Hi I'm Wendy Zukerman and you're listening to Science Vs from Gimlet. Today on the show. Was this coronavirus made in a lab? Since the early days, we’ve been hearing that this virus came from bats, and jumped to humans….but another claim has popped up that is getting a lot of attention

And developing tonight the US government is looking into the possibility COVID 19 originated in a chinese laboratory

The killer coronavirus probably originated from a laboratory in Wuhan

A Pew research poll recently found that 30% of Americans believe this virus was made in a lab...and not in nature.[1] 

TRUMP ...we’re looking at that. A lot of people are looking at it. It seems to make sense.

And here's what some people are pointing to as evidence[2] [3]. You see, early in the pandemic, some news reports were saying the outbreak took off at this animal market in Wuhan[4]. But then

Announcer: Chinese researchers say the first known patient had no exposure to the market.[5] 

Adding fuel to the fire…  it turns out that some 10 miles away from that market -- is a high security lab - the Wuhan Institute of Virology. [6] [7] [8] - where scientists study some of the most dangerous viruses around [9] [10] [11] 

<<Pompeo: This is a laboratory that contained highly contagious materials[12]>>

I would note that, Wuhan also has China’s only biosafety level 4 super laboratory[13]>>

So is it possible that someone cooked up this virus on purpose? And how could we even know? Today on the show the story of how scientists are tracking down the origins of this virus…  delving deep into its genome to look for human fingerprints.  And even if you don't think this virus was made in a lab… stick around for this one...because it's quite the adventure.

Science Vs Coronavirus Escape from the Lab? Is coming up just after the break…

BREAK

Welcome back. Today on the show. Did this coronavirus escape from the lab? To answer this question… we called up Benhur Lee from Icahn School of Medicine at Mount Sinai in New York City[14]... Behur studies how diseases emerge… and was like… yeah the fact that this high level virus lab in Wuhan is so close to the outbreak[15] ?

<<I mean the optics of it, right, I mean the optics of it doesn't look good>>

But scientists don't need to rely on optics to find out where this virus came from…  They use science. More specifically. Genetics.

You can think of the genetic code of this virus as the scene of a crime -- and we're looking for evidence of foul play. By early January, scientists all around the world were invited to inspect the crime scene.

On January 10th the full genome sequence was released and from that you can infer the origins in terms of of like where it came from[16][17]

The first thing scientists wanted to do with that sequence was compare it with viruses we already know about -- to see if it's a match.[18][19] Over the years scientists from all around the world - in New York, London or Wuhan - they have collected samples of different viruses and  sequenced them and popped them up on big databases[20][21][22] .So basically we can just look and see - have we seen this before? Because if we had  this exact same coronavirus already on file then we’d know this wasn't some new thing cooked up in a lab. 

WZ Have we found the exact some coronavirus that's now infecting people in nature, in a bat in anything?

BL No, no we have not, so there's no smoking gun

No smoking gun? So where did this virus come from? Could scientists have made it in a lab?  

Well, we know it’s true that scientists are out there playing around with viruses in labs[23][24]. - and they do this for all sorts of reasons - like trying to understand how viruses work and how dangerous they might become.  

WZ If this was your job to make this coronavirus like - could you do it?

BL Not from scratch! To design a virus from scratch it's extremely difficult. It's like building a 30th story bldg where a thousand different items on each floor—beams, windows, doors, elevators, wiring, plumbing, fire-alarm, sprinklers, etc.— they all have to come together exactly right

Benhur says that when scientists are messing around with viruses, like coronaviruses, what’s really happening is that they’re tweaking viruses that already exist.[25][26][27][28][29] They're not building coronaviruses from the ground up. It’s more like redesigning the doors.

BL So I think that's the scale that we're talking about

This coronavirus for instance -- has  around 30,000 bits of genetic code -- called nucleotides[30]… all working in perfect horrific harmony. And Benhur says scientists just don’t know how to make all that from scratch. So you need a building. A starting place.

 

BL You need something from nature. You need a backbone,

A backbone, as in a really similar virus that you just need to tweak. So, our next question is: do we have a similar enough virus ... that you could just change a little to make this coronavirus? Well, a paper published in Nature looked through the virus database and they said the most closely related virus that we know about [31]-- is called R-A-TG13.[32][33]  It was found in China.[34] In a type of horseshoe bat.

BL Tiny little bats, they have very big ears, If you look at the face and use your imagination it looks like a horseshoe[35][36]

These two viruses - the one causing the pandemic and the one in the horseshoe bats- they are similar. On average, they share 96% of the same genes[37][38]. . And it's led some people online to argue… that this was the backbone that scientists tweaked to create our coronavirus.[39]...

WZ: Couldn't that backbone have been that horseshoe bat virus?

BL: It's not

WZ: it's not?

BL: It’s not

Benhur says - hold your horseshoe. The problem with the R-a-tg13 virus? It's not similar enough.[40]  In the land of genetics  96% similar - it’s actually a huge difference…

BL I mean it's different in all sorts of different ways

So when you zoom in on the two genomes of these viruses there are actually more than a thousand[41]  tiny genetic differences… and they’re sprinkled along the genome - and a scientist would have to go in and change them one by one.

BL YES YES And hope that it works. It's extremely difficult

And..  there’s another glaring piece of evidence that suggests this virus isn’t the work of some evil geniuses ….  And it’s this --  if scientists did design this virus - they made an unexplainably dumb choice in putting together. And it all has to do with this …

CS: This piece called the poly basic cleavage site and so that's a big mouthful[42][43][44] 

It is a mouthful! But I tell you by the end of this - you'll be spouting polybasic cleavage site after you've downed ten beers so let's go. OK that was Christian Stevens[45] you just heard …he's Benhur's PhD student …

So what's the big deal with these poly basic cleavage sites that Christian is on about? Well, viruses use these sites to get into our cells to infect us. But for them to work… a protein[46] from our body has to come along and snip something off that site[47] 

CS: As though a pair of scissors came through  

Once the virus has been circumcised it can then enter cells[48] and start attacking us. Christian says it’s useful to think about this site like a button.

CS: Only when you pushed that button is it ready to go   

And while other viruses have this button - our coronavirus has a button that’s bigger and easier to find. That's the poly basic cleavage site. The big button. And we think it means that the virus might be able to do more damage in our bodies.[49][50],[51] .[52][53]

But here’s the thing -- when researchers saw that big button in our coronavirus -- they didn’t see a big "MADE IN CHINA" sticker on it - and that’s because … for years  scientists have been studying what makes the best cleavage site[54] - the biggest button! The easiest to press![55][56][57] And they can spot it in the genes. Let me tell you. This one - it ain't it. Producer Rose Rimler talked to Christian about it.  

CS It's very easy to make that in the perfect way[58], and this is made in an imperfect almost seemingly random way.

RR: If you were an evil scientist training minions of evil scientists and you told them make a cleavage site to end all cleavage sites what grade would you give them?!

Hahah Not a good grade[59]

Benhur - Christian’s teacher - agreed.

BL The polybasic site is a crappy polybasic site, so if someone designed it you know they didn't do a very good job.  

So - here's where we're at. To have made this virus in a lab. Scientists would have needed to have a very similar virus on hand they could tweak. A SECRET virus. Sure. If I put my conspiracy theory hat on … I can buy that. But then when they went in to tweak it they could have easily made this polybasic cleavage site much more dangerous, but they didn't.…It doesn't make sense.  And if scientists wanted to cause chaos  they really didn't have to go to such trouble

BL  in my private moments I'm like there are much easier ways to make a deadly virus

If you didn't catch what he says - there are much easier ways to make a deadly virus… Benhur … and other scientists we talked to … said if you wanted to cause a lot of trouble - you wouldn’t do it this way. With this coronavirus… Here’s what you do instead ...  

You'll get me in trouble

Hhahahaha

And so you can make <BEEEEEEEEEEEP>> and super charge it I mean…

Bottomline if you know what you're doing you don't use a coronavirus

Yes yes  People could do stuff much more easily than making a coronavirus 

For Benhur and other scientists who study viruses[60] - they knew right away there was nothing suspicious here ... there's no human fingerprints in its genetic code[61][62][63].

WZ  As a scientist is it silly to ask if this was made in a lab or is this a legitimate question?

As a scientist - as a bona fide scientist um… it's a ridiculous question

Hahahaha

I mean… I would say it's a ridiculous question once we knew full sequence because you can just compare the sequence if you know anything about virology and coronavirus and how hard it is - it's just a non starter kind of questions

Even the Department of Homeland Security[64] agrees that this did not come from a lab. In China or anywhere else. So if this virus didn’t come from the lab - then where could it have come from? What other suspects do we have?

They got scales. They look pretty bad ass. they look a little bit like a dinosaur

That’s coming up.

BREAK

Welcome back. We just learned that the best evidence tells us the virus wasn’t cooked up by an evil genius …  So where did it come from? Scientists think it came from nature… an animal and then eventually moved to us.  And they think this because this kind of thing happens a lot. In fact, scientists say most new infectious diseases come from animals[65][66].  Ebola, HIV, SARS, Bird flu..swine flu...they all spilled over from animals.

So the big question is what animal gave us this coronavirus? And you guessed it - it’s PORPOISES. Just kidding. We think bats are probably involved -- and that's because we already know that bats get a tonne of other kinds of coronaviruses. One big study in this space collected pee and spit from bats[67] … all over the world - and found almost 100 different kinds of coronaviruses in them[68]. Now we haven't found our virus in bats yet - but Benhur Lee says we haven't tested enough bats yet. That big study? Only looked at 12,000 bats.

BL Sounds a lot but that's not a lot when you compare it to how many bats there are and how many species there are

There are lots of bats out there.[69] Seriously MILLIONS AND MILLIONS[70] of them. So what scientists are trying to do right now… is find the right bat. And it's a mammoth task[71]. This is needle in a haystack stuff [72]. It can take time. In fact… get this.  Science has known about Ebola for more than 40 years[73] … and we still haven’t haven’t found the exact same virus in animals[74] …

BL And That's not for lack of trying, I mean CDC sent teams over there over the years and sampled everything that moved from insects to shrews[75] to rodents[76] and they couldn't find ebola[77]

And further complicating this quest to find our coronavirus … there's a chance that another animal is involved here. [78][79][80] . Now we know from other viruses this can happen -- for example take the deadly -- Nipah virus.[81] We know this virus has jumped from bats to pigs[82][83] [84].[85]   before eventually reaching us. Christian, who you heard from before, told us how we think that jump happened …

CS Someone started a mango farm in the same place they had their pigs- these fruit bats would eat mangos over the top of these pigs, they’d drip saliva, occasionally urinate and that could fall into what the pigs are eating[86][87]there are a lot of different ways we can see that happening

The pigs got the virus from the bats - and gave it to humans. And something like this might have happened with this coronavirus…  

To help us find the possible accomplice here -- we called up Oscar MacLean. He studies viruses at the University of Glasgow[88]. And Oscar told us one possible suspect that has recently gotten some attention: is the pangolin.

OM Pangolins are a nice one, they're a cool animal. They got scales.  They look pretty bad ass. they look a little bit like a dinosaur with quite a long tail

RR To me they look like half dinosaur half cat

OM I bet they wouldn't be so cuddly though that's the problem

So the story of how these scaly dino-cats made their way onto scientists’ radar starts about a year ago … officials in China intercepted a shipment of 21 live pangolins that were being smuggled through customs[89]..  They sent them to a wildlife rescue center….The animals were really sick, and researchers at the rescue center wanted to find out why.

OM So they were sick and they decided to sample viruses on basis of what was making them ill

When the scientists took tissue samples[90]… they found that some of the pangolins had been infected with coronaviruses. They sequenced the viruses … and published the data. Now after this outbreak started, another group of scientists went back and looked at the pangolin coronaviruses[91] …  and found … that one of them was like a cousin to the virus infecting us now … In fact there was one piece of the pangolin coronavirus that looked very familiar … The spike protein.

OM The spike protein was much closer than anything we'd sequenced before[92]

Maybe you’ve heard about this spike protein, it’s the little nubs that surround the coronavirus and help it attach to our cells. And this pangolin spike is super similar to the one on the virus that’s infecting us[93]. So now scientists are wondering if maybe a bat and a pangolin got mixed up somewhere … shared some virus ... and that eventually led to the one we have now. 

Scientists are still out there looking for the source of this virus ... whether it's just a bat, or a pangolin, or some other animal. But big picture, we're pretty sure that this kind of thing is what happened. Some unlucky human came in contact with some animal. And the virus took off … unluckily for all of us. And of course there is one thing that all the evidence points to, which is that this virus wasn't cooked up in a lab. 

Given all this, Rose asked Oscar...

RR Why do you think this made in a lab idea is so appealing to people

OM I just think its a convenient scapegoat, I think we've come up with this massively complicated society, inconvenient truth that that 30,000 letters of RNA can sort of crumble it

Yeah, 30,000 little genetic bits making up this coronavirus … and that’s wreaking havoc across all the world …

OM it's just so awkward

RR awkward is such a great word for it… like how awkward that we think we're the most powerful things on the planet and something invisible to us is actually more powerful than we are

OM yeah exactly it's easy to ignore the reality of how vulnerable we are to these sort of things

 

And now… time for a little NCVC

JINGLE

Today...we’re going to a rainforest in Panama. If you look by the edge of a stream[94], you might see some tiny pug-nosed treefrogs. What makes these little guys so special is the sneaky way they attract mates - while avoiding predators. You see, the male frogs call out to attract female frogs - but when they do, nearby bats hear the call too. And for the bats, that call is like a dinner bell, cuz these bats love tasty frogs.[95][96]  So scientists have been studying this clever way the frogs have outfoxed the bats. Or rather. Outfrogged the bats. Here’s how it works: one little male will start the call.

One frog sound

This is an actual frog the researchers recorded, by the way![97] And almost immediately, he’s copied by all these other frogs...

Multi-frog sound

And scientists have figured out - that while the bats might go for that first frog[98] ... and the other frogs that chime in are safe to get the girls. Scientists still don't know how frogs decide who makes the first call[99]... cuz that frog is kind of a sucker, right? They think it might be sort of a game of frog-chicken. Poor little guy.

That’s Science Vs.

CITATIONS

CREDITS

This episode was produced by Wendy Zukerman and Rose Rimler, with help from Meryl Horn, Michelle Dang, Sinduja Srinivasan and Laura Morris. We’re edited by Blythe Terrell and Caitlin Kenney. Fact checking by Diane Kelly. Mix and sound design by Peter Leonard. Music written by Peter Leonard, Marcas Bugulah, Emma Munger, and Bobby Lord. A big thanks to all the researchers we got in touch with for this episode, including Dr Kirsty Short, Dr Ximena Bernal, Henry Legett, Dr Muhamed Amin, and Professor Kristian Andersen. And special thanks to the Zukerman family and Joseph Lavelle Wilson. I’m Wendy Zukerman, i’ll fact you next time.


[1] Nearly three-in-ten Americans believe COVID-19 was made in a lab 

[2]https://www.washingtonpost.com/opinions/2020/04/14/state-department-cables-warned-safety-issues-wuhan-lab-studying-bat-coronaviruses/ 

[3] Some examples: US officials confirm full-scale investigation of whether coronavirus escaped from Wuhan lab 

https://www.reddit.com/r/Coronavirus/comments/g26t55/us_explores_possibility_that_coronavirus_started/fnjt8br?utm_source=share&utm_medium=web2x

Don't buy China's story: The coronavirus may have leaked from a lab

Microbiologist claims the coronavirus was man-made in Wuhan lab

Inside the Viral Spread of a Coronavirus Origin Theory

[4] Novel human virus? Pneumonia cases linked to seafood market in China stir concern 

[5] Phyloepidemiologic analyses indicated the SARS-CoV-2 source at the Hua Nan market should be imported from other places. The crowded market boosted SARS-CoV-2 rapid circulations in the market and spread it to the whole city in early December 2019.

[6] e.g. The S, E and M genes of SARS CoV were amplified from the WH20 strain (GenBank accession no. AY772062) by reverse transcription polymerase chain reaction (RTPCR) with the following primers: …

 https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2567.2007.02676.x

[7] directions to wuhan institute of virology from the wet market (by car) 

[8] http://english.whiov.cas.cn

[9] (2017) A laboratory in Wuhan is on the cusp of being cleared to work with the world’s most dangerous pathogens. The move is part of a plan to build between five and seven biosafety level-4 (BSL-4) labs across the Chinese mainland by 2025, and has generated much excitement, as well as some concerns.  …. Future plans include studying the pathogen that causes SARS, which also doesn’t require a BSL-4 lab, before moving on to Ebola and the West African Lassa virus, which do

[10] (page 10) The microbes in a BSL-4 lab are dangerous and exotic, posing a high risk of aerosol-transmitted infections. Infections caused by these microbes are frequently fatal and without treatment or vaccines. Two examples of microbes worked with in a BSL-4 laboratory include Ebola and Marburg viruses..

[11] In August 2017, the National Health Commission of China approved research activities involving Ebola, Nipah, and Crimean-Congo hemorrhagic fever viruses at the Wuhan BSL-4 laboratory

[12] https://www.youtube.com/watch?v=upkldowZjaI [0:52]

[13] Hearings | United States Commitee on Armed Services  [2:02:17]; https://www.armed-services.senate.gov/imo/media/doc/20-02_01-30-2020.pdf

[14] Benhur Lee, MD

[15] If coronaviruses were the culprit, she remembers thinking, “could they have come from our lab?” https://www.scientificamerican.com/article/how-chinas-bat-woman-hunted-down-viruses-from-sars-to-the-new-coronavirus1/

[16] From Jan 23: By January 10, 2020, Chinese researchers released a full genomic sequence of 2019-nCoV to public databases - this analysis suggested that it shared something with SARS

[17] https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/

[18]e.g.  Jan 24: https://www.nejm.org/doi/full/10.1056/NEJMoa2001017?query=featured_home 

 “more than 85% identity with a bat SARS-like CoV (bat-SL-CoVZC45, MG772933.1) genome published previously.”...”Although 2019-nCoV is similar to some betacoronaviruses detected in bats (Figure 4), it is distinct from SARS-CoV and MERS-CoV. The three 2019-nCoV coronaviruses from Wuhan, together with two bat-derived SARS-like strains, ZC45 and ZXC21, form a distinct clade.

[19]  See also Fig 1: https://www.nature.com/articles/s41586-020-2012-7

[20] Virus Pathogen Database and Analysis Resource (ViPR) - Genome database with visualization and analysis tools

[21] NCBI Virus is an integrative, value-added resource designed to support retrieval, display and analysis of a curated collection of virus sequences and large sequence datasets. We are a community portal for viral sequence data, and our goal is to increase the usability of data archived in GenBank and other NCBI repositories.

[22] GISAID - Initiative  and note: nextstrain.org gets its data from GISAID

[23] Experimentally, the –RRRKK– insertion was also found to increase in cleavage of hemagglutinin by FR. The simulated data provide a clear answer to the question of why inserted H5 is better cleaved by FR than the other subtypes, explaining the high pathogenicity of avian influenza H5N1  

[24] Generation of wild-type MERS-CoV, MERS-Uganda, and HKU5-CoV viruses utilized reverse genetics and have been previously described (6, 16, 19). For MERS-Uganda chimera expressing RFP, we utilized the MERS-CoV backbone, replacing ORF5 with RFP as previously described (19). Synthetic constructions of chimeric mutant and full-length MERS-Uganda and HKU5-CoV were approved by the University of North Carolina Institutional Biosafety Committee.

[25] Natural viruses and their capsids do not have all of the several properties and functionalities required, or at least are not optimized, for any of their contemplated applications. Thus, viral particles have to be modified by direct chemical means and/or by protein engineering using genetic techniques.

[26] Build your own virus: One goal of vaccine developers is to create strains of pathogens that are less virulent, or attenuated. To accomplish this feat, scientists tinker with the virus's genetic material, a step that, for RNA viruses, is most easily done by making a DNA copy of the viral genome. This DNA version can be put back into cells, which then manufacture a tailor-made virus.

[27] https://www.pnas.org/content/96/16/9345.short

[28] Even when viruses are “built from scratch”, they are copies of existing viruses: Venter's team cobbled together the virus, called phi-X174, following its published genetic sequence. They stitched up its DNA from ready-made overlapping fragments called oligonucleotides, each built from 40 chemical building-blocks, or bases.

[29] The above was still true in 2018:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171941/

[30] 29903 bases in one SARS-CoV-2 strain: see Fig 1 in this paper (March 17): https://www.nature.com/articles/s41591-020-0820-9

[31] Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. …. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus...Simplot analysis showed that 2019-nCoV was highly similar throughout the genome to RaTG13 (Fig. 1c), with an overall genome sequence identity of 96.2%

[32] PREPRINT: https://www.biorxiv.org/content/10.1101/2020.02.27.969006v1

[33] Furthermore, if genetic manipulation had been performed, one of the several reverse-genetic systems available for beta coronaviruses would probably have been used19. However, the genetic data irrefutably show that SARS-CoV-2 is not derived from any previously used virus backbone.”

[34] We then found that a short region of RNA-dependent RNA polymerase (RdRp) from a bat coronavirus (BatCoV RaTG13)—which was previously detected in Rhinolophus affinis from Yunnan province—showed high sequence identity to 2019-nCoV.

https://www.nature.com/articles/s41586-020-2012-7 

[35] Rhinolophus affinis (Intermediate Horseshoe Bat)

[36] Taxonomic implications of geographical variation in Rhinolophus affinis (Chiroptera: Rhinolophidae) in mainland Southeast Asia: “All members of this family are characterized by the presence of a horseshoe-shaped anterior nose leaf, the morphology of which can be diagnostic between species.”

[37] Comparative genomic analysis has been greatly assisted by the availability of a related virus from a Rhinolophus affinis (i.e., horseshoe) bat sampled in Yunnan province, China, in 2013 (Zhou et al., 2020). This virus, denoted RaTG13, is ∼96% similar to SARS-CoV-2 at the nucleotide sequence level.

[38]  Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus.

[39] Examples of talk/rumoring online: https://www.reddit.com/r/China_Flu/comments/g4gx5m/expert_reaction_did_covid19_come_from_a_lab_in/fnytnwv?utm_source=share&utm_medium=web2x

https://www.reddit.com/r/conspiracytheories/comments/etpd0q/coronavirus_update/?utm_source=share&utm_medium=web2x

https://www.reddit.com/r/Coronavirus/comments/ewoneg/at_first_i_thought_the_virus_escaping_a_bsl_4_was/?utm_source=share&utm_medium=web2x

"What Are The Odds?" - A Timeline Of Facts Linking Covid-19, HIV, & Wuhan's Secret Bio-Lab

https://www.taiwannews.com.tw/en/news/3880475

[40] In addition, although sequence similarity values of 96%–97% make it sound like the available bat viruses are very closely related to SARS-CoV-2, in reality this likely represents more than 20 years of sequence evolution (although the underlying molecular clock may tick at an uncertain rate if there was strong adaptive evolution of the virus in humans). It is therefore almost a certainty that more sampling will identify additional bat viruses that are even closer relatives of SARS-CoV-2.

[41] PREPRINT: https://www.researchgate.net/profile/Wen-Bin_Yu2/publication/339351990_Decoding_the_evolution_and_transmissions_of_the_novel_pneumonia_coronavirus_SARS-CoV-2_using_the_whole_genomic_data/links/5e5fa3ec92851cefa1dc62ef/Decoding-the-evolution-and-transmissions-of-the-novel-pneumonia-coronavirus-SARS-CoV-2-using-the-whole-genomic-data.pdf 

They found that in coding regions--that is, the regions that actually determine what proteins are made--there are around 1235 nucleotide substitutions between the two genomes. This number can change slightly depending on whether you are referring to SARS-CoV-2 sampled from a specific patient or the "consensus" SARS-CoV-2 as sampled from a set of patients.

[42] PREPRINT: the detection of a polybasic cleavage site in the fusion glycoprotein of SARS-CoV-2

[43] Acquisition of polybasic cleavage site and O-linked glycans. Both the polybasic cleavage site and the three adjacent predicted O-linked glycans are unique to SARS-CoV-2 and were not previously seen in lineage B betacoronaviruses. Sequences shown are from NCBI GenBank, accession codes MN908947, MN996532, AY278741, KY417146 and MK211376

[44] The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade: Despite a high similarity with the genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs.

[45] https://leelabvirus.host/team

[46] E.g., furin or another protease: Mammalian and low-pathogenic avian influenza A viruses (LPAIV) carry an HA cleavage site with a monobasic motif susceptible to trypsin-like proteases which confine viral replication to the respiratory or gastrointestinal tract. In contrast, HPAIV possess a polybasic HA cleavage site cleavable by furin which is ubiquitous and thus supports systemic viral replication.

[47] https://www.pnas.org/content/114/42/11157 Proteolytic processing at the S1/S2 and S2′ cleavage sites removes the covalent linkage between the two functional subunits and frees the fusion peptide, which allows shedding of the S1 crown and subsequent refolding of the fusion machinery

[48] https://science.sciencemag.org/content/sci/367/6483/1260.full.pdf 2019-nCoV makes use of a densely glycosylated spike (S) protein to gain entry into host cells… The S protein...undergoes a substantial structural rearrangement to fuse the viral membrane with the host cell membrane (7, 8). This process is triggered when the S1 subunit binds to a host cell receptor. Receptor binding destabilizes the prefusion trimer, resulting in shedding of the S1 subunit and transition of the S2 subunit to a stable postfusion conformation

[49] Example with influenza viruses in birds: https://www.nature.com/articles/s41598-017-10605-6  we demonstrate for the first time that the IAV(H7N9) viruses isolated from chickens in LPM in Guangdong province have acquired additional basic amino acids in the HA cleavage site, with only one amino acid difference from those found in HP IAV(H7N9) human strains, which can play an important role in increased virulence in humans.

[50] Here is an example paper early in the swine flu outbreak in 2009 where Dr. Peter Palese from Mount Sinai co-authored a piece about why this particular H1N1 outbreak may not be as lethal as other historic influenza outbreaks. One piece of evidence he uses is the lack of a polybasic cleavage site: https://www.sciencedirect.com/science/article/pii/S0092867409006357).

[51] a reassortant expressing the modified H9 HA with engineered polybasic cleavage site and all the other genes from an H5N1 HPAIV became highly pathogenic in chicken with an intravenous pathogenicity index of 1.23.

[52] https://sci-hub.tw/10.1159/000151618 In contrast to the local LPAI virus infection of the intestinal or respiratory tract, HPAI viruses cause systemic infection. As a result, virus can be recovered from many organs of infected animals. Large hemorrhages distributed all over the body, edema, and cutaneous ischemia are major symptoms of the disease. The final stage of the infection can be characterized by the emergence of neurological signs,….HPAI viruses have been found to specifically target lymphocytes and lymphoid tissues [5, 6], myocytes in the heart muscle [7], and endothelial cells [8, 9],

[53] https://www.cdc.gov/flu/avianflu/influenza-a-virus-subtypes.htm HPAI virus infection can cause disease that affects multiple internal organs with mortality up to 90% to100% in chickens, often within 48 hours.

[54] "Molecular composition of the haemagglutinin cleavage site motif: a key pathogenicity determinant." Here they talk about the effect of how many basic amino acids are at the cleavage site on the pathogenicity of the virus.

[55] We knew the effect of a polybasic cleavage sites from papers like this one: https://mbio.asm.org/content/8/1/e02298-16. There they look at avian influenza to compare the pathogenicity of different versions of the virus when there is a single basic amino acid, "R," to when there are a longer string of them: "RRKKR" (K being another basic amino acid).

[56] https://jvi.asm.org/content/jvi/78/18/9954.full.pdf Major factors affecting tissue tropism, systemic spread, and pathogenicity of avian influenza viruses are the amino acids at the cleavage site of HA0 and the distribution of proteases in the host…  The presence of polybasic amino acids at the cleavage site of HA is characteristic of HP influenza A viruses of the H5 and H7 subtypes.

[57] Here's another paper that engineered a polybasic site into avian influenza to see the effect: https://www.microbiologyresearch.org/docserver/fulltext/jgv/92/8/1843_vir031591.pdf?expires=1587512780&id=id&accname=guest&checksum=5A3BD3BC95BE604D9438D9293818422F . In this case they add 6 or 7 consecutive basic amino acids (mostly Rs and Ks) in a string. This is shown to be even more pathogenic than the canonical RRSRR.

[58] New polybasic cleavage sites have been observed only after prolonged passage of low-pathogenicity avian influenza virus in vitro or in vivo17. Furthermore, a hypothetical generation of SARS-CoV-2 by cell culture or animal passage would have required prior isolation of a progenitor virus with very high genetic similarity, which has not been described. Subsequent generation of a polybasic cleavage site would have then required repeated passage in cell culture or animals with ACE2 receptors similar to those of humans, but such work has also not previously been described. Finally, the generation of the predicted O-linked glycans is also unlikely to have occurred due to cell-culture passage, as such features suggest the involvement of an immune system.

[59] Back in 2006 there was a paper looking specifically at what happens if the single "R" in the original SARS-CoV is replaced with a polybasic cleavage site. They cite the canonical polybasic furin cleavage site to be "RRSRR" and replace the single "R" with that longer sequence: https://www.sciencedirect.com/science/article/pii/S0042682206000900?via%3Dihub The paper effectively shows exactly what we might expect to see in the case of an engineered virus; a known backbone (SARS-CoV in this case) with a known canonical site added to it. Had SARS-CoV-2 looked more like this, it would lend more weight to arguments of the presence of human design.

[60] https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30418-9/fulltext

[61] We have seen, with other viruses, the ability to develop polybasic cleavage sites when put under just the right conditions for long periods of time. While unlikely, this piece of the virus could plausibly be developed through selection in a lab setting. However, what is near impossible is the development of the o-linked glycan addition motif. This is because the pressure to develop this glycan shield requires avoiding an intact immune system. This type of selection cannot occur using cell culture, and there is no known animal model that would allow for selection of human-like ACE2 binding and avoidance of immune recognition. This strongly implies SARS-CoV-2 could not have been developed in a lab, even by a system of simulated natural selection.

[62]While not discussed in this episode - another argument is: “the RBD of SARS-CoV-2 is optimized for binding to human ACE2 with an efficient solution different from those previously predicted.”This is strong evidence that SARS-CoV-2 is not the product of purposeful manipulation. " https://www.nature.com/articles/s41591-020-0820-9 

[63] PREPRINT: https://arxiv.org/pdf/2004.02158.pdf “Our results also indicate that the SARS-CoV-2 is unlikely a lab engineered virus.”

[64]  All current evidence supports the natural emergence of SARS-CoV-2 via a bat and possible intermediate mammal species. https://www.dhs.gov/sites/default/files/publications/mql_sars-cov-2_-_cleared_for_public_release_2020_04_07.pdf

[65] Scientists estimate that more than 6 out of every 10 known infectious diseases in people can be spread from animals, and 3 out of every 4 new or emerging infectious diseases in people come from animals.

[66] It’s estimated that 75 percent of emerging human pathogens are zoonotic in origin. (Note that this paper is from 2001 -- we’ve known this for a long time)

[67]  All samples from swabs (e.g. oral, urine, rectal), fluids (e.g. saliva)

[68] https://academic.oup.com/ve/article/3/1/vex012/3866407; We identified sequences representing 100 discrete phylogenetic clusters, ninety-one of which were found in bats, and used ecological and epidemiologic analyses to show that patterns of CoV diversity correlate with those of bat diversity.

[69] In one cave there were a more than one million Mexican free-tailed bats: https://www.researchgate.net/profile/Donald_Mcfarlane/publication/272169859_A_proxy_population_record_for_the_Mexican_Free-tailed_bat_at_Eagle_Creek_Cave_Arizona/links/54dd30ba0cf28a3d93f89605.pdf

[70] In Thailand, a total of approximately eight million individuals of the wrinkle-lipped free-tailed bat, Tadarida plicata from 17 caves were reported, making it the most abundant mammal in Thailand...

[71]  https://sci-hub.tw/https://link.springer.com/chapter/10.1007/978-3-540-70962-6_13 The genetic diversity observed among bat-derived SL-CoVs together with the high prevalence and wide distribution of seropositive bats, as revealed by two independent groups, are consistent with bats being the wildlife reservoir host of SL-CoVs

[72]  Although no animal coronavirus has been identified that is sufficiently similar to have served as the direct progenitor of SARS-CoV-2, the diversity of coronaviruses in bats and other species is massively undersampled.

[73] First spillover event for Ebola was in 1976 https://www.fic.nih.gov/News/Pages/2016-ebola-outbreak-1976-lessons-relevant.aspx

[74] Researchers believe that Ebola virus is animal-borne (zoonotic) pathogen, and that bats are the most likely natural reservoir. However, the exact reservoir species that harbors the virus has yet to be determined.

[75]Search for the Ebola Virus Reservoir in Kikwit, Democratic Republic of the Congo: Reflections on a Vertebrate Collection...Animals were identified at the Museum Alexander Koenig, Bonn, Germany (shrews);

[76] Search for Ebola Virus in Animals in the Democratic Republic of the Congo and Cameroon: Ecologic, Virologic, and Serologic Surveys, 1979–1980: A total of 1664 animals representing 117 species was collected, including >400 bats and 500 rodents.

[77] In the years following the 1976 ebola virus outbreaks, trapping and bleeding of thousands of

animals and insects failed to identify a reservoir.

https://sci-hub.tw/https://doi.org/10.1146/annurev-pathol-052016-100506 

[78] The sequenced full genomes of NeoCoV, the novel betacoronavirus that was isolated from vesper bats in South Africa and MERS-CoV strains from dromedary camels and humans confirm that they all belong to the same species. NeoCoV is at the root of their phylogenetic tree, with evidence of genetic evolution of MERS-CoV in camels before humans. Furthermore, the high similarity of NeoCoV and MERS-CoV with genetic divergence in NeoCoV spike gene suggests that a recombination event within this region may have resulted in the emergence of MERS-CoV
[same source] MERS-CoV RNA and viable virus have been isolated from dromedary camels, including some with respiratory symptoms. Furthermore, near-identical strains of MERS-CoV have been isolated from epidemiologically linked humans and camels, confirming inter-transmission, most probably from camels to humans...Phylogenetic and sequencing data strongly suggest that MERS-CoV originated from bat ancestors after undergoing a recombination event in the spike protein, possibly in dromedary camels in Africa, before its exportation to the Arabian Peninsula along the camel trading routes.

[79] We identified the near-full-genome sequence (29,908 nt, >99%) of Middle East respiratory syndrome coronavirus (MERS-CoV) from a nasal swab specimen from a dromedary camel in Egypt. We found that viruses genetically very similar to human MERS-CoV are infecting dromedaries beyond the Arabian Peninsula, where human MERS-CoV infections have not yet been detected.

[80] Persons in Saudi Arabia with occupational exposure to camels demonstrated higher seroprevalence of MERS-CoV–specific antibodies (camel shepherds, 2.3%; slaughterhouse workers, 3.6%) compared with the general population (0.2%). A case–control study of primary human cases and matched controls also showed that camel exposure was more likely among case-patients than matched controls. Further evidence supporting camel-to-human transmission includes identical or nearly identical MERS-CoV sequences found in camels and humans

[81] NiV is a paramyxovirus that emerged in people in Malaysia in 1998 [7–9]. …..During the course of the outbreak investigation, nine cases of NiV encephalitis were retrospectively diagnosed in the Tambun area with onset dates between January 1997 and May 1998, five of which were associated with the index farm of the 1998 – 1999 outbreak in Perak (figure 1a). Six of the nine cases are considered confirmed cases on the basis of serological tests performed in 1999 [16]; the remaining three are probable cases.

[82] Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia 

[83] https://science.sciencemag.org/content/288/5470/1432.long

[84]  The origins of the outbreak were in a large, intensively-managed pig farm in Ipoh, Malaysia, where the first known pig and human cases occurred as early as January 1997, prior to the large outbreak in the Nipah region of Malaysia (Field et al., unpublished data). ...Both viruses appear to have fruit bat (Pteropus species) reservoirs, and both initially emerged via domestic animal amplifier hosts (horses for Hendra virus; pigs for Nipah virus),

https://sci-hub.tw/https://link.springer.com/article/10.1007/s11908-006-0036-2 

[85]  In phase I, early human cases were directly linked to the transmission of virus from bats to pigs, with all identified cases occurring in the immediate vicinity of the index farm where bat-to-pig transmission ignited rapid pig-to-pig transmission and produced a tight cluster of human cases on the index farm and an adjacent property. https://royalsocietypublishing.org/doi/pdf/10.1098/rsif.2011.0223 

[86] Transmission from flying foxes to pigs is thought to occur via saliva on fomites (discarded fruit pulp) or via faecal or urine contamination of pigsties https://royalsocietypublishing.org/doi/pdf/10.1098/rsif.2011.0223 

[87] ….the growth of large intensively managed commercial pig farms in Malaysia with contaminated fruit trees in proximity of the farms, created an environment, where bats could drop partially eaten fruit contaminated with NiV, laden bat saliva into pig stalls. The pigs would eat the fruit, becoming infected with NiV, and efficiently transmitted virus to other pigs.

[88] Research Institutes - Institute of Infection, Immunity & Inflammation - Staff AZ - Mr Oscar MacLean

[89] The Guangdong Wildlife Rescue Center received 21 live Malayan pangolins from the Anti-smuggling Customs Bureau on 24 March 2019; most individuals, including adults and subadults, were in poor health, and their bodies were covered with skin eruptions. All these Malayan pangolins were rescued by the Guangdong Wildlife Rescue Center, however, 16 died after extensive rescue efforts. Most of the dead pangolins had a swollen lung which contained a frothy liquid, as well as the symptom of pulmonary fibrosis, and in the minority of the dead ones, we observed hepatomegaly and splenomegaly. We collected 21 organ samples of lung, lymph, and spleen with obvious symptoms from 11 dead Malayan pangolins to uncover the virus diversity and molecular epidemiology of potential etiologies of viruses based on a viral metagenomic study.

[90] In our study, organ samples of lung, lymph and spleen were collected from dead Malayan pangolins at the Guangdong Wildlife Rescue Center

[91]  first detected the existence of a SARS-CoV-like CoV from lung samples of two dead Malayan pangolins with a frothy liquid in their lungs and pulmonary fibrosis

[92]  The amino acid phylogenetic tree showed that the S1 protein of Pangolin-CoV is more closely related to that of 2019-CoV than to that of RaTG13.

[93]  the receptor binding domain (RBD) of SARS-CoV-2 and RaTG13 are only ∼85% similar and share just one of six critical amino acid residues….The Guangdong pangolin viruses are particularly closely related to SARS-CoV-2 in the RBD, containing all six of the six key mutations thought to shape binding to the ACE2 receptor and exhibiting 97% amino acid sequence similarity (although they are more divergent from SARS-CoV-2 in the remainder of the genome).

[94] Smithsonian Tropical Research Institute-Smilisca sila Duellman and Trueb 1966: Males call from the edges of forest streams

[95] Interview with Dr. Ximena Bernal, co-author of this study: “Males of this species, however, are also preyed upon by frog-eating bats (Trachops cirrhosus) and frog-biting midges (Corethrella spp.), both of which use calls to localize the frogs”

[96]https://sci-hub.tw/10.1007/BF00300101

[97] Recordings courtesy of Henry Legett, corresponding author of this study

[98] Interview with Dr. Ximena Bernal, co-author of this study: “Both bats and midges, the non-target eavesdropping receivers, had a significant preference for leading pug-nosed treefrog calls over following calls. Frog-eating bats chose the speaker broadcasting leading calls on average 70% of the time”

[99]  Interview with Dr. Ximena Bernal, co-author of this study: “...pug-nosed treefrog choruses are characterized by long periods of silence punctuated by short, sporadic bouts of synchronized calls

...Whether certain males within the chorus always end up leading in this game is currently unknown.”