TWiV 364

Vincent Racaniello 0:00
This Week in Virology, the podcast about viruses, the kind that make you sick

From Microbe TV, this is TWiV this week in virology, Episode Number 364. recorded on November 20 2015. Hello, everybody. I’m Vincent Racaniello. And you are listening to the podcast all about viruses. Joining me today from southeastern Michigan Kathy Spindler. Hi, everybody. How you doing there, Kathy? And great. Thanks for doing this. And it’s an unusual hour again 1030 in the morning. So our feng shui is all messed up, but we’ll deal with it. Also joining me from North Central Florida Rich Condit

Rich Condit 0:55
Hi, not really. I’m in Austin, Texas.

Vincent Racaniello 0:58
Yeah, that’s right.

Rich Condit 0:59
My home is in North Central Florida, but I’m in Austin, Texas. And yeah, I’m even an hour earlier than you so but my feng shui is just dandy. dandy. Let’s roll.

Vincent Racaniello 1:13
All right. Today we have two special guests to discuss a paper published very recently, online was published ninth of November. This paper has generated a lot of press, in my opinion, much too negative, but we’ll let the author’s Tell us about that. We have the lastin first authors in the name of the papers as far as like cluster of circulating bat coronaviruses shows potential for human emergence. Joining us, he’s a professor at the University of North Carolina Chapel Hill, Ralph Baric. Welcome, Ralph.

Ralph Baric 1:48
Hello. Hey, Vince.

Vincent Racaniello 1:49
Thanks for joining us. And the first author on the paper is a postdoctoral fellow in Ralph’s lab, Vineet Menachery Thank you Welcome Vineet Did I pronounce it terribly?

Vineet Menachery 2:02
A I’ve heard much worse

Vincent Racaniello 2:04
manachery or menachery, is it menachery right?

Vineet Menachery 2:07
Menachery.

Vincent Racaniello 2:08
Thank you. I completely understand mispronounce names. As you might imagine. This episode of twit is brought to you by the Department of microbiology at the Icahn School of Medicine at Mount Sinai, composed of over 20 virology labs. All in one building in the heart of New York City. This department is a perfect fit for anyone with an interest in pursuing virus research. Departments presently looking to recruit prospective graduate students to apply to the program. The deadline is December 1. Not too far off, but you do have some time. If you’re looking for a postdoc, you’re encouraged to check out their website and contact your faculty of interest directly. For more information about the department, visit www.ssm.edu slash M I see. Once again, MSS m.edu slash NYC. Department of microbiology at Mount Sinai in New York City is where I got my PhD. Many many years ago, I graduated in 1980 20, 35 years ago, I loved that I worked for Peter palese. I was his first PhD student. And since then the department has gotten better, lots of great virology as well as immunology. They’re great place in New York City, and a big booming Medical Center. So if you want to do virology research, check it out. We appreciate their support of twith. I know Ralph has a has to leave in about 30 minutes and Vineet will will stay with us for a while. So let’s dive into this. And maybe, Ralph, you could start by telling us what were the little background for this paper and what were the goals of the experiment.

Ralph Baric 4:00
So one of the fundamental problems in emerging virus research is that viruses exist within animal species as a heterogeneous population of related viruses. In the case of the SARS like viruses, these can vary up to 50%. From the sequence of the SARS epidemic strain that emerged in 2003 caused 8000 infections and 800 deaths about 800 deaths and disappeared from human from from human infections around 2004 2005. But the problem with emerging viruses is that they they can re emerge and they do reemerge periodically over time from this animal reservoir. And when they do they’re not usually the same. The sequences are different. So that vaccines and drugs that were developed against The original epidemic strain may not be effective at all. So if you’re interested in developing strategies to protect the public health, against future emerging strains, you have to have an idea of the high risk potential strains that have high risk potential, that are circulating in the animal reservoirs. And so the goal of this paper was to try to identify strains that were existing in nature that had the capacity to replicate in humans. And that were variable from the 2003 epidemic strain. And if we could resurrect those strains and identify them, we would be much better positioned to to develop and design and test vaccines and antivirals so that they would have greater breath and protect against future outbreaks.

Vincent Racaniello 5:56
And my understanding is that you were interested in the Coronavirus as in bats that were SARS like, right?

Ralph Baric 6:03
That’s correct.

Vincent Racaniello 6:04
And you have for these a lot of sequences, but you don’t have isolate virus isolates. Is that also correct.

Ralph Baric 6:09
So over the past decade, there have been tremendous advances in meta genomics. And so many groups have been sequencing, in essence, bat guano or various bat parts, looking for viruses that are harbored within a bat population. And there’s about 1300 different bat species. And the current estimates based on that meta genomic analysis is that each bad species has somewhere between five and eight, novel bat Coronavirus. Some subset of those are group 2b coronaviruses that are related to SARS. And so over that past decade, sequences have emerged that range from 97% identical to SARS to 50%. Identical and that specifically within the spike glycoprotein. Yes, go ahead.

Rich Condit 7:01
The focus on bats is because one way or another depending on what story you subscribe to, but one way or another. SARS was supposed to have emerged from bats, right?

Ralph Baric 7:15
That’s correct. So the original hypothesis was that SARS was a bat virus that was spread into civets and raccoon dogs in the open markets of Southeast Asia, adapted to those species in the open markets which were being used for foodstuffs, and then transferred the virus adapted to humans. We actually think and several others have reported an alternative hypothesis is that SARS was originally a bat virus that jumped immediately into humans, and then adapted to civets in the open markets where a transmission cycle was set up. Obviously, in the first model, it’s a two step event requiring mutations at to adapt first of the civet and then mutations to adapt to the human. So it seems like future events would be unlikely. The alternative hypothesis was that viruses were harbored within bats that had pre potential for pre poised to replicate efficiently in humans. And perhaps civets, and so they could move directly from bats to humans. And then in within, in interacting with the civets in the open market set up a transmission cycle to maintain the virus in human populations. We actually, my own personal hypothesis is that the second model is more correct, that the bats actually harbor some subset of viruses that are generalists. That can use ACE2 molecules, ACE2 is angiotensin one converting enzyme two, which is the receptor for SARS docking and entering cells. And we think there are bat strains that can use the bat ACE2 molecule, the human ACE2 molecule and the civet ACE2 molecule with with high efficiency, and hence those viruses would be prepositioned to or high risk in terms of their ability to initiate the new epidemic.

Vincent Racaniello 9:14
Now in your paper, you use the bat virus called SHC014, why why did you pick that one? A sequence I should say because you didn’t have actually have the virus right?

Ralph Baric 9:27
We didn’t have the virus. In fact, no one has ever cultured the virus it only existed in in silico, in essence, as a full length sequence. So when you model the, and Vineet did a lot of this work in collaboration with some other postdocs when you model the receptor binding domain and you look at the residues that are present that interface with the ACE2 molecule bats civets or humans. There is some conservation. But there were seven of those residues were different, including I think five or six that occurred at really important sites for interaction. We knew from some studies in our lab that at least one of those sites were tolerated in terms of being able one of the mutations in that civet in that in that strain as SHC014 could occur within the context of a wild type virus, and still program efficient entry into human cells. So that was interesting to us. We were also interested in the strain because it was about 12% different in the spike glycoprotein sequence. And so it may function as a bookend. In other words, you know, if you’re trying to identify what cluster of strains were high risk that could use the human ACE2 receptor, this might be at the boundary of that of that cluster. And the reason that’s important is if you’re developing vaccines or drugs, or anti over immunotherapy immunotherapeutics against against SARS, like viruses, you really want to capture the maximal amount of diversity, that still would program efficient entry into human cells. Because those would be the most high risk. So it had a fair amount of variation. There was some conservation in the in the receptor binding interface sites, there was variation within some of those sites, at least one of those we knew probably could function and still mediate efficient interaction with the human ACE2 molecule. And so it was worth a try. There were other things we could do with that spike as well. Even if it didn’t program. In fact, we could use it to look at a cross neutralization, if we made antisera against that SHC014 spike could it neutralize SARS, that would be another thing we could do. If the spike was not capable of programming infection program, capable of programming infection in mammalian cells. We can use antigenic cartography, those types of things. So there’s a lot of things we can do with that spike. Go ahead, Rich. Sorry.

Rich Condit 12:12
I want to make sure I have my context right for the spike. glycoprotein. So this is the thing that sits on the outside of the virus and gives it its crown appearance, hence Coronavirus, right. And it’s the thing that attaches the virus to the receptor on the cells. Is it solely responsible for entry does it do fusion as well? Or are there other proteins involved?

Ralph Baric 12:42
The spike the spike glycoprotein is a and its interaction with cellular components is a major barrier for cross species transmission of coronaviruses. And it occurs it at least two levels. The first is the ability of that spike to program it’s the its ability to bind to receptors on the surface of cells of different from different species. So in the case of ACE2 for example, the SARS epidemic strain can bind to the bat ACE2 receptor, the civet ACE2 receptor and the human ACE2 receptor. It binds poorly, for example, to the rat ACE2 receptor the second major and so that first interaction event is key for virus binding to the cell and that begins to program the downstream steps that are critical for virus entry into that cell. The second major barrier for Coronavirus entry and cross species transmission is proteolytic cleavage of that spike. And so, the Coronavirus spike is generally broken into two pieces, and S1 N-terminal domain of about 90 kilodaltons and a C-terminal 90 kilodaltons domain proteases. And there are four or five depending on who’s doing the study different proteases have to cleave that spike to cause conformational rearrangements to expose a fusionogenic domain and that fusionogenic domain that then fuses the envelope with a virus to the surface of the cell. So over the surface of an inter intracellular vesicle to mediate entry into that cell. In the case of coronaviruses, there are proteases that that can mediate that event at the surface of the cell, and intracellularly. And so depending on the spike, the cleavage events may occur on the surface to mediate fusion to the plasma membrane or fusion to internal vesicles internally with the intercellular.

Rich Condit 14:44
Go ahead. So those are those are host proteases. And this proteolysis happens in the process of entry not before like flu, okay, okay, great. Got it.

Ralph Baric 14:57
So for example, in collaboration with a researcher at University of Minnesota named Fang Li. Fang Li has shown that HKU4 which is a relative to MERS Coronavirus that HKU4 strain can bind to the same human receptor that MERS does which is dipeptidyl peptidase-4 and it binds very well to the human DPP4 molecule. However, the barrier for entry is not in, in receptor binding. The human proteases can’t cleave the S2 spike and can’t activate the fusion domain. So the major barrier for HKU4, and the reason it’s not infecting them themselves is because of proteases host proteases not being able to process the spike appropriately.

Vincent Racaniello 15:46
So I think in the interest of time, we should continue talking with you, Ralph. And then we’ll we’ll have Vineet go back and go through the experiments in some detail. But your strategy was to take a human SARS Coronavirus strain and substitute it spike with that of SHC014. And you found that this this could infect human cells and mice. Now that that’s part of what’s generated a lot of controversy about this paper because because you made a new virus which and Simon Wain-Hobson said this virus doesn’t exist in nature, and who knows what would happen if it got out? And so some people have said, Why didn’t you simply make VSV pseudotyped with the spike glycoprotein of this virus?

Ralph Baric 16:36
So pseudotyping, is a common strategy strategy that’s used in viruses to ask particular questions about the functions of surface glycoproteins. And we did experiments with pseudotyping. So in collaboration with our, with our collaborators at Wuhan University, we pseudotyped lentiviruses with the SHC014 spike, and some other spikes from other bat coronaviruses and human coronaviruses. The other spikes could program entry into human cells and the SHC014 spike could not for that data actually suggested that SHC014 would not mediate efficient infection into human cells. Now, there are a variety of problems with pseudo typing experiment experiments. The first is that you don’t actually know whether the spike distribution on the surface is appropriate. You don’t know if the number of spikes on these are the surface glycoprotein with a number of those spikes on the surface of that pseudotyped particle are in the right organization. And you don’t know if they’re if they are functional in that context. And so, a positive is gives you some level of assurance that a spike and program entering into the cell. A negative doesn’t necessarily mean that the by the spike is not capable of mediating infection. So we felt that it would be prudent on our part to more rigorously test that and so we made the chimera.

Vincent Racaniello 18:22
So Simon Wain-Hobson has said that if this virus got out, you couldn’t predict what would happen in people. How do you address that?

Ralph Baric 18:32
So number one, it’s unlikely that the SHC014 spike, which as far as we can tell has never circulated through human populations would be capable of programming as serious an infection as the SARS epidemic strain was capable of doing. We have no evidence no idea no understanding of Coronavirus transmission or the genetic make the genetic factors that regulate Coronavirus transmission. Since SHC014 had never been transmitted through human populations, it would be unlikely that it would be as efficiently programmed for causing transmission between humans. SARS in all of its glory, both in 2003, and in 2005, four and five was controlled by public health interventions strategies through quarantine contact contract, contact tracing, was able to stop the epidemic to stop the reemergence of the virus in 2004. Third, I think what many people don’t understand is the Coronaviruses undergo recombination at fairly high frequencies. So in any kind of mixed infection 30% of the progeny can be recombinant. And in the case of many of the bat viruses in the meta genomic sequence databases. There is lots of evidence that recombination has affected the population genetic structure of Coronavirus family. And in fact, modular evolution occurred where individual genes can be swapped from one strain to the next. Not only not only the full length gene but also functional component parts of the Coronavirus spike can be transferred from one strain to the next. So, even the receptor binding domains, to some extent may be interchangeable among closely related strains. And since bats many some bats not all bats, obviously but but some bats live in these large populations, with several species co inhabiting with them harboring up to five or eight different bat coronaviruses at a time. recombination is fluid in Coronaviruses and the spike is is well adapted to handle recombinant molecules. So anyone’s argument that these kinds of events can’t occur is basically not knowledgeable about Coronavirus. So these this, this isn’t something that would not ever occur in nature. It probably occurs in nature and can occur quite frequently.

Vincent Racaniello 21:24
What kind of containment do you use to do these experiments?

Ralph Baric 21:28
So we use BSL3, we work under BSL3 settings. This is a high containment research lab under negative pressure. All the researchers in the lab wear PAPRs and Tyvek suits these are personal air breathing apparatuses that scrub the air of virus particles so they are in essence in containment suits. All research is done in a safety cabinet what’s called a BSL2 safety cabinet. The virus is not free outside of that cabinet in any moment. And then we have fairly vigorous reporting standards in terms of infection control, that anyone who does come down with a cold immediately reports to occupational health and safety for them see a group of physicians as well as EHS officers become engaged and track the infection and in these individuals, and most of the time, this is like common cold. In fact, every single time it’s been a common cold. And then, in some instances, they may be quarantined at home for two or three days. In other instances, if they don’t break fever will just be watched. And if a serious infection occurs, they can be hospitalized. So sorry, Yes, go ahead.

Rich Condit 22:56
So these experiments began before the gain of function moratorium, if I understand it correctly, and you’ve had some communication with NIH during the course of the experiments since Can you describe that?

Ralph Baric 23:12
So we had submitted paperwork to do these experiments, I believe, in April of 2014, and started the experiments in May of 2014. We had actually contacted our program officer under which this grant under which we were doing these experiments in the summer of 2014, to talk about them in more detail. When the moratorium hit, we immediately called them to talk to them about what our responsibilities would be under this under this pause. And I had several grants at the time and they so that different things happen. So for example, we were one of the groups that were asked to develop mouse models to MERS Coronavirus, which required passage in the mouse and those experiments were immediately halted until appropriate paperwork could be submitted. And then we were released to continue those experiments right at right before Christmas of 2015. We had other grants that were funded that they would not release the funds until the paperwork associated with experiments that NIH had identified as potential gain of function were addressed to a committee’s satisfaction, the committee that actually makes these decisions, I am not privy to its organization or how they deliberate. So it’s a black box to the researcher. And they will either decide that experiments do not represent a gain of function. They will or they will decide that this could be a gain of function. And they will ask for a risk assessment in terms of what we would do. So for example, if the SHC014 virus had replicated more efficiently than wild type SARS virus in human airway epithelial cells, in terms of about a log titer, or was more pathogenic in mice, the way we were given approval to do these experiments, they, we would immediately if either of those events happened, we would immediately notify our Environmental Health and Science Department and the IBC, we would then immediately notify NIH, we would stop all experiments and a group would be convened to make a decision about how to proceed, that decision would be obeyed to the letter of the law. So if that group had decided that these experiments were too dangerous to proceed, then and they wanted us to destroy it, then we would destroy it. And that would have been the end of the experiment. Fortunately, in this case, SHC014 replicated depended the full on virus replicated less efficiently in human airway epithelial cells. And, and even the chimera was attenuated in mice, and it didn’t replicate any better than a wild type SARS. So that data suggested that we really hadn’t gained function.

Vincent Racaniello 26:25
In your, in your discussion, you write that the data are at a crossroads of gain of function research concerns, maybe in the last few minutes that you’re here with us, maybe you could expand on that a bit.

Ralph Baric 26:41
So at this point, the actual the final rules that determine gain-of-function have not been established and are being deliberated by a group. I believe the NSABB, but I’m actually less sure exactly what the group is the makeup of the group that’s making that decision.

Vincent Racaniello 27:05
I am too I have no idea.

Ralph Baric 27:09
So So anyway, that group is going to come down with a series of rules and regulations that people are going to have to follow the boundaries of which I don’t believe has been set have been set. So it’s quite possible that these experiments would never be allowed at all. And I think that would be tragic, because the only way that that so we have a resource problem with emerging pathogens in it. And this is the same resource problem with SARS. And with MERS and with Ebola, or Nipah, or Hendra virus, these viruses are highly heterogeneous, they exist in nature, they spill out at periodic intervals, and they cause significant morbidity and mortality and economic hardship. A single vaccine is not going to solve the problem. A single human monoclonal antibody therapy is not going to solve the problem. We have to have the reagents in hand that allow us to develop broad based therapeutics and vaccines and drugs. And we can develop drugs. But if we don’t have highly variant viruses to test them against, we’ll never really know whether they work until an outbreak occurs. And so that leaves I think the research community, in kind of a catch 22 we have two choices. We can allow. We can take a reactionary response, or a reactionary approach, where we wait for outbreaks to occur, families lose their loved ones. And the research community then initiates massive and parallel efforts to develop a drug and a vaccine that would work against this new unknown. That’s a very painful setting for humanity. And that’s the standard that has been used for responding to new outbreak disease outbreaks, for most of our existence on this planet. The other approach is to take a proactive response where we try to identify representative strains within this family, or this swarm of related viruses. And as we develop drugs and vaccines, we now have reagents to ask whether they have breadth of activity. And so we actually have some very interesting data with new drugs that may work in SARS and MERS. And it looks like we’ll be testing soon whether they would work against these related group 2b SARS like viruses. And if that it turns out if they turn out to be effective, then we actually will have a reagents on the shelf that could be available to respond in future outbreaks. If they don’t work. We have reagents now available so that second generation compounds could be modified and targeted for that they’d be more entire cluster. And so we’re interested in in that as a conceptual approach to provide an alternative future, in the case of an outbreak of highly pathogenic respiratory coronavirus.

Vincent Racaniello 30:32
Seems to me that as science progresses, we have to change our approach, as you say, you know, we don’t use the old approach we, we get proactive in your paper, and we’ll hear the details from Vineet. In a moment, it’s so beautifully shows that there’s a spike out there in bats spike genes that are not neutralized by monoclonals they’re not taken care of by vaccines, SARS vaccines, and we need to know what’s out there. And as you said, make broadly active inhibitors, it seems to me that anyone who says this, this work isn’t doesn’t tell us anything simply hasn’t read the paper or doesn’t understand it.

Ralph Baric 31:13
I agree completely. And I think it’s the other thing that needs that, sort of that, that individuals need to think about is that in the 21st century, we’ve had two major new emerging highly pathogenic respiratory coronaviruses appear in human populations, SARS coronavirus is one and MERS coronavirus is currently the second one and that outbreak is still ongoing. In the animal world, porcine epidemic diarrhea virus, which emerged 20 years ago, 25 years ago and slowly moved across Europe became highly pathogenic in the early 2000s, in Southeast Asia. It then spread to the US in 2013. In the US, it killed 8 million pigs in the span of, you know, just several months. All told the viruses killed a billion swine since it arrived in Southeast Asia. So the pathogenic potential and the scope of an outbreak from a highly pathogenic Coronavirus is potentially enormous. And from a policy point of view, it’s critical to know that there are viruses in nature that are pre programmed to move fluidly from one species to the next. And if you know that, you’ve know you need to begin to at least put some resources into developing effective drugs and vaccines. And there are no effective drugs and vaccines against SARS or MERS. And that’s the situation we’re in 12 years after the SARS epidemic emerged in 2003. And I think that’s unacceptable.

Vincent Racaniello 33:02
Ralph, I want to thank you for joining us. I know you have to go now. I’m arranging a trip to Chapel Hill next year, maybe we can sit down and talk even longer about variety of interesting virological questions.

Ralph Baric 33:15
I look forward to that.

Vincent Racaniello 33:16
Thank you, Ralph. Okay, bye bye.

Ralph Baric 33:19
Thanks Rich. Nice talking with you.

Rich Condit 33:20
Thanks. Bye, Ralph. Thanks. Bye bye.

Vincent Racaniello 33:24
This episode of TWiV is also sponsored by ASM education department, grad students, postdocs and early career scientists are invited to apply to attend the ASM scientific writing, and publishing webinar series. This is a three month six part program that takes place from January through March of 2016. So it’s a webinar you can stay in the comfort of your home or office and learn great things about writing. You’ll learn from ASM journal editors about the whole scientific writing and publishing process. These are people who have been involved in this for many years and know what they’re talking about. And the things that we’ll be discussing include titles and abstracts, how to write them and make them attractive figures, figure legends, and the whole manuscript review process. Once again, it’s a three month six part series two parts a month from January through March of 2016. They are taking applications for the program you can find more about it at bi t.ly slash s WP AI online 15 all one word, bi t.ly slash s WP AI online 15. We thank ASM education for their support of TWiV. Alright, Vineet.

Vineet Menachery 34:51
I’ll move over into the chair for the moment.

Vincent Racaniello 34:54
Let’s circle back to that first series of experiments with the SHC014 spike in the background of SARS, tell us what you did there.

Vineet Menachery 35:07
So, as Ralph said, we have these spikes and our collaborators of Wuhan Institute have developed, they’ve isolated a number of sequences. So I think in their original paper in 2013, they reported 18 spike sequences and a couple of full length viruses, one SHC014, one they called WIV1, which was the virus they were able to isolate, that virus was able to use human ACE2. And so that’s, that’s the clinical isolate that they were able to actually isolate out of the bat guano, which, for those of you who have ever worked with that, it’s really an amazing thing that they were able to isolate that virus at all. Building from that SHC014 is the next virus down. In terms of closeness to the epidemic strains, it’s about 12% different. And when we first started looking at the strain, we had looked at the modeling and we have a number of people in our group, and other groups that have done modeling of the RBD. A lot of those studies were based on escape mutants and other interactions, the majority of the antibodies that have been generated against SARS, monoclonal antibodies that have been thought to be therapeutic or used for therapeutic purposes, focus in the receptor binding domain. And so a lot of the changes that we see there, we actually think we understand the interactions pretty well. And so, when we look at SHC014, there are 14 contact residues that are thought to interact between SARS spike, and the human ACE2 molecule. Of those 14, SHC014 has changes at seven of those positions. And of those 14, there are five that have been shown to be critical for binding and species, species specificity. All five of those had been changed in SHC014. This is in contrast to what we saw with WIV1, WIV1 only has three changes in those 14 residues. And of those three, they’re actually very conservative substitutions. And so we weren’t, we weren’t particularly surprised that WIV1 could bind, we would have probably potentially predicted that. And our collaborators at Wuhan shared with us their pseudotyping data, where they’d taken in the spikes, and using a lentivirus system had shown that with their SARS wild type spike, with the WIV1 spike that the virus could enter using human ACE2. And it showed pretty clearly that SHC014 in the pseudotyping experiment could not. And that was they use HKU3, which is at the other end of the group 2b spectrum in terms of the depth of the width of the Coronavirus group. And so from those studies, all of the predictions are that this virus shouldn’t be able to grow. And so

Vincent Racaniello 38:04
in human cells, you mean

Vineet Menachery 38:05
In human cells in using human ACE2 to at a minimum, and they’ve used human and then their pseudotyping experiments, they use a number of orthologs. And I can’t remember if they use bat, but for what they found was they had no evidence that ACE2 bat, civet, or human could use that, it could use the ACE2 molecule. And that kind of made sense based on the fact that there are seven amino acid changes. Now, Ralph mentioned there are changes that we see in SHC014 that we’ve seen in strains that combined humans to a couple of those mutations had been seen in civets, civet strains, but as individual so of the 14, we’ve seen one change that corresponded to the SHC014 but that’s one out of the seven. So there is evidence that binding could happen, but I don’t know that it was, was or is conclusive in that way. And so when we made these viruses, it was under the premise that it is probably likely that this virus wouldn’t grow. It doesn’t mean that we couldn’t use it for other aspects. And Ralph talked about the cartography and making, you know, making antibiotic or making vaccine responses against this spike would actually be interesting, because it may actually lead to antibodies that are outside of the receptor binding domain that may actually be cross protective against the entire group. And so that was the premise of the studies. And so we built the chimeric virus with most Ralph and I had a discussion on whether we thought that the virus would work or not, I, when I was originally doing the experiments, I wasn’t thinking that it would. And so when we were able to recover virus from the Veros that we transfect into, it was surprising to me. Ralph claims that he wasn’t surprised

Rich Condit 40:00
So, so the chimeric virus you’re talking about is that you’re taking the SCH014 spike protein. Yep. And swapping that into a mouse adapted Coronavirus swapping the spike protein on the mouse adapted Coronavirus for with this SCH014 virus spike protein. Correct?

Vineet Menachery 40:24
Correct. That’s that’s exactly right

Rich Condit 40:26
so so theoretically, you’re confining your attention there primarily to whether the spike protein is going to function in that context for binding an entry of a virus that is otherwise already adapted to the mouse. Right?

Vineet Menachery 40:45
Yeah, yes. And I think that’s an important point. When we think about the spike, a lot of a lot of the focus after the outbreak was and, and continue, and rightly so is on the ability of the virus to bind to the receptor. But Coronaviruses are really large they’re the largest RNA viruses, there’s, you know, 28 to 30 kb. And, for a number of reasons, the backbone may not actually support replication and in human cells itself. So this separates out whether the spike can mediate entry, and whether the whole virus so there may be aspects of the backbone of SHC014 that make it unable to grow, whether it be controlling the host immune response, or some interaction with cellular machinery, that the full length virus may not have been able to grow. But it has actually nothing to do with the spike protein. And so this, this controls for that in a way. And if we had known as SHC014 if we had predicted that it would grow, there there is value in making the chimera as well. I mean, we talked about that a little bit more as we go through the data.

Vincent Racaniello 41:53
So why did you decide to use the mouse adapted strain? Did you already think, well, we could do some mouse experiments if it does grow?

Vineet Menachery 42:00
Well, yeah. So I mean, I think, well, a number of reasons. The mouse experiments doing mouse experiments is gives us a lot of information on pathogenicity, it also allows us to ask questions, if the virus does grow, but doesn’t grow particularly well, we can ask questions about using an SHC014 based virus as a vaccine. It also while we don’t have any hard evidence for this, in general, we do believe that adaptation to the mouse is not particularly helpful to the human. So we’ve done the studies where we’ve compared the mouse adapted strain to the wild type strain in human airway epithelial cells, we don’t see a marked difference between them. If anything, the mouse adapted strain is not efficiently. It’s, it’s always less. But it’s twofold, threefold, fivefold differences that are not virologically important. And so we don’t have any, we don’t have any reason to say that this virus is, is more virulent. And we don’t have the data to say that it’s less virulent in humans, you know, the adaptational way? Yeah, unfortunately, the only way you could really do that is maybe non human primate studies or I don’t I don’t know that anybody’s volunteering for human challenge studies. But

Rich Condit 43:14
So this this mouse adapted strain that’s it’s a SARS strain that was adapted to mice, is that correct?

Vineet Menachery 43:21
Yep. So isolate of SARS was taken and passaged into mice. MA15 is the name of it, it was passaged 15 times and then gain virulence in BALB/c mice. And that is the strain that we almost always use on our mouse experiments. So in a BALB mouse, one times 10 to the fifth that on a 10 week old mouse will result in lethality. And that varies based on genetic background. So black six mice at 10 weeks are resistant. And there are elements of this model. As you age the mice, a year old mouse is much more susceptible, much lower LD50. And so there are elements that this SARS model recapitulate both the human disease aspects and are affected by both genetic components as well as aging components.

Rich Condit 44:07
And do you know what the genetic correlates of that adaptation are.

Vineet Menachery 44:12
So there are six amino acid changes or six, there are six changes. There’s one in spike, so there’s a Y436H mutation, which we think increases the affinity of the virus to the mouse receptor. It doesn’t ablate interaction with the human receptor, but we do think that this virus is able to bind and the mouse receptor better and that mutation alone is not sufficient to increase pathogenicity to the the full MA15 virus but it captures a lot of that so that mutation in spike is required for pathogenicity, but alone it is not sufficient. So there are other changes in the backbone of the virus that are required. And we’ve done several mouse adapted strains, trying to figure out if what, what components and how their interaction with host components are driving this response. And so there are mutations and other locations in the backbone. And we haven’t really figured out exactly what it is. So

Vincent Racaniello 45:21
This is the kind of experiment you could not do today. Right?

Vineet Menachery 45:25
Mouse adaption of SARS would not be I mean, it’s a it falls under the pause so it’s an increase in pathogenicity in a mammalian model.

Vincent Racaniello 45:36
Right.

Vineet Menachery 45:37
Is it making it more pathogenic in humans? I The answer is we don’t know. And there’s really no great way to test it.

Vincent Racaniello 45:45
Yeah.

Vineet Menachery 45:47
But we also have to remember that this the epidemic SARS strain, passed through a lot of people, and did plenty of adaption in humans already. So I don’t we don’t know if we can gain more adaptation from.

Vincent Racaniello 46:01
You know, traditionally, you think of you pass a virus in a nonhuman host attenuates it for humans. That’s how you make live attenuated vaccines. So if I,

Rich Condit 46:10
you know, I’m almost ready to volunteer for this, you know, stick me in a BSL3 facility. And I’ll snort some of this stuff and see what happens.

Vincent Racaniello 46:17
I’m afraid you can’t do that Rich, not go.

Vineet Menachery 46:20
I will mention that to Ralph.

Vincent Racaniello 46:24
I don’t think your family would like that. That’s not up to you Rich. But you know, if you look at all the information we’ve gained from studying this mouse adapted SARS virus, it’s tremendous. So you know, it’s been around for years. It hasn’t done anything, it hasn’t escaped, it hasn’t infected people. Why can’t we use that as some data and say, This is good stuff to do? And it’s safe. I just don’t understand why that’s overlooked. I don’t expect you to answer that Vineet. But I’m just complaining.

Rich Condit 46:51
The other interesting thing that came up is, is that you adapted to one strain of mice, and it’s not necessarily adapted to another strain of mice. So these adaptations are really highly specific, not only just to species, but even strains of varieties.

Vineet Menachery 47:08
Yeah, if Ralph was on, I’m sure he would talk about the collaborative cross. So that’s, uh, yeah, for those of you who are unfamiliar, the collaborative cross is a genetically diverse mouse panel. And it takes eight founder strains. And over the past 20 years, they’ve been developing mice to capture genetic diversity using these founder strains was have five lab derived strains, and then three wild derived strains. And while it’s not equivalent to human genetic diversity, it captures the same level. And in those mice, when we put SARS in those mice, we get the full range, we have mice that are completely refractory and gain weight after infection. And we have mice that rapidly lose weight and are highly susceptible. And one of Ralph’s major projects, in addition to what we’re doing with these coronaviruses, is identifying host factors that drive susceptibility, because it may not just be the virus that infects a person is important and the genetic background of that virus is important, but the person itself and their physiological conditions and their genetics and how old they are really matter in these kind of emergence questions as well.

Vincent Racaniello 48:18
Yeah. Okay. Vineet tell us what you found when you made this virus?

Vineet Menachery 48:22
So we’ve taken the SHC014, we made the chimeric virus. And we we started with Vero cells, we found that it replicated equivalently in Vero cells, which don’t have a strong immune response, and so our next step was to look in.

Vincent Racaniello 48:36
For those who don’t know, there’s a monkey cells, right?

Vineet Menachery 48:38
Yeah, money, African green monkey kidney cells. And then we went to respiratory cells. Calu-3 cells are a cell line that we’ve used, we use the 2B4 version of these, these have been sorted for ACE2 expression. And

Vincent Racaniello 48:52
They’re human right?

Vineet Menachery 48:53
They are human cells, type two alveolar cells are closest to alveolar cells. And then primary human airway culture. So these are derived from lung transplant donors. So the upper branches of the airway are available, they’re not always used and so a researcher here in the cystic fibrosis, Scott Randell, harvests these and is able to make cultures and takes weeks to make these and he’s been doing it for several years, where you you plate the cells, and you can actually differentiate them into a ciliated airway, primary human airway. And so there are air liquid grown on air liquid interface. So you have an insert, the top portion is exposed to the air, you can actually see the cilia and they beat and the lower layer has the basal lateral media. And so they mimic the human airway as as well as any cell culture modern model could. And we’ve done a number of studies with SARS over the years with these, these cultures and we started with those with the SHC014 in the SARS backbone and compared it directly to the epidemic strain their epidemic urbani strain and found that it could replicate equivalently to the epidemic strain. And from there, we took that same virus and infected mice, this is 10 week old BALB/c mice and found that it was not as pathogenic as the mouse adapted MA15. So those mice challenges one times 10 to the fourth dose died between four and six days post infection. The SHC014 mouse adapted virus, the SARS backbone virus causes about 10% weight loss in biopsy mice significant disease but not particularly lethal. What was interesting to us was that the titers were roughly equivalent two days post infection between the viruses. And it appeared that the viruses was able to carry out a lot of the same program that the epidemic the mouse adaptive strain could. And so we have these virus, this virus that the full length by the spike had mediated ability to replicate in human cells. And while it didn’t cause equivalent disease, it was causing disease in mice. It wasn’t equivalent to the mouse adapted strain, but it was definitely not attenuated.

Vincent Racaniello 51:24
Right. But it’s, you know, it, it has reduced virulence and that whether you can use that as any kind of comfort, I don’t know, because mice are not people, as we often say, on this show. But like, in many cases, when you when you when you make artificial viruses, and I would say this is an artificial virus, we don’t know if it exists out there. You you interfere with the ability of the virus to cause disease. So it’s not surprising, right?

Vineet Menachery 51:53
Yeah.

Rich Condit 51:53
So let me let me make sure I understand this, the the the hybrid virus with the spike glycoprotein from from the bat, in the mouse adapted background are

Vincent Racaniello 52:05
not bat, but bat virus

Rich Condit 52:07
bat virus sorry, is not quite the same as the mouse adapted virus itself in animals, correct?

Vineet Menachery 52:17
Right. It’s not, not at all, and in a lot of ways, and I think there’s actually a lot of interesting aspects of that. And we didn’t, we didn’t really have an opportunity to expand on this. But one of the observations in the paper, but we didn’t have a, we didn’t go into length about this was we actually see a tropism difference between the SHC014, and the epidemic strain, the mouse adapted epidemic strain. So the MA15, the mouse adapted SARS wild type infects, both the airway and the parenchyma. When you look at the lung sections, it’s very clear the virus is in all areas of the lung. In contrast, SHC014 in the, in the SARS backbone only is replicating or only stains in the parenchyma. And the airways are largely absent, the large airways are largely absent of any viral antigen, which is really interesting in terms of pathogenesis, and exposure. And considering that our titers were very similar, it argues that there may be actual tropism differences. And these tropism differences may have to do with protease cleavage, and the ability to infect those cells. And so it’s actually something we’re following up on now. We’ve been looking at host proteases, that may be differential between the two in those locations. And so it actually argues that one of the reasons that SHC014 may be attenuated relative to the mouse adapted strain, is that it doesn’t affect these airway cells, or these, these large airways are not getting infected. It’s a real opportunity, I think it’s something we’re really interested in. So much of the focus on the spike protein, and rightly so has been focused on the ability to bind. So the receptor binding domain is where a lot of the a lot of the focus has been really focused on this RBD region. And there’s a lot of difference in this region. Less focus has been focused on the protease cleavage site and the importance of that in emergence. And one of the things that this virus actually gives us an opportunity to we can actually look at this attenuation and ask, are we going to be able to attenuate the epidemic strain the mouse adapted epidemic strain by doing a domain swap? If we take the receptor binding domain of SHC014 and put it into the SARS backbone, will that reduce its ability to replicate or if we take a chunk of the protease cleavage site and find that it’s not as efficient that may be a critical step in emergence that we’re we’ve never even considered before and we couldn’t have done without a virus that actually binds to human ACE2. And it’s it’s something that we’re pursuing now we’re asking these kind of questions that, well, the SHC014 virus is able to grow. It’s not equivalent to SARS. And that can actually inform how the epidemic became an epidemic. I mean, I think that’s where we’re still very early in this response. But there are lots of tools here. In telling us more about how these viruses emerged by going from the bat to the human, and comparing them between the two.

Rich Condit 55:36
Although although it’s a logical I still find it fascinating that subtle changes in the spike protein could affect what tissues in the host are infected, and thereby effect pathogenesis. That’s really interesting.

Vincent Racaniello 55:56
Kathy

Kathy Spindler 55:57
I was just gonna say that, I think in this figure where you have you do show that histopathology it’s, it’s a really nice convincing figure about the the difference in tropism with the 014 strain compared to the epidemic strain. And I understand that at some point, something on the order of 2000 words had to be cut from this manuscript. And, and so you showed this one figure about tropism difference. Are there other things, you know, that are gonna end up coming out somewhere else in terms of this? That that you could mention at this point? So differences between the two viruses.

Vineet Menachery 56:39
So I don’t know how much I should say. So this is, this is work that your former student Lisa Gralinsky is working on. We have a number of mice that have protease, we have protease knockout mice, human, host protease knockouts, and we have one where the epidemic mouse adapted strain MA15. There’s no phenotype, it looks exactly the same as the wild type. And that the SHC014 virus, which caused about 10% weight loss has no pheno, like has no replication, is very low. So we think that that protease may be critical. I’ll let Lisa, if you if you want to talk to Lisa.

Kathy Spindler 57:24
Sure. And the the, what you mentioned about the collaborative cross, I know you’re a co author on this paper that’s in PLOS Genetics that maybe we can stick it in the show notes. It’s one of the cases where the collaborative Cross has been used for infectious virus. Looking at genetic differences into disease, disease susceptibility, it’s one that’s being cited now. So it’s it’s open access, and we can show that.

Vincent Racaniello 57:54
Can you stick that in the show notes?

Kathy Spindler 57:55
It’s already there.

Vincent Racaniello 57:56
Very good. All right, Vineet can you now, what do we do next? Let’s move so

Vineet Menachery 58:01
So we we’ve built at this point, we’ve built the chimeric virus. At that point, we heard. Once we knew that it was viable, we’d ordered and we started process of getting the full length viruses made. Obviously making the case the SARS, making the spike is not trivial, and it’s length making a full 28 kB genome is even less. It just it takes time. And so we initiated studies, asking questions about the therapeutics that we had available to us. And so we think vaccines and we think monoclonal antibodies. And so, the monoclonal antibodies, we’ve had a long standing collaboration with Wayne Marasco’s group at Harvard, and then Antonio Lanzavecchia’s group making antibodies for SARS. And then a number of studies that we’ve done over the years where they’ve given us antibodies, and we’ve created escape mutants, and then they’ve made new antibodies against the escape mutants. And the idea was that we could drive drive antibody response or make a panel of antibodies that could really block anything out there. And we spend a lot of time making chimeric viruses with civet strains and diverse strains trying to figure out ways to improve this antibody and its breadth. And so, with SHC014 and how different it was in the receptor binding domain, we saw an opportunity to ask the question, well do the antibodies that we would pick and the four antibodies that are in the paper are what we would have used if we were designing a ZMapp the ZMapp equivalent for SARS, these are the four antibodies that we would have picked. And so in vitro we took these antibodies and we asked whether they were able to neutralize SARS, which it does, and SHC014. And in all of them, the neutralization was weak at best three of them Were outright failures that we didn’t get over 50% neutralization, one of them approaches 50%. But you would have to use in a human, grams of antibody. So, and part of the reason is, and we’ve mapped a lot of these antibodies, we know exactly where they bind, almost all of them are binding within the receptor binding domain. And why did we make antibodies that bind receptor binding domain? Well, there, they are highly neutralizing, and they’re the, they’re the strongest of them. And for the most part, the expectation was, the viruses that are SARS like that can bind human ACE2 to wouldn’t tolerate major changes in the ACE2 binding region. And here, we find very clearly like that SHC014, have these had a very different RBD, and could escape these antibodies because the residues are different, and whether the tract of the binding is slightly different. And that’s the difference that we see. Whatever the reason that these antibodies don’t bind, despite the ability of this virus to replicate, it really clearly showed that our antibody responses, what we’ve developed are not sufficient. And I think what’s important is that Lanzavecchia’s group and Marasco, they have a number of antibodies that we’ve we have tested in the initial phases, but we kind of went with what worked. And we have an opportunity to go back and ask questions on to go back to those antibody stocks, the ones that maybe we didn’t go through first and ask which ones are there? Which ones can we expand upon and look at with this SHC014 virus and actually maybe find something a better treatment model. And they may be just adding another antibody that has more broad response. Unfortunately, we couldn’t have done this without without this SHC virus that’s kind of, as Ralph talked about earlier, the book ends. Without a virus that was in the middle, we really didn’t have an opportunity to do that we needed something that was different enough to really chase down antibodies that are outside of RBD.

Vincent Racaniello 1:02:09
So this result scares a lot of people. But you need to know this, right? It’s like Ralph said, you’re going to wait for something to arise that you can’t treat with monoclonal or you’re going to be proactive. And here, this shows you that there’s there’s spike genes in bats for SARS like viruses circulating that are really different. And this tells us we should try, as you say, make something more broadly neutralizing, right.

Vineet Menachery 1:02:36
And I think, you know, and Ralph’s right about that, you know, SHC014 may never emerge. But the via, but we can improve the cocktail with this. And it’s not that we don’t have these resources. It says that. If and if you would ask me whether the antibodies would work. I mean, I don’t want to appear that I’m always wrong, but consistently, paper, consistently in this paper, I really thought one of these four antibodies would have worked. And I was surprised that one of them didn’t work. And, you know, I think often some of the most interesting science is when you’re wrong. But I think this is an opportunity. I think this is why we want to do it. We are we are in better position to deal with a group 2b coronavirus, because of this SHC014 virus. And moving forward, there’s a lot of opportunity to learn about emergence and therapeutics from that information.

Rich Condit 1:03:38
I’d rather be scared than ignorant. You know?

Vincent Racaniello 1:03:41
yes, no, I think that’s this is a good point, you have to take some risk when you do science. Anything that doesn’t have risk is not going to be very good all the time. So I think that you know, you mitigate the risk, and you do the experiments. I just think that people criticize this. Don’t understand. That they don’t, as Ralph said, they don’t know the whole field of coronaviruses. And it makes it hard for them to realize what’s going on. And I recognize that that’s an issue. And I think they should listen to this episode. They won’t. That’s the problem. Vineet, then you did some vaccination studies, right with this virus.

Vineet Menachery 1:04:15
Yeah. So we have lots of different approaches for vaccination. In the field, a lot of effort was made to make a inactivated vaccine, this is a SARS particle that was doubly inactivated. I think they call it DIV in the in the manuscript. And it has been shown to be effective in young mice. And then our group had gone back and done this in older mice year old mice and found that there was immune pathology and as as the mice got older, the vaccine responses waned and actually were partially pathogenic. It was actually worse to have this vaccine than to not be vaccinated. Another aspect was that if you challenge the virus, that heterologously different, you amplify those immune pathology responses. And so the DIV vaccine itself was not particularly well received after that point, and isn’t really a viable candidate going, moving forward. But we tested the ability of that because it is a full length SARS virus, and we wondered if this DIV vaccine could protect against this SHC014 virus. And what we found in both young and old mice is that there was no protection offered by the live attenuated vaccine, which was not particularly surprising. But we would have expected some neutral, we could have expected some neutralization. I guess this is an example of we didn’t think it was going to work. And I was we were I was actually proven correct. The other aspect was, we did the inverse experiment. So we have a number of live attenuated vaccine platforms that work. Whether we knock out particular aspects of the virus or efficiency of the virus, we knocked out different components, our fidelity mutation virus has been used before as a vaccine platform. Luis Enjuanes has a mutate delta E protein deletion that he’s used as a vaccine platform. And we wanted to ask if we took SHC014, and we infected with a dose that was that caused disease but wasn’t lethal. And that’s the maximum dose, could we offer protection. And what was interesting was we did see protection if we infected the mice year old mice with a dose that cost 10% weight loss. When we came back at 28 days and challenge with the lethal MA15 challenge, those mice were completely protected. And so SHC014 the spike components of SHC014 are able to confer protection against SARS infection, the SARS infection there are overlapping epitopes between the spike proteins. When we looked at the actual level of the antibody, the sera, what we found was that the neutralization is that 1:200. So, the dilution is very low. I mean, we need a lot of that sera to neutralize, for comparative sake, our our vaccine platforms are 1:1600. And so the expectation was that even though this vaccine works in the short term, long term immunity is probably not established. And this tells us a couple things. One, there are common epitopes between this SHC014 spike and the SARS spike that we may be able to manipulate when we make antibiotics or antibodies to this region that they may actually have cross protection opportunities. I think the other important aspect is when we went back and we challenged the mice with a lower dose a dose that didn’t cause weight loss, this was 100 pfu. This actually didn’t offer any protection. And so it it signals that this spike protein that there is overlap between the SHC014 spike and the SARS spike, that we can drive protection, but this may be an issue that the major antigen long term, it may be really difficult to tease this out. And so we may have to design new approaches to actually pick for broadly neutralizing antibodies as opposed to ones that are specific to the receptor binding domain which is not common between SHC014 and the SARS spike.

Vincent Racaniello 1:08:37
Alright, so Well, after all, this was done your virus, your bat Coronavirus, was ready, right?

Vineet Menachery 1:08:42
Yeah. So.

Rich Condit 1:08:44
So I want I wanted to, there’s a practical issue here is the the initial swap of the spike glycoprotein into the mouse adapted background. That’s the logic of doing that, rather than going for the full length virus to start with is just that it’s a lot easier. And and if it didn’t work, there’s no point going any further is that the thinking?

Vineet Menachery 1:09:10
So I mean, there are a number of there are a number of reasons. One, you have to remember, based on the evidence in the data, this is a virus that shouldn’t have grown.

Rich Condit 1:09:22
Right.

Vineet Menachery 1:09:23
And so you have the practical aspects of you can make the spike protein and prove in the mouse in the chimeric virus that doesn’t grow. And so that’s both the fastest and the cheapest option. So

Rich Condit 1:09:35
and then you’re done and you don’t have to make the whole virus right.

Vineet Menachery 1:09:38
Well, and the other aspect is even without that, if we had known the virus, the full length virus could grow. There are a number of reasons that it wouldn’t grow that have nothing to do with spike itself. So, those background changes in NSP9 or other components that are adapted. You may actually have a compatibility issue with the host that either the immune response stops it or some interaction with some cellular machinery is required. And this virus may not grow, but it actually has nothing to do with binding and entry, it actually has to do with replication or the immune response.

Rich Condit 1:10:13
Okay, so the constructs are really complementary in that regard.

Vineet Menachery 1:10:16
Well, and I think, you know, if we had known the SHC014 the full length virus could replicate, it’s likely we still would have asked about the Chimera, because the chimera tells us about the spike itself and isolates the difference of the spike. And as Ralph said, these pools exists and recombination is well established in coronaviruses. And in data that we’ve seen from our collaborators of Wuhan Institute of Virology, there’s tons of recombination hotspots in the spike and they have these all these spike sequences they’ve compared and they actually see regions where this is directly cut from another spike. And the only way is that the only way this could happen is through recombination. So I understand, I mean, this gets into the hindsight of the results, if you know that the SHC014 virus is going to replicate. You should probably would probably start with that. At the same time. We didn’t know that at the front, and the second is the chimera still is has value in these responses.

Rich Condit 1:11:19
Right. Okay.

Kathy Spindler 1:11:20
Also, it’s a huge resource expenditure to do that. And like you said, it took time, whereas you could do this, it sounds like, chimera much quicker.

Vineet Menachery 1:11:31
Yeah, I mean, you’re talking about, you know, 1200 1300 base pairs versus 28 kb, and a bunch of different constructs, you were talking a month versus a couple of months.

Kathy Spindler 1:11:44
So and I have a question about the fact that you did this. Made this full length virus, and you compared it to the SARS epidemic strain virus in mice. So I thought that you had to mouse adapt the virus. So what can you do with the viruses without mouse adaptation? Or

Vineet Menachery 1:12:10
So we are comparing in the mice we compare to the mouse adapted strain in the human cells we compare to the epidemic strain.

Kathy Spindler 1:12:19
Oh, well, I guess maybe I misinterpreted the figure then. I thought that you just put the non mouse adapted virus in and the non mouse adapted full length. 014 in

Rich Condit 1:12:38
it looks. It looks to me like in 3d, you got all three? You got Urbani?

Kathy Spindler 1:12:42
Right.

Rich Condit 1:12:42
You got the reconstructed, you got the reconstructed bat virus, and you’ve got the mouse adapted virus, if I’m reading that correctly.

Kathy Spindler 1:12:50
Right.

Rich Condit 1:12:53
So you’re comparing all three?

Kathy Spindler 1:12:55
Right. Okay.

Vincent Racaniello 1:12:57
So your question is, why does the bat virus replicate in mice without adaptation? But

Kathy Spindler 1:13:04
Right

Vincent Racaniello 1:13:04
but it’s not the human virus. So maybe that that, Vineet if you just take human SARS isolate like Urbani, and,

Vineet Menachery 1:13:11
yes

Vincent Racaniello 1:13:11
you put it into intranasally into mice, BALB/c, is it replicated at all?

Vineet Menachery 1:13:17
Yes, that replicates with roughly equivalent. The titers are lower than the mouse adapted strain, but there’s, there are a large amount, there’s a large amount of virus in there.

Vincent Racaniello 1:13:27
Okay.

Vineet Menachery 1:13:28
And, and, and we have a number of reasons we’ve we, we have more depth in this question as well, we have a mouse that expresses human ACE2 under a Clara cell promoter. And we’ve done comparisons between the epidemic strain the Urbani from with no mouse adaptation to the SHC014 and we have interesting data there that shows clear attenuation but there’s complicating factors of that mouse is not as clean as we would hope in terms of where it’s expressed.

Rich Condit 1:14:06
So but the the epidemic strain the Urbani strain infects the mice, but it doesn’t kill them. Doesn’t look like it makes it very sick, right?

Vineet Menachery 1:14:16
No. So yeah, you’re talking about in figure three, I was like, I have to I had to pull up the paper cuz

Kathy Spindler 1:14:24
right.

Vineet Menachery 1:14:24
So figure three so figure three is the full length SHC014 verses the epidemic SARS, so

Vincent Racaniello 1:14:34
Urbani

Vineet Menachery 1:14:34
I didn’t include Yeah, the Urbani and so the expectation the wild type, MA15, which isn’t included in the paper paper, but those mice do die completely. So looking at the paper, it’s the gray line is mouse adapted, green line is the full length SHC virus. The black line is epidemic SARS, not mouse adapted.

Vincent Racaniello 1:14:57
So the bat, isolate SHC014 replicates in Vero cells, it replicates in human epithelial airway epithelial cells. And in mice, it causes more weight loss than does Urbani or the mouse adapted strain, right?

Vineet Menachery 1:15:16
No. So the SHC full full length, doesn’t cause

Vincent Racaniello 1:15:20
Oh, that’s the gray is the mouse adapted, sorry.

Rich Condit 1:15:22
Right.

Vineet Menachery 1:15:22
So then the full like virus is it grows equivalently in Vero cells is attenuated in primary human airway cells, and is attenuated in mice in terms of replication. Neither the Urbani strain or the full length SHC strain caused any weight loss or pathogenicity.

Vincent Racaniello 1:15:41
Got it. Yeah, I have a problem with with gray versus black here. I can’t see.

Kathy Spindler 1:15:47
Yeah, it’s it’s subtle. Okay.

Vincent Racaniello 1:15:50
All right. So, so that’s the full length bat SARS related Coronavirus, you made the entire genome as DNA and then you rescued virus out of cells, right, and then uses that to do these experiments.

Vineet Menachery 1:16:04
Yeah.

Rich Condit 1:16:07
And also, another thing that comes out of the same mouse experiments is that the full length bat virus does not replicate as its pathogenicity in terms of weight loss in the mice is the same as the epidemic Urbani strain. But in terms of replication in the lungs, the full length bat virus doesn’t replicate as well as the Urbani strain. So by a couple of different criteria, replication in human airway epithelial cells, and replication in the mouse lungs, it is not as robust an infection as the epidemic strain, correct?

Vineet Menachery 1:16:48
Correct.

Rich Condit 1:16:49
Okay.

Vincent Racaniello 1:16:49
So, Vineet, I was, I’ve been on Twitter all week defending this paper.

Vineet Menachery 1:16:54
Yes, I saw.

Vincent Racaniello 1:16:55
Because I know you need to do experiments. So I’m happy to do that. And at one point, I got an argument with someone who said, Why, why was this recombinant virus made? Why didn’t they just study the bat virus? I said, they did. It’s in the paper. And, you know, he said, eventually, sorry, but I think it just shows that most people don’t even read the paper. And they just read either something, some newspaper article or some online article, and then they make a decision based on that. And, you know, I bet. So two people have been quoted widely in the press, Simon Wain-Hobson, and Richard Ebright. And they have been notable anti gain of function advocates, and their quoted negative sayings. And I would bet, I mean, I could be wrong, but I would bet they have not read this whole paper as much as we had, and, therefore don’t appreciate what’s in it. And I think this is really bad, because this is driving the whole argument is that people that don’t read the papers are participating. Does that drive you crazy?

Vineet Menachery 1:17:57
It’s, you know, I think there’s a there’s a certain amount of knee jerk reaction to anything that’s gain-of-function. And I think, I think it’s important to consider and Ralph and I’ve talked about this, there is a fundamental difference between pursuit of gain and being prepared for gain. I don’t think any are anyone would argue that we expected this virus to be more pathogenic than the epidemic virus. And it hasn’t been. I think being prepared for that possibility is important. And I think it’s a consideration that we have to consider. But this is very different than mouse adaptions, or transmission studies where the gain is what we’re seeking. Here, we’re just, we don’t know anything about these spike proteins. And it gets to it’s very easy to be paranoid about the gain of function aspect to this and being prepared for it. And we really don’t want that to be captured in any kind of rules that, well, we don’t know what’s going to happen. So you shouldn’t do it. I think you could argue that any adaptation of SARS any manipulation of SARS, could result in a gain say we shouldn’t work with SARS is the down you know, that’s the logic goes down that way that oh, well, you added GFP to SARS, that could lead to Yeah, and it ignores the data that’s available and ignores scientific insight. And we have been rightly proven wrong before. But I guess that gets to, you know, the stuff that we have in place. And I think it’s hard. You know, the, as Kathy mentioned, we we cut a number of words from this manuscript. And I’m not even sure that we would have covered the things that people are interested in. So and I think this is a great opportunity to talk about the process that we went through to come up with these viruses and we’ve spent a lot of time figuring out the best way to get at these questions, and so we have local approval, we asked about these viruses putting them in the backbone. We had the pause, we, we had these interactions with NIH. And I think there was an aspect and Ralph talked about it, that there’s a black box when this stuff goes out, and we don’t know how it’s reviewed or who’s reviewing it. And we made the point that when we sent our materials to them, but we didn’t think that this was a gain, it could have been a gain. And if we had a gain, we specifically have criteria for what we define as a gain and what we would do and,

Kathy Spindler 1:20:34
and I’ll describe that, that preparation of who you would notify, and, you know, then what would happen. So that was clear that you were prepared for that.

Vineet Menachery 1:20:42
Well, and I think, you know, Ralph went into and we’ve talked about these studies, it’s really hard to talk about, you know, like, there’s not a place in the paper to talk about what we do and how these countermeasures are in place, I think, you know, we hear about these, these safety breaks, and all of that, but we don’t know each individual lab has its own protocols, we actually have gone through and we’ve counted the number of things that our lab does in terms of safety. And so we have nine levels of PPE that we use in the containment lab, we in our lab operating procedures, we have 31, 31, different things that we do in terms of our protocols, and lots of these things are overlapping elements, that if you don’t do one, you should be captured by two or three of the other things. Outside of the lab in terms of you know, if we have a cold or, or something like that we do drills, we have another 18 things. So you you have all of these layers, because you know, there are accidents, there are mistakes, there is human error, you you can’t you can’t exclude that no one is perfect. And expecting perfection is problematic. But we we spent a lot of time designing this approach. And I think I think that’s often in the gain of function criticism, ignored. That, we’re, you know, I think it’s very easy to think about the movies and you have scientists are on the one end of the spectrum, you’re the crazy scientists that never considered the ramifications of their work and made something that will destroy the world. Or you’re the lone scientist that saves the world with your with your cure. And there’s no in between. There’s no gray in the movie. So often. There’s no gray applied to us. But I mean, we know what we’re working with. We know. We know probably better than anybody what the risks are. And I think Ralph talked about it. We don’t want to be in a situation where a Coronavirus emerges. And we think we could have done something about that. Or we could have been more prepared. At the same time. We know that. Is this a risk? Yes. What do we need to do to mitigate this risk? We put all those things in place we consider all of these things, I think, to imply that there’s arrogance, and we just want to do this because we want to do it is really painful. And I think, you know, scientists have to be aware of what they say because you know, what you say when you have a position at a university, or you’re a researcher, can be misconstrued and taken out of context, or used in ways that you wouldn’t really think you can. You can justifiably, I mean, I think we can have differences of opinions, but it’s important that we consider the impact of those one set that one sentence can be taken out of context. And I think, you know, in our discussions, it’s important to consider that critics and proponents on both ends of the spectrum have to consider that oftentimes, you’re not you’re getting non scientists or people who are reporting it that may not fully understand the width of the statement.

Vincent Racaniello 1:23:52
I think this is so right on, because, you know, you can’t expect people to read your paper, that’s fine. I think it’s great to have a discussion, but no, the anti-GoF people are simply negative, you cannot do this, you’re going to kill the world. That’s not a discussion, right? That’s just saying you can’t do these experiments. And I think the key here is, you know, we try and provide a forum for virology here on TWiV, you can talk for an hour and a half about your work. You’re not going to get that anywhere else in the world. No radio station, no interview, nothing, not even a talk at ASV goes that long. And it’s not back and forth. So I think this is important, but unfortunately, you know, Wain-Hobson, and Ebert will not listen to this and get a real appreciation of, of really the amazing things we’ve learned from this work. But the other key here, I think, and part of the problem is the press release from UNC was really bad. I don’t know.

Rich Condit 1:24:51
Yeah, I thought they kind of blew that.

Vincent Racaniello 1:24:53
The headline was picked up directly by several websites. And it’s they say something like Investigators show that bad viruses can can go right into people. And that, of course, I don’t think you’d agree that that’s correct. And this is a problem. And, you know, I know we’ve talked about this a lot on TWiV, and I wish Alan were here. But often press releases get that the wrong message. And you know, investigators are not supposed to be writing them, but they should be more closely involved. So I think that’s part of the problem here as well. Right.

Vineet Menachery 1:25:25
I don’t disagree. I think I think part of it is the the press release. I mean, it gets to we have, you know, the press office at the university has a different purpose than the researcher.

Vincent Racaniello 1:25:38
Yeah.

Vineet Menachery 1:25:38
You know, if it. I mean, you may argue it is. When Ralph and I had a discussion, is it inaccurate what they said? I mean, you could argue that they didn’t say anything that wasn’t true. Is it inflammatory? Absolutely. Is that what brings eyes to the paper? Yes. I mean, I think, fundamentally, when we looked at that, if you read the headline, and took that, and just took the headline, it’s bad. If you go into the paper, Ralph talks in that in the, in the press release about, we’re further than you think, but like, there’s important aspects, but this virus, you know, like, there is aspects in the actual text that minimize that headline. But if you’re taking it with the context that you want to, if you want, if you want eyes, the headline did not. The headline drove that and it, it probably drove a lot of the negative press.

Vincent Racaniello 1:26:30
Yeah, the Daily Mail took it, new SARS like virus could jump directly from bats to humans without mutating. This

Kathy Spindler 1:26:36
I think, I think they could have come up with a headline and a lede line for the article that were equally catchy, but not so inflammatory. I, I wanted, because you talked about the biosafety and biosecurity stuff, there’s almost a full column of discussion of it in the online methods, which again, if people don’t read the paper, they’re not going to recognize some of the things and you’ve even elaborated some more of them. I wanted to know whether in submitting this paper, you had to go through any additional hoops, or was it simply submitted to Nature Medicine.

Vineet Menachery 1:27:13
So we, we submitted it with paperwork from our, so our final approval letter from the NIH. So the kinetics of the approvals when such that we initially applied for approval do the work in the spring of 2014, to make the viruses. When the pause went into effect, we were not, this project was not under the stop order that the MERS MERS mouse adaptation was. So we had until the roll date of the grant to respond. And we sent our paperwork where NIH asked for, you know, our process and this grant covers a number of, it’s a large collaborative grant that has Ebola and other groups in it. And so there was a number of things that we had to answer. And at the end in May, before we, before the final approval, we we basically got asked for a letter saying whether we could do these experiments continue this experiments or not. And we actually submitted that paperwork to the journal with the understanding that they they wanted it.

Rich Condit 1:28:29
That was that was before it even went out for review. Is that correct? Did you get any? Was there any comments from the reviewers that required extra action?

Vineet Menachery 1:28:41
We we did it on the front end, just so that it would be available to the editors and to the reviewers as needed. So we were proactive in that sense. We expected that this would be a question and had been Yeah, I I think if we hadn’t included it, people would have asked,

Rich Condit 1:29:00
Okay.

Kathy Spindler 1:29:00
Sure.

Vincent Racaniello 1:29:02
Well, I do hope and in Ralph mentioned this, the people who are making these decisions about the future of these gain of function with flu and SARS viruses, I hope that they look at the science and really disregard all the hype. They disregard Ebert and Wain-Hobson and everything they say in the press, because if you look at the science, you understand that this is important work. And I’m afraid that this is going to be lost in the shuffle. And and we’re gonna and science is gonna lose out in the end. I’m afraid of that. But I just don’t know what’s going on. Because as he said, We don’t even know who’s making this decision.

Vineet Menachery 1:29:40
Well, and I think, you know, I I don’t want to characterize, I mean, while they we don’t know the NIH process, they have been very supportive of this work.

Vincent Racaniello 1:29:50
Great. That’s great.

Vineet Menachery 1:29:51
They’ve, they’ve been very complimentary. And, you know, in a lot of ways, it’s important to realize that while NIH is administrating, they, they they’re at the front lines of this, the policy came down from the State Department, the pause is not an NIH directive.

Vincent Racaniello 1:30:08
Right.

Vineet Menachery 1:30:09
And so at a certain point, they have to they’re tasked with applying metrics that are not necessarily scientific. That may be more political than anything. And so they are in as difficult as a position as we are. And we’re all operating, I think, everybody I’ve interacted with on this at the NIH and with oversight, we’re doing our best. And we we want to minimize any risks that there is. implications that, you know, I mean, it’s really, you know, I expect the Daily Mail or I actually showed Ralph, the daily sheeple. Which was, I mean, I think so, I mean, we kind of laugh at these, I mean, you kind of expect that the internet is what it is, I think it’s, it’s really disheartening when you have scientists who have the opportunity and can look at the paper and just fully look at it, making these comments and, as you’ve said, I don’t know how in depth, they’ve looked at it, but there were, I mean, it’s been said words matter, especially when you’re in positions of power. And so I would encourage critics and proponents to really consider that when you make comments that you have to have a full understanding. And if if you don’t fully understand, and I think there’s no, there’s no problem in asking, and having this discussion I’ve had, I had Twitter discussions about this. I’ve talked to a number of people. And, you know, I, where was I surprised that it happened this way, I think in the culture that we live in, I think part of this was expected. But dialogue is really important. And it’s important that we talk with people and get their feelings. I mean, I, I really value scientists that engage with me on this, because I have my own biases. You know, I think this work is really important. But and I don’t know that I mean, part of me thinks that it’s probably good that I’m not making the final, you know, that NIH, it makes it easy that NIH makes this final decision, because I have I have real interest in this one way or the other. Now, that being said, I have lots of ideas and lots of interesting things that I can follow up on. It’s important that this work be done. A, but if if society or scientists scientific society thinks that it’s too risky, I can understand that.

Vincent Racaniello 1:32:35
Well, if you have, if you have a good discussion, and that’s the outcome, that’s fine. But you know, Ebright says, the only impact of this work is the creation of a new non natural risk, is absurd. It’s patently absurd to say that he can’t have read the paper, right? This is what makes me crazy, but you’re very level headed Vineet. So I’m glad that you’re here doing this work. And I do think that people need to talk about this. And we, you know, I’d like to have Wain-Hobson on TWiV, and talk about it. And we you know, he’s supposedly a smart guy. Let him tell us why he’s really concerned. And we can we can go over it. I think we have the best forum for that here. To do that

Rich Condit 1:33:15
We’ve done we’ve done several of these episodes where we’ve gone over a paper that is controversial in this regard. And I have to say that for me personally, every time we do this and go through an episode when we’re done, I come away thinking First of all, that there’s not nearly the danger that or none that people make a flap about. And second, I put myself in the position of how much I’ve learned from this paper. And he’s and what what I would miss if we didn’t if we didn’t have this work, and it’s amazing. Okay, because you learn so much from this stuff. And without it, there’s this this vacuum, it doesn’t make any sense.

Vincent Racaniello 1:33:57
Kathy, what’s your schedule? You need to leave?

Kathy Spindler 1:33:59
And no, no, I’m fine.

Vincent Racaniello 1:34:00
Okay, cuz

Kathy Spindler 1:34:01
Yeah, yeah, no, I’m just gonna skip that.

Vincent Racaniello 1:34:05
But you have a flight to leave at 130.

Kathy Spindler 1:34:06
That’s at 115.

Vincent Racaniello 1:34:08
We’ll be done by then.

Kathy Spindler 1:34:08
Yeah, I hope so.

Vincent Racaniello 1:34:10
But I think

Vineet Menachery 1:34:12
I just want to I just want to add, I think, you know, it’s important, and I think part of it has been Yeah, we, we’ve had a lot of questions and Ralph gets questions about the transmissible flu studies and it’s, it’s really hard to, to comment on them because, you know, we work with respiratory viruses. There are similarities between SARS and flu, but the ins and outs of flu are, are are difficult understanding all the literature and all that and these people who are working in these areas really know these areas really well. And so I was I my wife is an educator. So she’s a teacher and I said that it’s kind of like asking a kindergarten teacher what to do with senior level English class. It’s not that they can’t. It’s that and like, a really like, I can comment on flu, but I don’t have the experience or the understanding that someone who has all the experience and understanding would. And I think that’s it’s important. And I think often we look at these these groups and say, Oh, well, you should you, you know, you guys are biased. You guys are working on the literature.

Vincent Racaniello 1:35:27
Exactly, exactly.

Vineet Menachery 1:35:29
But at the same time, it’s hard to find people that aren’t biased in some way to comment on it and who have who have, you know, I read, I read these journals. And honestly, if I wouldn’t work, if I wasn’t working on this paper, I may have looked at the figures, I don’t know that I would have read or read the abstract, I would have looked at the figures, and then I would have moved on. It’s hard to make strong statements one way or the other. But I think it’s also important, you know, my opinions, my thoughts are often discounted by gain-of-function because I’m, I’m right there. I, I do have a conflict, rightly, but at the same time having a conflict is it’s also how people say, Oh, well, you know, these people who study climate change, have a conflict. So they can’t talk that’s kind of a ridiculous

Vincent Racaniello 1:36:21
Who’s better, as you say, who’s better qualified. But so the the gain of function, opponents, some of them are scientists, Wain-Hobson is a virologist. And but he may not have read these. And so who who’s going to make the decision? That’s what I’m concerned about. And, you know, to circle back to something you said earlier, you know, these, these newspaper, online articles don’t matter too much. But you know, what, I think Congress sees them, unfortunately, I think their staff gives it to them. And that informs their opinion, you know, and it’s really unfortunate. And I don’t know how to combat that. It’s a big communication issue for sure.

Vineet Menachery 1:36:55
Yeah. And I think, you know, it gets back to, there are a number of things that the gain of function arguments, Drew clear lines, and it’s, it was interesting. Seeing, I could have predicted, you know, I think we all know who the critics are, and who would have been quoted in these stories, I think. And it gets to, you know, they point out a conflict for, for me or Ralph, in these studies. But if you’re on record as being against gain of function 8, 10, 20 times. If that’s your claim to fame, aren’t you also in conflict? I think that is not considered.

Vincent Racaniello 1:37:35
Yeah.

Vineet Menachery 1:37:35
When? I mean, I don’t I don’t know, Dr. Wain-Hobson at all. I’m not familiar with what his scientific work is. But I do know, he’s a gain-of-function critic. And if that’s what you’re known for, is that is that relevant? I don’t know. But I think

Vincent Racaniello 1:37:54
I agree. I think that they have an agenda. They just don’t like this, the idea of this. And so they will not even read the paper to understand it. They would say, No, you shouldn’t be doing these experiments, period. And the public says, Okay, this is a scientist, this is a virologist. So it must mean something. And, but to their credit, some of these articles did quote, Ralph, which is good. You know, they have both sides what you’re supposed to do, of course, the headlines don’t reflect that at all.

Vineet Menachery 1:38:22
Well, and I think, in Ralph, Ralph, and I’ve talked about it, you know, you have these interviews, that can be long, but

Kathy Spindler 1:38:31
they take a sound bite out of it.

Vincent Racaniello 1:38:34
Yeah.

Vineet Menachery 1:38:35
Well, there’s, you know, I think in having this, this platform is great for it. There’s a lot of nuance to this. In in, if you don’t know anything about virology like the the background behind it. Why didn’t we make the full length virus to start with? is a reasonable question based on that. And I think we’ve we’ve talked about why we started with a chimera here. But there aren’t great platforms for explaining that.

Vincent Racaniello 1:39:01
I think TWiV is a great platform.

Vineet Menachery 1:39:03
And I am happy to chat about this, whenever you like.

Vincent Racaniello 1:39:06
I just don’t know why more people don’t listen, I realize we’re long. But and we do have I have a bias. But I think it’s founded in good science. But there’s nowhere else as I’ve said, where people could talk in depth about this and get the real story, right. Am I wrong about that? Anyone?

Kathy Spindler 1:39:24
I think it’s good to talk in depth. I’m just gonna go out and say that I’m probably a Pollyanna about this, but I really hope that Simon Wain-Hobson and Richard Ebright read this paper. If they didn’t, then they really don’t have any business talking about this. As scientists, they should do that.

Rich Condit 1:39:45
Study it. You know

Vincent Racaniello 1:39:47
I doubt it. Well, so I understand your position. But I doubt it from what he said. How can you say this doesn’t give us any information. We’ve just talked about this for an hour and a half of all the cool stuff we’ve learned, right?

Kathy Spindler 1:39:59
I think I see your point. And I agree. But you know they can they can read it and like Rich said, if they have if they just read it and didn’t study it if they just read the abstract and look at the figures and read the press release. It’s not enough.

Vincent Racaniello 1:40:13
Yeah, I agree I that’s what we’re trying to do here is to supply people with, you know, a digestible form of the information. I wrote a blog post yesterday where I tried to make it simple for people.

Kathy Spindler 1:40:23
It’s a really good blog post, people should go to it. virology dot ws

Vincent Racaniello 1:40:27
I just don’t think people will read it. People will not listen, we have a lot of great listeners and readers, but not enough to inform this. This whole debate, I’m afraid. So I think in I would love to dedicate this episode to just this one topic. I don’t want to we have another topic planned. But we’ll put that for another time. Vineet. How much more time do you have in the Baric lab? What’s your plan?

Vineet Menachery 1:40:54
Well, I am, as Ralph, Ralph has told me I am transitioning he is he’s like, oh, aren’t you on that email? And I’ll be like, I wasn’t on that email for that meeting, and he’s like, Oh, I am transitioning you I keep forgetting that. So I am currently in the job search mode. And so I expect I will be here. I I work on this stuff. And I as I mentioned earlier, there’s there’s lots of interesting host components about genetic, the CC and having how much is driven by the host genetics and the aging aspects of Coronavirus disease. I think what’s interesting is a virus that is circulating in bats, it just as important is that it actually interact with the right human for emergence. The genetic components of the human can drive it and the age of the person that’s there, you know, coronaviruses, SARS, and MERS. The lethality goes way up as you get older. And so susceptibility and changes in our host responses as we get older are really driving this response. And that’s, that’s one of the things I’m really interested in finding the host factors both in the genetic component, the genetic side. And I think Kathy mentioned in Lisa’s PLOS Genetics paper, she really drives into the genetics aspects and I’ve got some funding for doing aging work, looking at aging in mice and asking why susceptibility is changing as we as the mice get older, and hopefully we can apply that to human disease.

Vincent Racaniello 1:42:32
Very interesting. So do you want to have an academic position? Is that your goal?

Vineet Menachery 1:42:36
That is the plan at the moment, anything I knew, I think I want academic

Vincent Racaniello 1:42:40
and you’ll continue Coronavirus work right?

Vineet Menachery 1:42:43
Yeah, primarily using Coronavirus work using Coronavirus as as as a platform, but I think relating it to flu and other other viruses is really a great opportunity to interact with other groups. really driving that, you know, our understanding of it, I think often we’ve we’ve worked in silos of our own individual viruses. But comparing and work we’ve done with Yoshi Kawaoka’s group, comparing SARS and high path flu and MERS has really given us real great insights into how the host is controlling viruses globally and how different viruses use similar approaches to drive these different responses. And so I think there’s, there’s great virology there. There’s great, great interest in host immunity. And of course, you know, we have these emerging viruses that are really interesting too.

Vincent Racaniello 1:43:38
Where did you do your PhD and was it virology?

Vineet Menachery 1:43:40
Yeah. So I went to Washington University in St. Louis. I was in the immunology program there as I came in through and I worked with David Leib on herpes simplex virus, working on how looking at host components that were driving differential disease I focused on, there’s an innate component that people have used. People are familiar with IRF3 and IRF7 and the interferon responses. And so I was looking at how different tissues have different requirements. So the brain really requires IRF3 to control the encephalitis portion and without IRF3, you could have control at peripheral locations. But once it got to the brain, the brain really needed this internal IRF3 more so than the systemic responses. And so that’s that’s kind of where I jumped into looking at. I was interested primarily in immunity I think, you know, I I operate on if I’m in a group of virologists, I say I’m an immunologist. If I’m with a group of immunologists, I say i’m a virologist.

Vincent Racaniello 1:44:43
That’s good. Matt Freeman told me that you would be a great spokesperson for this paper and he was absolutely right. You’re really articulate and calm and level headed, and I’m looking forward to a great career in virology from you.

Vineet Menachery 1:44:59
I appreciate that.

Rich Condit 1:45:00
I was thinking the same thing, great spokesman for the paper.

Vineet Menachery 1:45:03
I appreciate that. And I, that’s the nicest thing i think i’ve ever heard from Matt about me.

Vincent Racaniello 1:45:08
He’s a tough guy.

Vineet Menachery 1:45:10
He said

Kathy Spindler 1:45:10
He told me good things about you in person yesterday. So yeah,

Vineet Menachery 1:45:13
I imagine that our relationship is that we would never say that nice things that we think about those people to them Matt’s a great person. And he’s really helped me in understanding how the transition is working. And you know, I think what’s underestimated and I think Julie Pfeiffer kind of mentioned it about, you know, in all these gain of function stuff. One of the conversations I have with a lot of people that work with me, with Matt, with Tim Sheehan, who’s in our lab, other young people is, is it really worth this effort? We all want to do really good science. And we all have lots of ideas. Are we better off doing this with MHV? And not doing it with SARS? Because I mean, it’s a consideration. I mean, I think we worry, I worry, you know, Matt’s talked about this with me, you make a mistake, in your BSL3 lab, you know, an innocuous mistake, and you’re shut down. Yeah. So am I taking my career? Am I taking a huge career risk? It’s, you know, it’s very easy for me to do a lot of this work with the umbrella of knowing how well established Ralph is and how respected he is, and having these in place. But as a junior investigator, I have to ask myself the question, is it worth the effort? Is my department going to stand behind me if I make a mistake? Or if there’s a problem, or if there’s controversy? I think UNC is committed to Ralph in a way that he can, he can go out and make the you know, have these discussions and do these kind of works? It’s important to remember that this kind of work in the criticism and all that impacts people, I think it goes down the line. young scientists are really do we really do think about these things. And how they affect our career. For bad and for good.

Vincent Racaniello 1:47:04
That’s a great point. And this is something that Julie Pfeiffer brought up, originally with the moratorium, right? She wrote a little piece in mBio, about how it’s affecting the future scientists. And you know, and of course, you need to find a department that will support you. And you and you know, when you interview, you got to bring that up. You know, here’s this is, this is tricky stuff, and you’re going to support me. I tell you, Vineet, if I were a chair, and I hired you, I give you 100% support for this. So but I’m not so it doesn’t do you any good. It just makes you feel good. But that’s a great point. It really is. Now, I let me do a few administrative things here. I want to read just a couple of things. And then maybe we could do some pics and wrap it up. So I sound like a plan.

Rich Condit 1:47:47
Sure.

Vincent Racaniello 1:47:48
So the reason why we did this paper was I got an email, I think early in the week from Emily, who is a graduate student at UNC in the de Silva and Baric laboratories, you probably know Emily, Vineet. Right?

Vineet Menachery 1:48:00
I do know Emily.

Vincent Racaniello 1:48:03
She wrote, TWiV hosts, exclamation point, I want to suggest a new paper to discuss on the podcast published last week in Nature Medicine. And that’s the paper we did today. It has stirred up plenty of press about gain of function studies and the risk benefit, although mainly the risk of doing this kind of research and also made it some made some tabloid news sites that have completely overblown what the research says in exchange for a flashy headline. And she sent links to these, which was great, because I use that for writing my post and mentioned a few of them today, if commentaries throughout the internet, with bad headlines in it as well, she sent links to the UNC press releases, which we’ve talked about. So Emily, thank you very much for doing that.

Vineet Menachery 1:48:50
Thanks. Thanks. Thanks for nothing Emily.

Vincent Racaniello 1:48:54
No, I think I, I mean, I might have done it at any point, because it’s getting a lot of press, but she kind of made me do it this week. And then I thought of having you guys on

Vineet Menachery 1:49:06
Her excitement level this morning for it was very high

Great. That’s great. Yeah, I told her yesterday, we were gonna do this. And I I do think that we need to do more of this. Because you know, we typically talk among ourselves, and we have our opinions. And maybe people don’t care about that. But when you get the investigators in here, and you let them speak about their work, I think it’s more compelling in many ways. It’s great, but I do think that press, people need to listen to this stuff. You know, they want to ask their own questions, but they don’t often ask the right ones. I also want to read one follow up from last time. last page. Last week, we discussed two influenza virus papers. And one of them was from Kanta Subbarao’s lab at NIH and the lead author was Seema. And we had I had emailed her to clarify one of the figures beforehand. And I didn’t hear from her and then I heard from her over the weekend. She said, Dear Vincent, I apologize for the delay in responding to your question. I got your email after 1pm on Friday, wanted to make sure I had the correct answer from our collaborators doctors Rahm Seisikaran and Akila Jayaraman, who performed the glycan binding essay shown in figure 2B. After listening to TWiV yesterday, I realized there were some multiple concerns regarding that figure one What does present binding signal mean and two, what is the control that we are comparing the alpha to three h1 and one to two to G revert and virus to first in the glycan binding essay Akila normalizes each of the raw binding signals by dividing it with the maximal binding signal obtained in that assay. Typically, the maximal binding signal value is obtained for the strongest binder at the highest viral titer. In an assay, I believe this is as you suspected second in the text, we are comparing the revertant two to D glycan binding profiles shown in figure two B two the binding profile of the alpha two three h one n one virus that had a two to two g that we had published in the 2013 paper. Alright, so that’s the comparison to previously published data. So it’s normalized and compared to binding studies published before. I hope the answers that can hope this answers the concerns you and your colleagues had regarding figure to be pleased. Let me know if you’re any further questions. Kanta and I are thrilled that you picked our paper to discuss on TWiV and I was intrigued by your connection to the mammary gland transmission paper that came out recently. So Rich and Kathy, does that make sense?

Rich Condit 1:51:31
Yep, that’s all good.

Vincent Racaniello 1:51:34
This episode of TWiV is also sponsored by the 32nd clinical virology symposium of the American Society for microbiology. This is a meeting taking place in Daytona Beach, Florida in May of next year, May of 2016. Specifically may 19, through the 22nd. It’s the 32nd clinical virology symposium, and they’re now accepting abstracts, send in your abstract before March 17. to debut your latest research on rapid viral diagnosis, the clinical course of viral infections, and preventative and therapeutic modalities for virus infections, all before over 1000 researchers and primary care physicians interesting audience there. For more information, go to asm.org slash CVS 2016. That’s asm.org slash CVS 2016. We thank the American society, for microbiology for their support of 12. And is it okay, just to go to some pics now. So because we’re at an hour 20 and then it won’t be too

Kathy Spindler 1:52:53
might have to do an all email episode someday. But yeah, we should do one someday, right.

Vincent Racaniello 1:53:00
All right, let’s do some pics of the week. Kathy, what do you have for us?

Kathy Spindler 1:53:04
I picked linked to the California State Department of Health. Because I went to a fabulous lecture yesterday by the director of that state Department of Health, the State Department of Public Health, I should say, Karen Smith, and it was the susan b Meister lecture on child in child health policy. She gave a really good talk about the history of public health briefly, the history of vaccination briefly. And then she talked about measles, measles and Disneyland legislation in California. How outbreaks of pertussis led to some better legislation with respect to vaccination for children going to school, and and how timing is really important for that. And the Disneyland measles episode contributed to a complete elimination of the personal belief exemption in the state of California. And she did mention in the questions afterward that someone asked it was there any? Is anybody appealing that decision and, you know, the proposition, mode of government in California where you can get signatures and get something on the ballot, she said they didn’t even get enough, anywhere close to enough signatures to get that on the ballot. So that’s really good. Anyway, she talked about how press releases are static. And Twitter and Facebook are active. And so the California State Department of Public Health was very active on Twitter and Facebook within two days of knowing that there was this association of measles in Disneyland. They had it on Twitter. And interestingly Disneyland it first resisted this, you know who us and then they embraced it and she showed two slides of Mickey coats, they had it on their electronic Take boards that were really great, and I don’t think you’ll find them anywhere. So I want to read them to you. 1000s of vaccinated people did not get measles here. And then there was one with Mickey Mouse. And he’s in one of those big protective bubbles, that he might be in one of the Disneyland parades or something like that. And Mickey is saying, we can’t all stay in protective bubbles. That’s why Mickey vaccinates so from going to who us Disneyland fully embraced the, you know, you need to be vaccinated and come here, there’s 24 million visitors a year, and a large part of them come from out of the country, and places where measles is still an endemic problem. So that was really good. The webpage has a really cool link on the far right to data. And you can click down and look at all kinds of data for the state about infectious disease, West Nile virus, various other things. And so that was my way to have something physical that you could look to it. There’s a bio of Karen Smith on the website. And just it was amazing if anybody ever has a chance to hear her speak. Don’t miss it. It’s good.

Vincent Racaniello 1:56:12
Excellent. Nice. Great. It’s really good story to reach. What do you have?

Rich Condit 1:56:18
First of all, since we didn’t do the weather coming into this, I have to say that in Austin, Texas, it is clear blue skies. And I’m getting it I’m getting it as must be about it’s 69 degrees. So it’s a beautiful day. I have a pic from NASA TV. It’s a couple of months old, but I’ve had it on my picklist for a while called meals ready to eat expedition 44 crew on the space station members, expedition 44 crew members sample leafy greens grown on the space station. And this is about growing food plants in space. It’s a little deceptive because you see this picture up on the top. And it looks like they’ve got a whole greenhouse on the space station. But that’s a greenhouse somewhere else. And they’re growing these things. If you get into the article, you can see the conditions under which they’re growing these things. They have what they call plant pillows, which I guess are bags of dirt and nutrients and seeds and stuff that they grow lettuce and other things in including zinnias they grow flowers. And they’ve been knocking on these things recently. So this is important for the future long range missions and stuff. And so I just thought it was interesting. Sorry Dixon’s not here. I thought he might be interested as well. But I thought particularly interesting that they thought that the thinking is that growing plants in space is all has also Vincent pay attention psychologic psychological benefits, okay, if you’re gonna be up in space and going to Mars and stuff, you’re going to be better off if you’re growing some plants and houseplants and growing flowers and stuff like that. It’s good for you. So there you go. Well,

Vincent Racaniello 1:58:15
that’s great. I’m not going to Mars. So I’m not growing plants on Mars, and I hope to the people who do go there grow plants. Yeah. That’s pretty cool. The weather here is really nice. It’s beautiful. Today, it’s a cloudless blue sky, 14 degrees Celsius. And no rain in the forecast. Beautiful. It’s actually been warmer than than usual. Vineet, what’s it like down there in Chapel Hill?

Vineet Menachery 1:58:42
It is also beautiful here. It’s sunny, about 60 degrees. I didn’t. I didn’t put a coat on my kid today. And I think it was okay.

Vincent Racaniello 1:58:50
Nice. How about Kathy out in Ann Arbor, which is

Kathy Spindler 1:58:54
sunny, not a cloud in the sky that I can see. And it’s 45 Fahrenheit, which is seven Celsius. Nice.

Vincent Racaniello 1:59:02
Well, all over the place. It’s pretty nice. Sorry.

Kathy Spindler 1:59:04
Supposed to get snow tomorrow, though.

Vincent Racaniello 1:59:06
You are really? We have good weather tomorrow. This morning, by the way, guys. A stump grinder appeared on my property. The town. My town took down a big tree at the edge of our property. You know, there’s this strip of land which is eminent domain, right? The town owns that in theory. And this tree was a big old oak about three foot diameter and it was dying. So they took it down. And then today the guy came with a grinder. And I was gonna go out and take a picture for everyone. You know, it’s this big wheel with spikes on it. And this is this was a about an eight inch high three and a half foot wide stump. He was done in five minutes.

Rich Condit 1:59:46
Yeah. Great machines are

Vincent Racaniello 1:59:49
unbelievable. And he left his big pile of chips which I have to move away this weekend. But I thought of it all of you guys on TWiV, because we had a stump grinding episode right

Rich Condit 1:59:59
now. We had a stump grinding episode and that was it. Coincidentally, that revolved around Buda. As I recall, and I had lunch in Buda yesterday with Niva

Vincent Racaniello 2:00:10
cool how’s Niva doing well,

Rich Condit 2:00:12
he was doing fine, great. It’s just great and beautiful fine place to be.

Vincent Racaniello 2:00:18
I have two picks for you today. The first one has to do with computer keyboards. Now, I do I figure I spend most of my day in front of a computer in some way. And I’d like to have a nice keyboard. And a few years ago, I learned all about nice mechanical keys. And there’s an article at life hacker called How to Choose the best mechanical keyboard and why you’d want to I like a clicky keyboard. That gives you a lot of tactile feedback. And I love typing on that I have one at home. I don’t have one here because it makes too much noise during recording podcasts. But the original IBM keyboard years ago was clicky. And one of the apple ones was clicky, too. And now they make them again. And this article will tell you about all the switches. So every key, of course has a switch under it. And the clicky ones are called cherry switches. And they have cherry black, red, brown, blue, green and clear. And they all have different tactile feedbacks. And some of them are good for typing. Some of them are good for gaming if you need to have a really fast pressing of the key, right. I love this stuff. So very few people will care about this pic. But there’s going to be some of you out there who are into keyboards. So the one I have home is made by dos. It’s called the das keyboard and it’s very clicky. And I feel like I’m working. When I hear that click click click click. So that’s a cool article.

Kathy Spindler 2:01:48
They had typewriters in the olden days.

Vincent Racaniello 2:01:51
And they clicked a lot. Yeah.

Kathy Spindler 2:01:53
And had the little bell.

Vincent Racaniello 2:01:54
Yeah, the bell is the return. I used to use those for sure. And you’d hit the metal thing to push the carriage over. Right, right. That was called a carriage return. And that and that stuck out key here.

Rich Condit 2:02:06
It says return you and if and if you push too many keys at once you jammed all the little hammers up. Boy people there’s going to be a lot of people who don’t know anything about this, we need to include a picture of a typewriter as the episode or something like that.

Vincent Racaniello 2:02:21
Well, supposedly the the QWERTY keyboard was made to slow down typist so that things wouldn’t jam, right? Because it’s not the optimum layout of keys. And now they’ve stuck Of course, well, you can buy a keyboard that’s arranged in the optimum way. But you’d have to learn how to use it. One of the best things I ever did. I think in my freshman year of high school, I took a summer typing course. And I learned how to touch type, which to this day has stuck with me. And one of the best things I ever did. Anyway, that’s cool article and the other one. Earlier this week or last week, I was contacted by a fellow named Mike Carruthers. And Mike is out in LA he is he’s been a radio guy for 20 years. He’s got a radio show. And he decided not too long ago to make something called perfect your podcast. It’s a website and he does podcasts called perfect your podcast. If you don’t know it, podcasting is really hot right now everybody is getting into it. And, and at the same time people want to teach you how to do your podcast. So there are dozens of sites all over about how to make a podcast how to do this and that and the other, which I find really amusing. Because when I started with, I don’t know 2008 there’s none of this around. I had to figure it all out myself, which is actually part of the fun. So anyway, Mike interviewed me. He interviews people about their podcasts. And that’s my other pick, Episode 12 discussion with with Vince American ello this week in virology. And he was amazed that I have these niche podcasts that do so well. You just listen to it because he says how do you make it not boring, and he actually had listened to an episode and he said it’s really entertaining and easy to listen to. And he said you have great co hosts too. That’s what makes it really interesting. He writes here if you think virology sounds too Nishi or academic, he has also pad cast on parasites and one of microbiology. Those are topics would be very easy to make boring or appeal only to academic types. But those people do not make up the majority of instance audience. So if you’re interested in I talk about the art and science of podcasting with Mike and I also told him about the twitch bump and he said I really would like to feel the twitch bump. I said okay, I will pick your website in the podcast. So folks go and listen to it and show him what this twitch army can do. We have two listener picks one is from Jessie. The twist. I’d like to submit a listener pic of the week a book called networking for nerds by Elena Levine. It’s a great resource. for academics and also those pursuing other career paths, and discusses what networking is connecting with others for mutual exchange of value, promoting your own brand, how to interact with others and find people to interact with and more. For those that institutions with subscriptions to Wiley publishing, it may be downloadable as a PDF for free gives a link to that. For others, it’s available from Amazon Keep up the great podcasting. So Jesse is Jesse nor have bacterial files that’s a very nice podcast about bacteria and sometimes viruses. Jesse’s a PhD student down south and he this bacterial files is on microbe world.org. It’s supported by ASM so you should check it out. Give Jesse the twist bump.

Kathy Spindler 2:05:44
It’s spelled b a c t e r i o f i l e s,

Vincent Racaniello 2:05:50
right.

Kathy Spindler 2:05:51
That’s very good. I Ph.

Vincent Racaniello 2:05:53
That’s a nice. I really like that title. I think that’s very good. As opposed to auto immunity, which, which I didn’t even say right.

Rich Condit 2:06:00
That’s right. Audio immunity.

Kathy Spindler 2:06:03
Yeah, there you go. Rich. Alright. The

Vincent Racaniello 2:06:05
other pic is from Russell, if you haven’t done world mapper, you should great series of maps, including those showing the global importance of a large number of diseases, including infectious diseases using a unique map format. The higher the prevalence in a country, the bigger it is on the map really interesting to look through them. Look at disease, and cause of death. Russell is a professor of clinical pediatrics at Tulane University School of Medicine. And finally, last week, I mentioned a contest to win a brand new to volume copy of principles of virology, the fourth edition just published. And hardly anyone responded, which tells me Nobody listens to the end.

Kathy Spindler 2:06:51
We got it. We got some.

Definitely we got a couple right. I would say, Well, I probably had 10. People really had me. Yeah,

Rich Condit 2:07:01
yeah, at least problem is Vincent, you have a really high background of Twitter followers. You got to you got to go with guys like me who have hardly any followers, and then I can I can see the signal.

Vincent Racaniello 2:07:14
Right, right. Well, I just think that, uh, I can’t imagine you wouldn’t want to have a free, you know, textbook set, and I have a lot to give away. But I’m not going to do it on Twitter. It isn’t, you know, it’s not. I want to try to promote TWiV on Twitter. But obviously, it’s not working. But you know, I’m going to let it go a little more and see what we collect. And then we’ll figure out another contest to give away a book set. So you know, that they say, of all the listeners, maybe less than 1% ever respond to anything. But I figured if you give things away, they would remember the Drobo contest we did that was pretty fun.

Rich Condit 2:07:48
Yeah. Yeah, that was fun.

Vincent Racaniello 2:07:50
All right. You can find this in all the other episodes of TWiV at twit.tv. Also on iTunes, and if you have questions or comments, send them to twiv@twit.tv. Today, we had two very special guests, Ralph Baric, from the University of North Carolina who has left us a long time ago because he has important things to do. We thank him for joining us and a postdoc in his laboratory, Vineet Menachery, right.

Vineet Menachery 2:08:16
Yep.

Kathy Spindler 2:08:17
Menachery.

Vineet Menachery 2:08:19
Menachery.

Vincent Racaniello 2:08:19
Menachery

Vineet Menachery 2:08:21
You can call me Vineet

Vincent Racaniello 2:08:22
Vineet Menachery. Thank you so much Vineet.

Vineet Menachery 2:08:26
Yeah, it was great, thanks for the opportunity.

Vincent Racaniello 2:08:28
It was really good. And I’m sure we will hear from you again. Are you going to go to ASV next year?

Vineet Menachery 2:08:33
That is the plan, in Virginia Tech?

Vincent Racaniello 2:08:35
Yeah.

Vineet Menachery 2:08:36
Yep. That’s the that’s the plan at the moment.

Vincent Racaniello 2:08:38
Seek us out and say hello, we’ll do a twig there. But seek us out at some point say hello. I’d like to meet you.

Kathy Spindler 2:08:44
And we know you have important things to do to

Vincent Racaniello 2:08:46
Yes.

Kathy Spindler 2:08:47
But we’re happy that you were able to stay a little longer. Yeah,

Vineet Menachery 2:08:50
I am excited to be described as special. So

Vincent Racaniello 2:08:53
You are a special guest for sure. Kathy Spindler is at the University of Michigan in Ann Arbor. Thank you, Kathy.

Kathy Spindler 2:09:00
Thanks. This is a lot of fun.

Vincent Racaniello 2:09:02
Rich Condit is usually in Florida. He is an emeritus professor at the University of Florida in Gainesville. Thank you Rich.

Rich Condit 2:09:11
Sure thing is always a great time.

Vincent Racaniello 2:09:12
You had a good signal there from Austin.

Rich Condit 2:09:15
Oh, yeah. This is a good podcasting. Good podcasting plays and it turned out that the change in time was just right. This fits. fits great into my day.

Vincent Racaniello 2:09:25
Are you at your daughter’s home?

Rich Condit 2:09:27
I am at my daughter’s home sitting at the breakfast table. Yes.

Vincent Racaniello 2:09:30
It’s really quiet there.

Rich Condit 2:09:32
That’s because all the kids are in school and all the parents are in work and it’s just me and my wife. Here. It is. I

Vincent Racaniello 2:09:37
know it’s right. It is Friday. So you’re hanging out there till Thanksgiving.

Rich Condit 2:09:42
Hanging out there through Thanksgiving. Part of what we’re doing is grandparenting here because the parents are extraordinarily busy the Harper age seven is got a singing part in their Texas state’s production of Evita which is outstanding I can’t believe what a What an amazing theater program they have here and so my my daughter Let the show and Harper’s in it. And my daughter’s working on another show every night so it’d be and I are helping out taking care of the kids cuz it’s pretty crazy around here. I’ve seen Evita three times this week so far, and I’m gonna see it another four.

Vincent Racaniello 2:10:24
Nice. I’m Vincent Racaniello, you can find me at virology.ws you’ve been listening to this week in virology. Thanks for joining us, and we’ll be back next week. Another TWiV is viral.