philippmarkolin.bsky.social, (still exist on Twitter as well but maybe not long)https://medium.com/@protagonist-scienceReferences45PROTAGONIST SCIENCE | APRIL | 2024HTTPS://WWW.PROTAGONIST-SCIENCE.COM/ProtagonistS C I E N C E