TWiV 1353: Phage bet-hedging and EBV antibodies

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

0:10From Microbe TV, this is TWIV. This Week in Virology, episode 1353, recorded on August 28, 2026. I'm Vincent Racaniello and you're listening to the podcast all about viruses. Joining me today from Austin, Texas, Rich Condit. Howdy. Sunny and 93 degrees. Shockingly cool. We had a little mini cold front come through yesterday. It was very windy. Dropped a little bit of rain. Much needed. So it's a little cooler today. That's good. Well, I'm in Washington, D.C., and it's a little warm here, but my phone is being used as a camera and I can't get the weather on my, here we go, Washington 29C and mostly cloudy.

1:04There you go. And I forgot to bring my computer glasses so everything is fuzzy. Oh, wow. I've got, some time ago after I got my cataract surgery, I've got stuff going on in my eyes that can't be corrected with cataract lenses so he just gave me some sort of intermediate distance and says you're going to have to wear glasses and I got myself for the first time progressive lenses and man that solves a lot of problems yeah that's what I have here I can read the dashboard I can see the computer and see all sorts of other stuff at the same time the problem is that to look at something close you have to tilt your head i don't like that so i get computer glasses but some i didn't somehow that doesn't doesn't bother you somehow that doesn't bother me but i see you looking up all the time like there yeah i can see up your nose yeah okay so i guess i do that but it doesn't bother me doesn't bother you so what's the difference Also joining me from College Station, Texas, Jolene Ramsey.

2:15Hello. Good to be here. It's 96 degrees Fahrenheit here at 36C. It's warm. I walked across campus and still drying off. But, you know, it's the height of summer right now, so it's okay. And from Madison, New Jersey, Brian Barker. Hi. Hi. It's a pretty lovely 82 Fahrenheit, sunny today, so not too bad. And I also vote for progressives. When I had to get glasses a couple of years ago, because I couldn't read tubes in the lab anymore, I went with progressives and they've been great. Are you wearing them now? I am. Because I find for the computer, I use a single magnification that works so I don't have to tilt.

3:07I don't like tilting. But it's a personal preference. So I'm in Washington for an influenza meeting. Tomorrow is Saturday. I'm going to be doing a TWIV at this. Options for the control of influenza. But it's pretty dead here in D.C. The Uber driver, she was telling me there's nobody around. I almost went home when I got your call. And the airport is empty. Newark Airport this morning was empty. So everybody's away, I guess. It's pretty interesting. If you enjoy these programs, we'd love your support to do them. You can go to microbe.tv slash contribute. The big news is that this week was both Alan and Angela's birthday on Wednesday, the same day.

3:52How about that? Happy birthday. Yay. I have almost everyone from TWIV's birthdays in my calendar to send out a message. Yeah. Almost everyone, Jolene. That's proprietary information? Yeah, I don't have Jolene. Actually, yeah, I got everybody else, so that's why I knew this. But it's pretty interesting that they both came on the same day. I don't know what the likelihood of that is. I think in a room of 30 people, it's likely that two people have the same birthday. That's the way that thing goes, right? But of course, also this week, Dolly Parton passed away. And I wouldn't normally talk about it here, except that she was a big advocate for vaccines, for public health, for literacy, for marginalized communities.

4:46And of course, the first cloned sheep, Dolly, was named after her. So there's a nice article in Nature. Unfortunately, it's paywalled. But you can see part of it. And it goes through some of the things that she did. You know, when the pandemic started, she donated a million dollars to Vanderbilt to help them do stuff. That was great. And she gave money left and right. And they say here, basically, she chose not to be a billionaire. Yeah. Yeah. It's pretty remarkable some of the choices that she's made and the way that she's impacted communities. Most of us, me included, who, you know, make it big after you come out of a place, then you don't go back and you don't always help to lift out the place that you left.

5:39But she definitely did not do that. She made concerted efforts over time, not just in her family, but in her community to make available infrastructure and resources for people to be able to study. And then she always used her platform to advocate for people and their health, which I find amazing. I've been sort of, I don't know if inspired is quite the right word, but I've been sort of inspired this week as we keep hearing about stories of all of the wonderful things she did as we're reminded of them. And, you know, both thinking, yeah, that was, that's great. And like, we all need to do that, but also being really excited about how broad the respect was and how many people are easily able to recognize how good these things were.

6:28Yeah, cool. I'm really happy to see somebody on the radar who seems like nothing but good. Yep. And for everybody. You know, nobody has trouble with this. Except the fact that she died. Cool. Great stuff. Someone said, I didn't like her music, but I liked her. She sang quite a range of different kinds of music over her lifetime So I can't say that I always listen to her music But there was one song in particular that I heard quite a bit And still do Nine to five? No That's not the one Jolene Jolene All right, sorry about that We had a guest on Twin a couple weeks ago. Her name was Melissa. And I said, did you ever listen to the song Melissa by the Allman Brothers?

7:31And she said, yeah. We played at my wedding when my daddy gave me away. So cute. Melissa. Who put in the Scientific American? Me. Go ahead. So, yeah, my wife, she reads the paper to me every day. and she came across this. On this day, August 28, 1825, 181 years ago, Scientific American, the first issue was published. So Scientific American is 181 years old. Not only that, but it is still the oldest continuously published magazine in the United States. So that's cool. I like that very much. I think this is also the anniversary of the I Have a Dream speech. I have to look that up to confirm it

8:24and if you're just counting deaths yeah Tim Curry died this week but I don't think he did anything science-y so yeah August 28th 1963 the I Have a Dream speech August 28th how about that right here in Washington on the reflecting pool before it turned green right Right. Well, I mean, the reflecting pool has a history, apparently. Yeah, it's unusual. It's very shallow, right? Because they... Why did they make it shallow? They didn't want to have ripples or something? They wanted to reflect, I guess. Yeah. So, and that makes it susceptible to microbiological growth, I suppose. All right. Speaking of microbes, Jolene is going to tell us about viruses that infect microbes.

9:11That's right, as I tend to do. So today I picked a paper from Nature Microbiology called Phage-Encoded Contingency Loci Enable Bet Hedging Against Host Defense Mechanisms. It was just published this month, and there are three authors, Jasper Gomez, Jeffrey Barrick, and Christopher Waters. They are all at Michigan State University. And so I thought this was a really interesting concept. The paper itself is relatively short, and it's not something that's well studied in phage, but it's actually observed all across various different kinds of DNA and RNA-based organisms. So this is the idea that the way that heterogeneity or kind of individual differences is encoded in the genome can be based on the kinds of DNA that's there.

10:20So we know that the DNA encodes for proteins by having triple nucleotides that can be interpreted into an RNA that gets made into an amino acid, and that is considered the genetic code. And we also know that if you then change a nucleotide, then that will change the amino acid. But there are other ways to get variability in the sequence that comes out of an RNA. And one of those is often frame shifting. So that's like during the translation of an RNA to a protein, you can have the ribosome move and then keep reading. And so instead of being able to read the sentence, as it were, in order, then you get something else.

11:12But this is different than that. So that's what I thought was cool. This is the case where a gene has a region of the DNA that is several of the same nucleotide in a row, a homopolymer, or what they call here in this paper a simple sequence repeat. And so instead of having a translation frameshift, what you have is during replication of the DNA, you can get one or more changes, additions or deletions of that base. And that can then change whether or not the sequence is coding for a functional protein or not. And so that's what this contingency locus is meant to represent. And the bet hedging aspect is something that I hadn't heard of in terms of genetic heterogeneity.

12:07So in this case, the sequence is actually changing because you go from having, say, like six of the same nucleotide to five or having seven. More I've heard bet hedging in the context of having the same genetic sequence but having a different phenotype expressed. And that might be just due to slightly random differences in which genes happen to be on or a programmed change in what is being expressed. Go ahead, Brianna. I was just going to say that this way of getting genetic variation is more dangerous in some ways than the other mechanisms that Jolene talked about. It's one thing if you make one RNA and then the ribosome frame shifts, you still keep the piece of RNA.

12:58And importantly, you also still keep the same piece of DNA so that if, say, the product is non-functional after your frame shift, you still have the original DNA. You can go back and make the functional product. Here, making the change in the actual DNA itself during replication is a potentially much more costly strategy because if it results in you making a non-functional protein, you're out of luck in terms of having the gene encoding that protein. And so it's a really interesting mechanism to think about in terms of how it might act towards populations versus individuals. And, of course, particularly interesting to think about with haploid organisms that only have one copy of their genome, unlike us, where we've got two copies.

13:56So if one gets messed up, there is that other one as a backup. Yeah. So I was going to ask you to explain why it's bet hedging because that's something from gambling, right? So why does it apply to this? Yeah. So the idea being that the population has some variation naturally due to this slippage that occurs. And so most of the sequences are going to be favorable for that environment because they're already replicating there. And then you have these variants that now maybe can't make the protein or can make the protein. And those ones might not be as fit in that particular environment, but as soon as the environment changes, they may be more fit.

14:36So that you have a low-level variation always there that could be the better fit for the next environment. Got it. Yeah. And so just to be clear, the change is at the DNA level, and it does involve kind of an error during replication that is due to the DNA polymerase slipping. And we'll see that this has a tendency to be one type of base versus another. Okay, so that's kind of just in general the bet hedging idea. So it's to help the population survive better in case of a changing environment. And in this particular study, they were studying this in the context of the co-evolutionary arms race between bacteria and phage.

15:26This group had previously reported the discovery of a new defense system that the bacteria used to defend against bacteriophages, and that was a type 4 restriction modification system. So restriction modification systems are proteins encoded by usually bacteria that recognize DNA, and they then are directed to cut that DNA. And so bacteria use this to defend against bacteriophages, in this case bacteriophages with modified DNA. So this type 4 restriction system can recognize modifications that are very common on bacteriophage genomes. And the modification in this case is a glucosylated 5-hydroxymethylcytosine.

16:18This is something that's really common in the specific family of bacteriophages that T4 is a member of. These are called the T-even bacteriophages. T4 is one of the most well-studied phage, has been studied for a long time. It has a big genome, and all of its cytosines are modified in this way. So they are then susceptible to this type 4 restriction system. So why does it modify a cytosine? Is that not an attempt to protect itself from other restriction modification systems from the get-go? Yes. Well, that is one effect of modifying the DNA, certainly. Okay. And yeah, I'll leave it at that. So the phage has a method to evade this defense system.

17:12It encodes a gene that allows it to get around the restriction system. So in this paper, they first found that the gene that encodes the defense against the defense has a short sequence that is a repeat, and that's the contingency locus is what they're going to tell us. And so what they first did was they took advantage of the fact that when the phage had the anti-defense gene, then it could survive just fine in the presence of the defense. But when it did that, it couldn't survive in the presence of another defense that would target the DNA. So there was this tradeoff that it was making by not modifying its DNA, just like Rich mentioned.

18:06So maybe you're going to go here, but the phage defense gene is in at least one of the enzymes responsible for that base modification, correct? Yes. So the alpha-glucosyltransferase gene, or AGT, is what helps add the modification to that DNA. So you can get around this particular bacterial defense system by not doing that, but then you make yourself susceptible to some other. Right. So it'd be nice to be able to sort of say, hey, what are we facing here? Which one of these tricks do we need? Mm-hmm. Yeah. So when they lose the ability to modify the DNA, now it can survive on the type 4 restriction system defense, but not on the other one.

19:00And when they looked at the part of the genome that seemed to be responsible, the AGT gene, specifically at the sequences, they noticed that there was a repeat of adenines. So there are seven, sorry, one, two, three, four, five, six, seven, six or seven, depending on the wild type itself has six. And then there's a mutant that had, I don't think I'm counting those right, one, two, three, seven. The wild type has seven. And then one of the mutants, one of the sets of mutants that they found had six. And then another set had eight adenins in this spot in AGT. And so those deletions or insertions were changing the ability of the RNA to make the protein, changing the reading frame.

19:54And you can imagine, if you think about those numbers, 6 versus 7 or 8 is pretty important because something that's 7 or 8 is going to be a different reading frame than something that's 6. Exactly, because of the triplet nature. So like 3 makes or codes for an amino acid. Okay, so they saw this in multiply independently isolated mutants. They also saw a few mutants that were not in this homopolymer of adenines, and those actually changed the sequence of the protein. And so what they did was they then compared the mutants that were in the homopolymer or the contingency locus, as they call it, and then they compared that with a mutant that would change the base to something else in a different spot in the gene.

20:50And they looked at how often did they get reversion, and they could put the selection pressure for going back to the original sequence or the ability to make the AGT that could modify the DNA based on plating it on the bacteria where they otherwise couldn't grow that had the other defense system. And they looked at how often did they get a change in the sequence for the contingency locus, and that was actually quite high compared to the frequency at which they got a change back in the regular mutation that changed the coding sequence. So they saw that the frequencies were quite different. I think they said it was 10 ,000-fold different reversion frequency.

21:43Now, that was interesting to them, and they wondered if this kind of rapid ability to evolve would be a benefit in more than just this case, where it could switch back and forth between which defense it was actually able to defend against. and they so what they did was they took their they sequenced to get these original frequencies they took that information of the sequencing and they looked not just in the phage that they're working with so I told you that these are the t even phages the really famous one is t4 they actually did their studies in t2 and there's also another one called t6 all of these are part of an original, sometimes called set of seven dwarves of phage that were isolated and studied by a number of groups a long time ago.

22:40And so they compared these T2, T4, and T6 genomes, and they saw that this locus had this contingency locus in the same place. And they then looked across the entirety of the genome, and they compared how often did they get the difference in reversion across them. And so when they propagated these three different phage, and then they isolated five of them from each of them that had been propagated, and they sequenced them, they saw that sequenced to 2 ,000-fold, so like every single spot they checked 2 ,000 times. They found every sequence within their genome that had at least six bases that were the same nucleotide in a row, these SSRs, and they saw a lot of them.

23:44There were some that were encoded between genes, but they focused on the ones that were coded inside of genes because then you know that if you get a change one nucleotide more or less, that you're going to affect the protein product that would be coming out. And they saw the majority of them were about six. They had some that were seven and eight base pair long. And they also noticed that the majority of them were AT homopolymeric runs and not GC homopolymeric runs. And I think the significance of that, as far as I understand, is that AT base pairs only have two bonds that hold them together in their complementarity configuration, and Gs and Cs have three.

24:36And so when the polymerase is going along, the DNA polymerase is going along and replicating, it's easier to break AT bonds and slip than it is to break GC bonds. So as they were analyzing where all of these simple sequence repeats were found, they wanted to know how often were they not just in the T-even phages, but were they found in other phage? Is this something unique to these ones? So they took advantage of a set of phage called the Basel Collection. This is something that was isolated, I believe, in Alexander Harms' lab. And it is representative of many of those original phage that have been studied as well as others.

25:24So it kind of has representatives from many different types. They took 41 of these from the Basel Collection, and they looked at their genomes to find any of these SSRs. And they did find them. They definitely found them across these genomes. They noticed that the amount that was in the different genomes varied. So for example, a phage called P2 seemed to have two SSRs per kilobase, and that was a little bit unusual. There were a few of them, including T7, that had none. And then again, they saw this same pattern that the GC SSRs were not nearly as prevalent as the AT SSRs. I'm really amused by the fact that T7 has none.

26:15T7, of course, is my old buddy. I did my thesis on T7. And I have always, I think Bill Stadier even described T7 as business-like, okay? No nonsense, okay? None of this fancy stuff. We're just going to go in and trash the joint and get out. Right. I mean, that's really what it does. So maybe it has not been an advantage for T7 based on its chosen mode of infection. So the next thing that they did was they wanted to know what kinds of genes had the SSRs, and that they binned all the genes that had them and saw what they do. They noticed that they, in general, they were fewer than you would expect in the genes that were involved in replication.

27:13So polymerases, for example, were the least common to have an SSR. Those are essential. I mean, if you lose your polymerase, then you're done for. So not surprisingly, the regulatory genes, or what some people might call accessory genes, tend to have less or more. They were less depleted in SSRs. They had more than other categories of genes. So then they selected another example from the T4 genome to see if they could replicate their contingency locus observation that they had seen with the first gene, the AGT gene in T2. And they selected the gene called R2A in T4. And this is one of a pair of very famous genes in, not just in phage biology, though, in the entire field of genetics and molecular biology.

28:11And that's because this gene R2, which is actually two genes, R2A and R2B, were the ones that were used to determine that there is a triplet genetic code. And they were also used for demonstrating homologous recombination. So they have a lot of literature about them, even though we don't entirely understand how they work. and turns out that R2A had a simple sequence repeat of six adenines in it. And conveniently, R2A also has a phenotype associated with it, so they were able to take advantage of that for their studies. So first thing that they did was they checked their sequencing reads And they looked to see whether there seemed to be an elevated frequency of insertions or deletions in the SSR.

29:12They didn't really see much of a statistically significant change compared to what they saw everywhere else in the genome. So they then took that information that's in the literature and they just made a change in R2A in those six adenines. And they also picked another spot that was already known to be a mutation that would make it not functional. And they took those two mutants and they compared them. And the way that they compared them, I have to give you a little bit of background here, but just a little bit. So R2 is known to be important for the plating of this phage onto a particular host.

30:01So in this E. coli host, it has a lambda bacteriophage already in the genome. And lambda encodes basically an antiphage defense system called REX. So when REX is there, then you need R2 to be able to plate, to infect and make a plaque. But if R2 is not there, then it won't make plaques on this E. coli. And so this is setting up a similar system to what we saw with the AGT gene, where if you have the gene, then you can plaque. If you don't have the gene, then you can't. And so they took advantage of that to look and see if they got a change in their frequency. And they did. They did see that there was an elevated rate of reversion in the mutants that were in the SSR.

30:59and there was not nearly as much for the other change that they had made that was what I might call a normal mutation that changes the coding sequence. And that's not surprising, I think, based on the way that we think that the SSRs are working, but it was really cool that they could demonstrate it with their actual phenotype of can the phage plate or can the phage not plate. Here, most of the time, accessory genes don't have a phenotype that's easily trackable, but the R2 genes do if you plate them on the host that makes that RECS defense system. And their overall conclusion is that this region of the R2 gene, as well as it seems many other genes, may have these contingency loci.

31:51They are genetic heterogeneity that allows for the phage to be able to adapt rapid, the phage population to adapt rapidly to a change in the environment. And this is also what is known as the red queen co-evolution between bacteria and phage. So they're constantly having to go back and forth. And just slipping the DNA polymerase here and there is way easier and faster than having to acquire a new gene or fix a gene that is broken. You just always have some that's not working and some that is. So I thought this was really cool. It's involved in some phenotypes that I'm interested in, and it seems like it's pretty widespread.

32:42I know that viruses of eukaryotes do this as well. And I think that there are also cellular genes that do this, but cells grow at not quite the same rapid rate that viruses do. Yeah, that was sort of the thing that really surprised me in this paper was both in sort of the intro and the discussion where they talked about how much contingency loci were sort of understood in bacteria systems and eukaryotic systems and were not really well known as much in phage and even were not known quite as much in archaea. and so um i guess when i was reading and thinking about this contingency locus it made sense to me i sort of was like oh yeah i've heard of things like this before um and the idea that it wasn't known in phage um when it sort of makes sense as a really useful way to evolve rapidly amongst the a population and not have to exactly, as you said, gain new genes or make nearly so many changes.

33:50It sort of fits with our understanding of phase, or at least my understanding of phase really nicely and allows a population of phase in these cases to sort of get around multiple bacterial immune defense mechanisms. And so I thought it was kind of interesting that it hadn't been described in phage before. Yeah, I think it's really based on the idea that so many of the genes are, we don't know what they do. So it's like, okay, so you've got some variation here. How do you show that it's conferring an advantage in a changed environment? So as I was reading this, I think to myself, this is really familiar.

34:36And I sent, I looked in my own archive, because what I was seeing in my head was a string of Ts in some gene that we studied. Okay. And it turns out that, yeah, I was able to dig this out of the archive and I sent it to Jolene. I did see that. This is a really weird gene in vaccinia called J3 that has in our hands at least three different, well, our hands and others, three different activities. It's a 2 'O-methyltransferase. So it methylates the ribose of the second base in an mRNA cap. And so that activity, in fact, makes vaccinia messages look like cellular messages to subvert some of the cellular mRNA-based defense mechanisms.

35:32In cell culture, so biochemically, this thing also has some sort of transcription elongation function that we never fully understood. And it also biochemically affects the length of poly-A tails. It's an accessory protein on the poly-A polymerase that affects the length of poly-A tails that the virus makes. All of these functions I could envision as being sort of contingency functions. And if you blow this gene off completely, and we see, so we found the gene, because of the transcription elongation function, if you don't have it, it doesn't grow on normal, the normal cells and culture we use.

36:17But there's a drug that enhances the transcription elongation that has the effect of correcting that phenotype. So the mutants were drug dependent. but you know who knows in other cellular environments uh whether this might have a oh so when we asked isolated drug dependent genes drug drug dependent mutants we got a whole bunch in this uh string of t's okay um because they're easy to make uh for for the virus so there may be some circumstance in vivo where this is more a contingency thing rather than a black and white grow or not grow in cell culture. So it made me think of that. And Jolene, you said eukaryotic viruses have these things.

37:06Has somebody done anything like this phage paper where they take a whole bunch of different viruses and look for these sequences? I don't know. Yeah. Yeah, it was not really described in depth here, but I did a little bit of other reading. And there are some people who are very interested in these in pathogens. And I was looking at it because there's a phage that we study that has one of these. It's not in a coding sequence, or maybe it is. But anyway, the reading that I had found about it was more looking in pathogens, antibiotic resistance genes apparently tend to have these kinds of sequence repeats.

37:50Yeah. I'm not sure which genes in eukaryotic viruses have them, though. Okay. So apparently eukaryotic viruses do not have these. They don't have any? Contingency sequences, but they achieve the same effect in other ways, basically. Like high mutation rates. It feels familiar, though. Yeah. But, Jolene, this is probably present in other phages too, right? Because they just chose to look at this set. Probably, yeah. I think this seems to have been a follow-up from their type 4 restriction system work. I'd like to see, and maybe it's already been done, but I'd like to see these guys or some other eukaryotic virus person do this same kind of thing where they do extra deep sequencing.

38:44To see if there are sequences in a population of viruses that show sort of hypermutation in one of these stretches of sequence. I thought that was a clever way to approach this. I also am blown away still by the way genetic mapping is done nowadays. It's just sequence stuff. Sequence the genome. Unbelievable. I didn't emphasize this before, but it's kind of hard to – you have to do that deep sequencing because these homopolymeric repeats are the regions that sequencing doesn't resolve well. When you have those runs, they make errors. You're not so confident in the sequence that you get back. For the same reason that they're immutable, right?

39:33Exactly. It's polymerase-based or there are so many of them. Thank you, Jolene. Very cool. I like that. So the Red Queen told Alice, now here you'd see it takes all the running you can do to keep in the same place. Yeah, I remember very vividly in an evolutionary biology class having our professor actually read from Alice in Wonderland, the section leading up to that quote. And I imagine it every time I think about Red Queen evolutionary dynamics. I imagine that same passage over and over. All right. Now we will move to eukaryotes. And this is a paper in Cell Reports Medicine entitled, Transgenic Mouse-Derived Human Monoclonal Antibodies Targeting EBV, the Epstein-Barr virus GP350 and GP42, provide basis for therapeutic development.

40:40Yeah. So the authors are, first author is Crystal Chan and last author, Andrew McGuire. They're all either at the Fred Hutchinson Cancer Center or the University of Washington in Seattle, several different departments there. every time i see a paper from the hutch or uw vincent i think about our visit there yeah that was just awesome i came away with such a wonderful impression of the place as you know just a wonderful place to be the the the either institution on its own uh is amazing uh and the overlap and collaboration between the two is amazing yep so i wanted to just give a super brief overview of this, and Vincent will give the details.

41:34So this is Epstein-Barr virus, and the idea is they wanted to have antibodies that could be used prophylactically to reduce diseases that come from Epstein-Barr virus. And so they did a mouse study where they made it make human-like antibodies, and then they tested whether they would protect from EBV, and they were honed in on the right kind of antibodies to protect from the disease that they were testing. And they found a couple. So now we can hear the details. So most people have... We've talked a lot about EBV in the past year. A lot of cool things have been happening. Most people have been infected with EBV, and the most common disease that you can get is mono, infectious mononucleosis, but it's also associated with multiple sclerosis.

42:30We've talked about that work, SLE, rheumatoid arthritis, complications of COVID-19, and it was also the first virus shown to cause cancer in humans. It is associated every year with 358 ,000 cases of new cancers and 209 ,000 deaths globally. So not insignificant. Now, this virus infects primarily epithelial cells and B cells. And so those cancers are typically originating from those cell types in people. We'll talk about that a bit later. So the virus is a herpes virus, a large DNA-containing virus. It's enveloped with a double-stranded DNA genome in an icosahedral shell within the envelope. And there are a bunch of glycoproteins or spikes in the envelope that help infect B cells and epithelial cells.

43:33And what's interesting is that you have two different kinds of cells, and so the glycoproteins have different functions. There's a fusion machinery. So this envelope is going to fuse with that of the membrane of the host cell. And that consists of the glycoproteins are called G and then a big letter. So G for glycoprotein, GH, GL, and GB. GH and L make a complex, a heterodimeric complex that regulates membrane fusion. and then when the virus binds to self-surface receptors, which we will get into, this GHL relays a signal to the actual fusagen, the fusogenic protein, which is called GB, cause fusion of the virus with the cell.

44:20Now, the virus infects B cells by attaching to complement receptors, CD35 and or CD21, complement receptors one and two. And the viral protein that does that is GP350 or a splice variant of it called GP220. So you need GP350, but you also need an additional viral protein called GP42 that binds GH and GL, right? The fusion complex that I've just told you about. Binding of GP42 to class 2 HLA on the B cell surface leads to triggering of fusion by GB, and that's through the GHGL-GP42 trimeric complex. And so that's the receptor for EBV on these B cells, class II HLA. There's also a hydrophobic pocket on GP42 that's different from the HLA binding site, and this is the target of neutralizing antibodies, as we're going to see.

45:36So when you're infected with, the first time you get infected with EBV, the virus establishes a latent state in resting memory B cells. And these cells can reactivate, but most of the time they're eliminated by cytotoxic lymphocytes. So you carry the virus asymptomatically for most people. will carry it asymptomatically for most of your life. But if you have immune compromise of some sort, reduced immune surveillance, you can have a disease called post-transplant lymphoproliferative disease, or PTLD. And this disease is higher in EBV seronegative people compared to seropositive people, which suggests that pre-existing immunity plays some kind of role in preventing it.

46:31people have wondered whether passive transfer of antibodies could protect against getting the virus in these EBV-negative people who get solid organ transplants, for example, and so we could reduce the risk of this disease, PTLD. And so people have done this. They've tried transferring EBV-neutralizing antibodies. These are monoclonals against GH, GL, GB, GP350, GP42, with all these proteins we've just talked about that are needed for entry, into humanized mice before EBV challenge. And that, giving those antibodies, can prevent viremia. It can prevent infection of the spleen. It can prevent viral-driven lymphoma and deaths to various degrees.

47:16And also monoclonals against GP350 and GHL can protect against a challenge with a rhesus lymphocryptovirus, which is basically the EBV ortholog that infects non-human privates. So that's kind of proof of concept that these antibodies could be prophylactic. But it's prophylactic in mice or in macaques, not in people. And so how do you make that jump to people? Yeah, people, they have tried some experiments in people. They use the murine monoclonal to treat people. And this was bad because people basically made antibodies against it and they had bad hypersensitivity reactions. So not a good idea to give people, at least in that case, mouse antibodies.

48:07So what they do here is they use a mouse called ATXGK. There are a couple of different mice that do the same thing. They have replaced the murine antibody, heavy chain variable, diversity, and joining genes, the kappa variable, and joining genes with the human ones. So all the mouse genes have been replaced with the human ones. So the mice make human antibodies. My understanding was that the constant region is still mouse, right? That's right. A small amount of the antibody, right? So most of it is human, which is better than any other. Well, and plus the fact that when you make those antibodies, you can fully humanize them in vitro.

48:52Yes. That was my understanding. So I am not quite picturing it. You have a chimeric gene or you don't have a chimeric gene because you can fully humanize it later. So you are only replacing the V, D, and J portions in the mouse genome with the human versions. You're not replacing the mouse consonant regions. So your first pass antibody for playing with is effectively a chimera of mouse in the variable region and, I'm sorry, human in the variable region and mouse in the consonant region. You can play with this in vitro to figure out whether this is what you really want. And then if you decide that's what you want, you can use recombinant DNA technology to splice in the human constant region.

49:47Have I got that right, Brianne? That is correct. And then that would be your therapeutic thing for humans because you're not going to make antibodies. Well, you're not going to make as many antibodies to that. So it's not like you're replacing pieces of the protein in a tube. You're making a fully human antibody later by moving the complementarity region to another clone. Yes. It's just this amazing technology, right? It's just great. The part that's the problem for us if we get a mouse antibody is the constant region, right? It could be any of them. Any of them, yeah. I see. It's foreign, right?

50:28So you make immune responses to it. I mean, and people before they had these mice, they chimerized them, right? They could drop the variable regions into a human antibody, right, Brianne? But then there are conflicts between the variable and constant regions still. Yes. Yeah, to get the sort of biochemistry to work correctly. And so people, so there's a guy who did that. I forgot his name. For years figuring out how to change the constant regions to make it compatible with the variable. and then you just have to make a humanized yeah so the whole point is in order to make a therapeutic antibody you need something with the right specificity that is human so how do you do that this is a method to do that they speak in the introduction of very briefly of human monoclonal antibodies that have been isolated that do this and I where do those come from so you can get people right and you can get B cells and sequence the heavy and light chains, right?

51:29And then express them. So if you have a way of selecting any B cell against any specificity, you can do that, right? Yeah. And so you're basically taking a B cell from a person and sort of making a clone and expanding in culture or taking out the heavy and light chain genes and putting them into another cell in culture. And it's sort of the same thing with the mice here. We're not going to use these mice and make enough monoclonal to treat the world, but we're going to use them to help us identify the B cell that we can then take heavy and light chain genes from and rearrangements from, put them in a saline culture and make lots of antibody.

52:15That was my aha moment right there. I was like, I don't get the mouse part. Okay, I get it. Yeah, so they're going to take the B cell out of the mouse once they find the right B cell. So the catch with human antibodies, you may not find the antibody that you want, right? Whereas in the mouse, you could screen a lot. Right. You could modify the antigen in mice where you can't. In humans, you just have people who are infected, right? Right. And they may not give you what you want. Right. So that's the beauty of the mouse. Yeah. And it's not the mouse per se. It's all the B cell clones that you get out of the mouse.

52:53Yeah. Yeah. It's the fact that you're getting a full sort of diversification process in the mouse. Yeah. The whole antibody production thing is normal. And then you have to do Cesar Milstein again. You have to take the spleen out. You have to fuse it with cells that will make the spleen cells immortal, the B cells. And then you have to put them in 96-well plates and screen supernatants with the monoclonal you want, right? That's the original hybridoma technology, which was subsequently bypassed by phage display and so forth. But it gives you what you want, right? So sometimes you have to do the harder work.

53:34I think the immunology students now don't even have to memorize what's in hat media. Like, I don't understand. Yeah. So you take the B cells and you fuse it with a myeloma cell line. Is that right? Yes. And then you have to kill off any unfused cells. Yep. And you have to make some kind of selection. So we use HAT selection, hypoxanthine, aminopterine, thymidine. And we used to have to memorize this because it was old technology, right, and how it worked. Anyway, so here what they do is they immunize these mice with, they have produced recombinant GP350 and GP42. right? And then they immunize the mice.

54:19They make hybridomas. They put them in 96-well plates, 384-well plates. Sorry. No more 96-well plates. Then they take the supernatant and they look for binding antibodies to GP43 or GP42. They have antigen coupled to beads, So you can do a high-throughput flow-based assay. And then they look for also inhibiting infection of cells. And so they take the antibodies that bind, and that inhibits cells, a certain infection of cells. And then they subclone them to establish monoclonal lines. And then they sequence them. They sequence the heavy and light chain genes. and they make the DNA and they express it in cells to make antibodies that they then use for the studies in this paper.

55:16So in the end, so they're making recombinant in human IgG1, they have two GP350 binders and eight GP42 binders, neutralizing antibodies. Right. And the reason why you have to do all of this is that antibody that is the binder has a particular variable diversity in joining, VD and J in the heavy chain, V and J in the light chain. But it also will have some base pairs added and subtracted when those VDs and Js are joined. So it won't be the exact sequence that's in the germline. There will have been basically this evolutionary process that happened with base pairs getting added and subtracted to make a perfect fit antibody.

56:03And so you have to let that whole process happen in the organism to get whatever the perfect fit antibody is with whatever base pairs and thus amino acids end up being added or subtracted to make the perfect fit antibody. So you want to let that whole kind of evolutionary process happen to generate the perfect antibody coming from the perfect B cell that's done all this adding and subtracting. Once you've done that, you've let the B cells do their adding and subtracting and evolutionarily come up with the perfect antibody. Then you can sequence out the DNA and figure out what additions and subtractions they made so that you can work on that more.

56:49But you first kind of have to let them go through this amazing process of the germinal center reaction and of also junctional diversity that we can't just model easily. So these monoclones are called ATX42 or ATX350, depending on which antigen was used. And they have other numbers in there to distinguish them, right? Because they have two and eight. Okay. So then they do a bunch of studies. I don't want to spend a lot of time on this because the most interesting experiment is later, but I'll just tell you a little bit about it. So they look at the affinity. They make FAB fragments, just the Y part of the antibody that's binding the antigen.

57:38They do affinity measurements to the proteins using what I used to call BACOR, but which is now called biolayer interferometry. It's just a really cool way of measuring affinities between a ligand and a receptor. And they can characterize. And these antibodies have different affinities. One of them, ATX352, has the highest affinity. And then they have others that are significantly less potent. OK. So then after affinity, they say, let's look at the neutralization of infection. They use a B cell line called RAGI. And then they have some other already existing human GP50 monoclonal antibodies, and they compare them.

58:25And ATX350-1, which had a very low affinity into binding assays, was also less potent. Then they go on to look at these monoclonals' effect on inhibition of infection in epithelial cells, right? So that RG was a B cell line. Then they look in epithelial cells, and you see some differences compared to the B cell line, which makes sense because there are different modes of entry. So what they conclude in the end is the GP350 monoclonals neutralize EBV, and I haven't told you the data, by inhibiting the interaction of GP50 with CD21, one of those complement receptor proteins. All right. Because they have CD21 negative cell lines and so forth where they can do all of those experiments.

59:20Which is great, right? Like the virus never attaches to the cell. Right. So altogether, the experiments that I just glossed over very quickly, they show that ATX350-1, that's one of the 350 monoclonals, and 350-2, they neutralize EBV infection of B cells at different potencies by inhibiting GP350 binding to CD21, and they say presumably CD35. They haven't looked at that. Then they also look at competition binding by biolayer interferometry, whether they compete for binding with GP350. and the data suggests the antibodies have unique footprints on GP350. They're slightly different. And they have one ATX350 that has the highest neutralizing potency.

1:00:16They determine the structure of that antibody together with GP350. And there's a lot of lovely stuff. You can tell exactly how the antibody is interacting with GP350, but I'm not going to talk about it. Well, and the reason why all of this part is important is that they've come up with these two antibodies that they've designed. And you could imagine trying to use them as a therapy together or using them separately. And if you found that the two of them bound to the same site, then delivering them together would be kind of useless. Waste of time, waste of money. Right. Whereas if they bind to two different sites, then they are going to be sort of helpful together.

1:01:06And so that's really what this is showing, is that they're binding to two different sites. So it is potentially useful to actually deliver them together. So next, they characterize the GP42 antibodies. Everything so far has been GP350. So they measure the binding affinity like we did before. All the 42 clones bind GP42 both alone and together with GHGL. Remember, 42 has to bind GHGL. And they look at the affinities as well, particularly this one antibody. So one of the previous antibodies, 4C12, had a reduced affinity to the GHGL-42 complex, which they think reflects steric occlusion of the hydrophobic patch.

1:02:02Remember, we talked about that before on GP42 by GHGL. So the idea is GHGL is occluding this hydrophobic patch on GP42. Then they asked, do these GP42 monoclonals inhibit infection of B cells. They all did. And one of them, ATX42-2, is the most potent. But none of them neutralized EBV infection of epithelial cells, right? There's another antibody actually previously described called AMO1 that had potent activity in that assay. And that's because the entry is different in epithelial cells. So basically, these eight GP42 antibodies, they actually belong to two clonal lineages that bind GP42 with high affinity.

1:02:55They neutralize EBV infection of B cells by preventing GP42, the EBV protein, from binding to HLA class 2. I want to make sure I understand a clonal lineage um so by that i understand it to be that the uh multiple uh individuals in the lineage of a clone would have had the same germline sequence but taken a different maturation pathway does that make sense yeah so i i might say that they they picked the same vd and j but they did different addition and subtractions right Okay. They also did some biolayer interferometry to show that these ATX42 monoclonals actually target the HLA binding site on GP42.

1:03:49Remember, GP42 is binding HLA, so these antibodies are binding it, and that shows it. All the work that they do shows that these antibodies against 42 neutralize EBV infectivity by inhibiting the GP42 class 2 interaction. All right. So now this sets up an experiment in mice. We want to know if these can actually have an impact on infection of mice and disease. So there's a humanized mouse model of EBV infection where you engraft human CD4-34 positive hematopoietic stem cells into mice. So you're basically putting precursors and you can find human B cells in these mice. And they show that. And then they give these mice, once they have the human B cells, they give them an intraperitoneal injection of the antibodies.

1:04:48So they get 500 micrograms of ATX42-2, ATX350-2, so the two different proteins, and AMO1, one of these other previously identified antibodies, and an isotype control. And then they give them an intravenous challenge with EBV. So they watch these mice for 11 weeks. They look for weight loss, survival. They're bled weekly, and they look for viral DNA in the blood. And then at the time of euthanasia, spleens are taken out. They look for tumors in the spleens, and they also look for viral DNA. And there's a lovely picture of, I know people can't see it while I'm talking, but there's Immunohestochemical sections of the spleens.

1:05:45And you can see the spleens, the H &E stain, which shows all the cells in the spleen. And then there's a stain for CD20 for B cells. And you can see how, in some cases, the B cells are going away because of the EBV infection. And then, finally, they stain for a viral protein called EBER. and you can see that in some treatments, so in the mice that aren't treated at all, there's lots of Eber foci. And in the mouse treated with ATX42-2, there are no Eber positive cells. In the mice treated with MO1, there are no EBV positive cells. And that's just one part of what they're looking at. So some of these antibodies really are effective at preventing, in fact, destruction of the B cells.

1:06:43So they see differences among the mice, right? Some of them survive, some of them don't, some of them lose weight. But the antibodies can prevent tumor formation, they can prevent viral DNA, they can prevent splenomegaly, the enlargement of the spleen. And then they use a different kind of mouse, basically, for the same kind of study. So you need to use an immunocompromised mouse to engraft the human B cells. And they use two different kinds of that, and they get the similar findings as well. So basically, you see that some of these antibodies really do well at preventing EBV disease. And now the nice thing is, unlike some of the antibodies that have been studied previously, these are ready to go into people.

1:07:39So now that it's been shown that they seem to have this activity in mice, these are potentially useful in humans, while the previous ones were not. So I wonder what the trial is. Probably post-transplant lymphoproliferative disease, right? See if they would be helpful in that situation. So you would give it before the transplant, right? Yeah, I think so. And then see, do they not develop? Yeah. Oh, and what – I was disappointed that they didn't try the combination of the two, 350 and 42. Mm-hmm. to see what that would do. Though it could be that 42 is so good that you wouldn't necessarily see an additive effect of 350.

1:08:29Right. Plus these mice ain't cheap. The humanized mice. So the human study where they used the mouse monoclonal, that was 72A1. So ATX 350-2 has a comparable neutralization titer. And so they say that would be a good one to use in the people.

1:08:56So one of the issues here is that in these mice, only the human lymphocytes are susceptible to infection, right? Right. Because in people, you infect also epithelial cells. And so you would like to know in vivo how that does. Right. Right. And that's particularly important since some of the ATX antibodies were really useful against blocking B cell infection. And AMO1 was the one that was more effective for epithelial cells. So you'd want to start thinking about combinations and things like that in a human system. So one of the things I was thinking about is whether you could engineer a vaccine that makes this antibody.

1:09:41right? And so there have been vaccine trials, right? So I looked into this. So GP50 alone, the subunit GP50 plus an adjuvant, aluminum adjuvant, has failed to generate protective immunity in trials. GP42 makes different confirmations, so it's hard to get the right one. and these yeah i was going to ask about that actually because these are surface glycoproteins right so when they're doing all these immunizations they're probably not including the anchor that would put it in the membrane and so it's maybe it's not quite confirmationally presented in such a way that you can induce exactly what you get in vivo in the in vivo context.

1:10:33But it does still work. Yeah. Well, you make antibodies, but not the ones that are not as many of the ones that are neutralizing that you want. You can't predict that you're going to get the same outcome every time. So there have been some human trials. There's a phase one of a GP350 ferritin nanoparticle vaccine, which was shown to be safe and immunogenic. And interestingly, they had seronegative participants in that trial. None of them acquired EBV through day 540 post-vaccination, so that's pretty cool. That's pretty good. Dr. There's also another phase one that's recruiting, which is a GHGL-GP42 ferritin nanoparticle vaccine.

1:11:22It targets the fusion complex. Moderna is testing phase one, a GHGL-GP42-GP220, four antigens in one mRNA vaccine. And so these, you try and present the antigens in a native-like confirmation in order to make the antibodies. But what this paper does, it defines the epitopes, right? You now have a structural target and try and reverse engineer a vaccine. You know, you have the antigen, can we design an antibody around this? And I think, as someone said before, I think it would be interesting to combine 350 and GP42 and get both simultaneously, right? And this would be more than a post-transplant.

1:12:14If this vaccine worked, it could be an MS vaccine, right? That could be cool. Anyway, so that's why this paper was very exciting because it's the first time that we've got these kinds of antibodies, human antibodies, right? So one of the things I was thinking as I was reading this is how difficult it is to make vaccines to some things. We've talked about this before. You know, smallpox vaccine was basically low-hanging fruit, as was, fortunately, the COVID vaccine. Turned out spike protein as an antigen worked just fine, but it didn't have to be that way. Some of these critters are really complicated and really difficult to make vaccines for.

1:13:07but you were getting better at it now getting better at it and you go back to our podcast you and i did rich with mcclellan right two years ago yeah he's doing that too yep okay let's do some email

1:13:27um brianne can you take that first one sure charles writes hello twivers it is a very nice Monday in Chapel Hill, North Carolina. Mostly sunny, 68 Fahrenheit 20 C and 76 % relative humidity. Nice evening for a walk. The discussion about religion caught me a bit by surprise. Saturday night before TWIV 1351 dropped, my partner Marie and I were discussing Trump and reincarnation. A conversation we had during the first Trump administration with a Hindu postdoc she worked with came up. He told us that coming back as a tree was reserved for those who did not do many, if any, of the human things expected of them, building up a lot of negative karma.

1:14:11That fit Trump. So we burned a few watts, sorry about that, and had Gemini AI make a drawing of Trump as a sad bristlecone pine tree. With luck, that should lock him up for 5 ,000 years or so. I tried to get the AI to add a miniature white poodle urinating on Trump, but it refused. And the picture is below. Dr. Racaniello, thank you for your blood, sweat, and tears. Child is the father to the man album suggestion. Interesting album cover. I did not remember the song. My loss. I did not realize that BST played that much brass. I don't know why, but the music reminded me of the TV show The Prisoner.

1:14:52about the same point in time. Continuing with the music arc, when I was at Murray State University in Kentucky decades ago, I listened to a lot of moody blues. Days of Future Past, In Search of the Lost Chord, On the Threshold of a Dream, A Question of Balance, and Seventh Sojourn were all really good albums. My favorite was Every Good Boy Deserves Favor. What do you expect from an 18-year-old? Off the music arc and going with an old book. On TWIV 1351, Dr. Dove mentioned Henry David Thoreau's book Walden, a book well worth reading. I suggest, as my freshman English professor insisted on, read Edward Abbey's Desert Solitaire, A Season in the Wildflowers, after you finish Walden.

1:15:40This is my favorite scene from the book set in Arches National Park in Utah. Quote, this would be a good country, a tourist says to me, if only you had some water. He's from Cleveland, Ohio. If we had water here, I reply, this country would not be what it is. It would be like Ohio, wet and humid and hydrological, and all covered with cabbage farms and golf courses. Instead of this lovely barren desert, we would have only another blooming garden state like New Jersey. You see what I mean? If you had more water, more people could live here. Yes, siri. And where then would people go when they wanted to see something besides people?

1:16:21I see what you mean. Still, I wouldn't want to live here. So dry and desolate. Nice for pictures, but my God, I'm glad I don't have to live here. I'm glad too, sir. We're in perfect agreement. You wouldn't want to live here. I wouldn't want to live in Cleveland. We're both satisfied with the arrangement as it is. Why change it? Agreed. We shake hands and the tourist from Ohio goes away pleased. As I am pleased, each of us thinking he has taught the other something new. That's the end of the quote. I get the symbolism and sarcasm, but I also have a good non-sarcastic answer to what arches would look like with water.

1:16:55Red River Gorge in eastern Kentucky is arches with water, at least as close to it as you can get and still be in the U.S. If you have been to Arches National Park, follow it up with a trip to Red River Gorge. A bit rambling, but I have burned enough watts on the AI drawing. I am not going to ask AI to improve this email. yes charles uh have you guys ever been to arches no yes it's amazing yeah arches really made a really strong impression on me did you hike up to the arch i did the delicate arch yeah yes absolutely it's beautiful and i really like the town moab have you stayed in moab i haven't really stayed in moab i don't like the people you know i'm not there for the partiers i'm there for the desert.

1:17:41Yeah, Arches is awesome. I highly recommend it. The best trip my wife and I ever took was a driving trip through the Colorado Plateau. You know, Bryce and Zion and Canyonlands and Arches. Man, it's just awesome. Just really cool. I haven't been to Red River Gorge, though. Neither have I. I understand there's good rock climbing there. Next one is from Scott. I'm going to take this. So last time Jolene was on, which wasn't too long ago, I think. Just a couple weeks. You had an email from Scott who had made a search tool for TWIV, right? I picked his search tool for TWIV as my pick. Pick. So I emailed him and I said, thank you so much.

1:18:33Could you do it for the other programs? And so he said, here you go. So we have TWIV, TWIM, TWIP, TWIVO, TWIN, Immune, Infectious Disease, Puscast, Matters Microbial, Urban Ag, and one for all of them. So you can search all the Microbe TV podcasts together. Very cool. Thank you, Scott. Thank you, Scott. Awesome, Scott. I got to bookmark these guys. I offered to pay him, but he didn't want to be paid. I bought him a coffee. That's good. His link. Mm-hmm.

1:19:06Rich. Anyway, so wait, this is a long one. I don't want to give you a long one. You don't like long emails. Just, Jolene, can you take the next one? Yes. Katija writes, hello, TWIV team. I don't know if you remember me, but during early COVID, I had written a letter that was along the lines of, hey, race is a factor or something. And afterwards, Brianne came on my since-defunct podcast, We Be Imagining, to talk about immunology and how she leads undergrads to become researchers with her. I remember. Hi, Khadijah. I'm reaching out to share a perspective on long COVID reporting based as a parent of a child diagnosed with autoimmune encephalitis in 2019.

1:19:44I generally don't share information about my kids, but the experience I witnessed with him was so unbelievable that in certain contexts it feels like necessary context from which people can appraise these long COVID self-reports and kind of inconclusive large surveys. He tested antibody positive with NACHR autoantibodies in 2019 within three months of his first seizure, albeit noting that I discovered upon retest last year that Mayo no longer even includes that on their AE panel. My son, who was 7 at the time of the initial diagnosis, had the most severe case in terms of initial neuropsychiatric presentation, but ONSAT began with what looked like tonic, clonic, and absent seizures, and longest duration of perceived neuroinflammation.

1:20:38A. His clinicians and I agreed that his relatively speaking fast diagnosis was a result of the following factors. One age at the time didn't index to any other major clinical alternative. She kind of goes into some more details about this seizure being another one. He had worsening behavior, cognitive decline, anti-epileptic drugs didn't seem to work. And they think that the deterioration was reported with detailed evidence by his teachers at a school that's the equivalent of primary school Ivy League in New York City. B, Vincent, I heard your comment that a test is better than self-report, and Brianne chime in that it will help being believed.

1:21:24Partly, it's a question of which signs fulfill initial clinical suspicion to initiate treatment, and then OFC, I don't know what that is. Of course. Of course, with the response to treatment. What is the response to treatment? But I would like to dimensionalize another aspect of that belief. what the clinician believes to be the baseline of the patient as a social judgment. After six or seven months of treatment with IVIG and IV steroids in accordance with the protocol, we saw return of speech, seizure cessation, reduced psychosis, and cognitive improvement, but it was incomplete. He had no response to rituximab, consistently had a clear response to steroids, but it wasn't sustained.

1:22:08I found a case report on a medical student who had a similar incomplete response, but given her background as a medical student, the assumption was she should be expected to make more improvement. This study successfully convinced his clinician to trial third-line treatment, cyclophosphamide, which once started we saw substantial dramatic improvement. Okay, see? Noting that my son is a dark-skinned former foster child on the fetal alcohol spectrum, so you can imagine the default assumptions about how much he could improve or what is his normal. Besides the social implications of FASD, there's some literature indicating that prenatally exposed people have a more permeable blood-brain barrier, and we speculate that this may account for his initial vulnerability and the duration of illness.

1:22:54I met Dr. Douglas Waite, the developmental pediatrician in New York City who specializes in FASD once he had already focused on foster and adoptive kids, but he always reminds me that he initially worked in the Upper East Side with a white affluent population with higher prevalences of FASD, but that they would proactively seek an autism diagnosis instead. Witnessing the dramatic overnight response year after year to immune modulators of both psychiatric and vaguer sick symptoms like fatigue and brain fog, my bystander speculation is that it will be hard to know whether or not COVID is the primary pathogenic mechanism in many of these cases, but if post-viral inflammation is more often suspected and treatment initiated, our miracle will be commonplace.

1:23:42My son often looked like the kinds of homeless people who are erratic and screaming outside, but who's giving them extensive medical testing? And D. Lastly, this is now changing, but for most of the time since his diagnosis, the Rankin was being used as a benchmark for appraising severity of illness and extent of recovery. Increasingly, I do see greater acknowledgement, including in the more common and therefore greater volume NMDA, encephalitis, that many people are experiencing a wide array of longer standing neuropsychiatric pain, cognitive, and other symptoms due to ongoing inflammation and what is speculated to be burnout aftermath once the inflammation has concluded.

1:24:25After reaching his lifetime limit of cytoxan, he was still experiencing symptoms.

1:24:34tocilizumab targeting IL-6 from a very small trial at Duke failed so he was trialed on asiathropine which does seem to work I know this is different than long COVID in particular his severity but even in AE many have low severity but dramatic responses to the same treatments nonetheless Kodija PS I think part of the conspiracy theorizing about lab leaks etc is due to how little is popularly transmitted about similar subsets of people who get post-viral conditions like HIV-AIDS, neuro-DO. So kudos to you for making thousands of episodes trying to do this kind of public education.

1:25:18Well, I think, you know, this is, it's helpful to have stories because that is what a lot of people learn from, more so than the data. So I guess thank you for sharing your story. Yeah. It sounds like you're getting better, so that's good. Yeah. I think that Khadija's points about, you know, how different patient groups are received and what kind of improvements are or are not expected and what normal looks like in different patient groups are important things for all of us to remember. To me, one of my reactions is that this is a confusing enough realm so that stories are relevant, okay? Because it's so heterogeneous that an individual story is quite relevant, it seems to me.

1:26:15I thought this point number B where, you know, we said we need a clinical test, and he says, partly it's a question of which signs fulfill initial clinical suspicion to initiate treatment and, of course, what's the response, but also what the clinician believes to be the baseline of the patient as a social judgment. I think that's really important, right? Well, thank you. Appreciate that.

1:26:41This next one, do you want to do a short one, Rich? Sure. Martin? Yeah. Martin Rice, dear Twivers. another piece of news which Dixon would have appreciated I'm sure and for those newbies out there Dixon was an avid fisherman and this is a link to a new scientist article that is titled burns improve more rapidly after treatment with fish skin I can't get the whole article basically I would have to subscribe to the Guardian to get it it's got a nice picture on top of a an emergency, a surgical procedure with it. It says fish skin may be the safest, most effective way of treating severe burns. Actually, the sub headline is severe burns are often treated with synthetic products ahead of skin graft surgery.

1:27:37Sort of meshes of one sort or another, I think. But there is evidence that a product made from minimally processed cod skin is more effective. And this article is about that. So that's pretty cool. Exposing burns to skin of Atlantic cod seems to result in patients spending less time in the hospital and experiencing fewer complications compared with other treatments. This may be due to cod skin containing omega-3 fatty acids that trigger just the right amount of inflammation and assist the healing process. Awesome. I love it. Thank you. There you go, Dixon. Yeah, Dixon would love that. Oh, Dixon's here.

1:28:19Look at him. He's up there in the corner. And then finally, Zoe writes, long time listener here. I was just listening to TWIV, 1351, when you started the episode talking about what you would do if you were reincarnated and what animals you would want to be. When you started speaking of wanting to be artists, musicians, I immediately started to write this. Because you can be. You should be. It's never too late to be an artist. My grandmother only started painting a few years ago. I also think that as scientists, you all have a unique insight that you could bring to art, whatever that may be. And that art can be an excellent form of communication for scientific issues.

1:28:58I'm going to count barbershop harmony singing as an art. It is, of course. And call myself an artist. Of course. Well, you said you'd want to come back as a musician, but you already are, right? Yeah, I guess in a way. It's funny because I don't think of it that way. But, man, I had a rehearsal yesterday with a group that I just absolutely love. And the harmony just blows you away. It's amazing. Anyway, that's a digression. Throwing my hat in the ring of your earlier conversation, I think I would want to be a peregrine falcon. Diving towards my prey at high speed seems very cool. On the topic of having empathy with animals, I would suggest the short story, The Secrets of the Last Greenland Shark, which you can read for free at this link, where the endlings of every species can share their experiences with each other.

1:29:50There's a quote from it. There was one left. She still lived. These creatures, the other still living lasts, were especially dear to me. I knew they sensed my presence when I visited them, just as I sensed theirs. I made sure to enjoy something when I felt one visit, a bite of grapefruit, a sip of rum, a smell of lily. I wanted to give them the best of what it was to be human. On the virology side, I would recommend reading In the Company of Men by Veronique Taggeau, which draws on real accounts from the Ebola outbreak in West Africa, including the perspectives of health workers, volunteers, those in the community, as well as parts of nature, a bobab tree and a bat, the virus itself.

1:30:37And some quotes, I myself, though I'm a doctor, have no idea how my body would react if I came down with Ebola. Chances are I wouldn't fare any better than my patients. A woman can survive, an old man can survive, a teenager can survive. And what about me? The virus respects nobody, makes no exceptions. Incapable of rational thought, it's the kind of enemy that instinctively wants to crush its opponent. The human race itself wouldn't be enough for it. Even in death, Ebola doesn't want to let go. Like bombs, it's corpses. So destruction.

1:31:13Ebola virus knows nothing about their beliefs. I'm not governed by any law. I'm here purely for the sake of existing. I am me, period, an organism that needs to reproduce itself. No compromise, no negotiation. I'm alive. I'm prepared to do whatever it takes to stay that way. My only needs are to feed and defend myself. A pile of flesh will do. Any kind of receptacle, animal or human, it's all the same to me. I'm neither good nor bad. Such judgments are useless. I'm like a plant that grows, like a spider that devours its prey. You may also be interested in Surgeon X, the path of most resistance, a comic set in a future London facing a crisis of antimicrobial resistant infections.

1:31:53Notable not only for being an excellent comic, but having a large team of science consultants from a variety of universities to make it accurate and educational. With so many of our issues today surrounding health being related to communication and disinformation, I suppose I can't help but hope that stories, whether real-life accounts or fictional interpretations, could help communicate important, often life-saving information and change conversations. I am a student of political science, an illustrator, and a writer. The scope of what I can write and draw is limited by my knowledge and experience.

1:32:28Everyone brings a unique life experience, style, and educational background to their works. Attached are two pieces that I made, one of my experience with long COVID and POTS, and the other of scientists racing to develop a vaccine. Once I got frustrated explaining what my body felt like to a friend, and I just showed her the image. Sometimes pictures and stories can speak louder than technical words and numbers, though facts are, of course, vital. Anyways, this is a long way of saying, please don't wait until you're reincarnated to make art. You have years of experience in a fascinating field.

1:33:07Write a story about it or paint or make songs about viruses. The possibilities are endless, and I think we would all be better off for it. I have also now made myself very curious as to how virus rock music would sound. Anyways, thank you for TWIV. It's excellent. Please keep doing science communication, whatever form that may take, Zoe. And her paintings here are lovely. Yeah. Yeah, they are. Thank you for the encouragement. I like art. I don't feel I'm good at it, but it's very therapeutic to make music, even when I'm not good at it. I wouldn't apply any judgments, Jolene. Just go for it. Yeah.

1:33:57I have been making crocheted art for, what, three months now, and I'm improving greatly. So I'm sure you're improving even more, Jolene.

1:34:12Well, I have an electric guitar behind me at home when I do my live stream. I've had it for 30 years. I used to play it. I resolved to learn how to play it again, again. So maybe I can do that. All right, let's do some picks of the week. Brianne, what do you have for us? My pick is an article from Scientific American. So I was extra excited when Rich told us about the Scientific American anniversary at the beginning of the show. This is an article called about life on Earth. And that says life on Earth may have emerged not once, but twice. It is talking about a recent science advances paper, look, trying to look at the last universal common ancestor of life on Earth.

1:34:59And this paper suggests that, in fact, life on Earth emerged twice, once for bacteria and once for archaea, and that previous ancestors before those would not have been alive, were in fact, perhaps sort of collections of compounds in hydrothermal vents doing metabolism. They note that when you look at all of these different groups and compare genomes of bacteria and archaea, you find that only about half of the enzymes that you need for the basic reactions are conserved. And basically, the other half are completely unique to bacteria and archaea, which they I think is an indication that this sort of transition to life happened twice separately.

1:36:02And I just thought it was an interesting thought. I hadn't really I guess I always imagine those trees that we see in a textbook or something like that going to that universal common ancestor. and hadn't really thought about multiple. I also thought I was really interested in where they were drawing the line of what's alive and what's not alive, which I thought was pretty relevant to virology in thinking about sort of this gray area of where that line of life and not life is. And so I thought it was pretty interesting to read. So my problem with this is that, I mean, archaea and bacteria have the same genetic code, right?

1:36:52And the same fundamental process. So I envision an ancestor to both of those that had that property in common. Though maybe that was some form that we wouldn't consider alive? That's kind of what they're arguing. Yeah. Okay. Weird. Okay. Have to have a look at this. Rich, what do you have? So this is an astronomy picture of the day pic. We hit on that frequently. I think we've seen something like this before. This is a flyby of Saturn by the Cassini spacecraft. If this is new, it might be considered remastered because they've got thousands of pictures. taken by Cassini in its mission to Saturn that have been sewn together into this flyby.

1:37:54So this is the real thing, okay? It's not digitally enhanced. This is as real as it can get. Cassini, by the way, I also put in here a link to the Wikipedia page on Cassini, was, you know, I got to look these up all the time, a very interesting mission that had two spacecraft, well, two spacecraft combined into one. One orbited Saturn taking, collecting data for like 13 years. And the other was a probe that it carried with it that actually went down to the surface and collected a bunch of data on the way. So this thing was launched in 2017. Took seven years to get to Saturn. Was it 2000? Oh, no, no, I'm sorry.

1:38:481997. Took seven years to get to Saturn. Make sure I got this right. Launch date. Yeah, 1997. Took seven years to get to Saturn. And then orbited it for a number of years. and finally ran out of gas in 2017. So we're talking, what's that, 20 years? Part of that time to get there and a lot of time orbiting the planet. It's got pictures of the moons. You can see it going through the rings. It's just awesome. That's very cool. I love the last sentence of the APOD description. After more than a decade of exploration and discovery, the Cassini spacecraft ran low on fuel in 2017, was directed to enter Saturn's atmosphere where it surely melted.

1:39:37But I guess we don't know. They actually, they talk about in an expanded article on this, that they deliberately crashed it into Saturn where they figured it would be consumed because they didn't want to take a risk of contaminating any of the moons. Of which there are 260 with any trash from the spacecraft. Right. And some of them are, you know, candidates for having potential life. Yes. Right. Good. That's cool what we do, you know? It's just awesome. Thoughtful people. It's neat. Jolene, what do you have for us? So today I switched my pick. I am picking a CBS News evening story about Dolly Parton when she first got her COVID vaccine.

1:40:37So she thought that vaccines were very important and decided that she would get hers live on camera. And at the beginning, she comes on with her classic style and talks about how vaccines are important and then masks up. And then the physician comes in and gives her the shot. But before he comes in, she does a new rendition of her song, Jolene, which I know the chorus very well of. And she sings it with the words, vaccine, vaccine, vaccine, vaccine. I'm begging of you, please don't hesitate. And then she says, vaccine, vaccine, vaccine, vaccine. Because once you're dead, then that's a bit too late.

1:41:27Oh, yeah. So it's just a really nice way for us to kind of remember the contribution that she made. And I think it's a good rewrite of the song. It's much more wholesome than the first one, actually.

1:41:49that nature article that was picked earlier makes the point that Dolly gave a million bucks to Moderna to assist in creating a vaccine and through a foundation I guess that she created which is cited in numerous publications on the development of the vaccine for funding. It's cool. Yeah. I think she gave the money, or at least a big portion of her money she gave to Vanderbilt, where they did the development. All right. My pick is apparently a pick I've done before, according to Jolene, but this is the book by Daniel Kahneman called Thinking Fast and Slow. And the reason I wanted to pick it again is the following.

1:42:43So this week I had some visitors at the incubator on Monday, and I was showing them the shelf behind me. And I have this little card that I got, I don't know, 50 years ago when I visited the Thomas Edison Museum in Edison, New Jersey. and it's a quote by Sir Joshua Reynolds, who was a 18th century philosopher. And it says, there is no expedient to which a man will not resort to avoid the real labor of thinking. And I was looking at it. I said, where does this come from? I've had it my whole career since I was in high school, college, graduate school, postdoc. It was in my office at Columbia. Now it's at the incubator.

1:43:28I just kept this little cardboard thing because it just impressed me so much, right? Because I think it's, I feel that people avoid thinking hard, right? So I looked it up. And of course, there is a known thing. It's called cognitive laziness. That's what that's about. Because the brain, you know, uses a lot of energy. And so it defaults to this type one thinking where you basically don't have to think much. You just react to things. And that conserves energy. But real thinking, real deep analytic thinking requires a lot of energy. It's exhausting. And most people don't like to do it. So I just read about this.

1:44:10You know, this is a thing that researchers have proposed, cognitive economy. And I just wonder if cognitive economy can explain like anti-science, anti-vaccine attitudes, at least to some extent, because it's hard to think about science data. You know, we read a paper like we do today. It's not easy to figure it out. And we have a lot of experience, so we can at least understand the terminology, the methods. But people without any background in science, it's going to be really hard. So they switch to type 1 thinking and gets us all in trouble. It's hard for me, too. Yes, I think it's hard for all of us.

1:44:53Yes, it is. I think that as I've been spending my time recently trying to get ready for the new semester and thinking about ways to talk to students about AI and what they should do themselves versus what they should have AI do for the semester, it ends up sort of being related about sometimes you got to do the thing that's hard to help out practice your brain. Vincent, I feel like you've talked about this card before. Oh, yeah. In my head, in my brain, I feel like it's somehow related to the Edison Museum. Oh, yeah. That's where I bought it originally. Yeah. Because Thomas Edison had this in his lab to try and motivate people.

1:45:36Yes. Because I've always been like really liked the quote myself and always thought I should go to the Edison Museum, but haven't done it. Yeah. So I think, I mean, we, thinking hard is exhausting, right? Sometimes you read a paper and you're tired at the end because your brain is working hard. It's using a lot of energy. to do that. So we have to do it as part of our field. But now AI can do it for you, and that's not good because I think thinking hard and deep type two thinking is really important for keeping your brain active and so forth. But I just had this idea that maybe it can explain a lot of anti-science skepticism, right?

1:46:18Anyway. This looks very interesting. Right. The book is really good because it gives lots of concrete examples of type one and type two thinking and also ways that you can push yourself to type two instead of being lazy and just saying, yeah. So someone says, Tylenol causes autism. Oh yeah. Okay. I buy that. That's lazy type one thinking instead of going to the literature and reading it yourself. And people will say, well, I don't have time. That's just, that's laziness. It's cognitive laziness. It makes me think, go ahead. I don't think you said what the book is, Vincent. Yeah, I said Thinking Fast and Slow by Daniel Kahneman.

1:47:00I think so. But anyway, yeah, Thinking Fast and Slow, Daniel Kahneman. I linked to the Wikipedia page about it, which you can go check out. I think about procrastinating a task. Like, you know, in my case, actually I don't have to procrastinate doing TWIF because I like that, preparing for TWIF. But, you know, I do other stuff like, I do administrative stuff for this music chapter that I'm in. So creating an agenda for a meeting or something like that. or doing some other task. And I will, it, I actually have, it takes a mental and emotional effort to sit down and say, okay, now I'm going to do this because I know it requires thinking, right?

1:47:55And I don't want to do that. But usually I find when I get into it, it's okay. I think the things that you have to do are the ones you want to put off often, right? Yeah. If you like to do something like this, I love doing this. This is not a chore ever, ever, ever. Well, it's almost as if the term have to implies that it's something you don't want to do. Well, we don't have to do this, right? It's not part of our job, although you could argue that it actually is, right? But technically, nobody has to do this. Rhianne has to go to her lab. Jolene has to go to her lab. So this is not like that sort of thing.

1:48:43There was an interesting article that I almost picked. I mean, these come out periodically, right? But just arguing for how it should be part of our job to communicate to the public the science that we're doing. I mean, the teaching part is an easy thing for us to say, like, yeah, this is my job. I have to teach the undergrads. But it's not just them. It's the responsibility we have with the training that we've had to communicate the information in a way that's relatable to particular audiences. We are the scientists. We know the science. We are the ones who have to communicate it. Yep. I cannot speak for all of us, but I at least was very much funded in that training through U.S.

1:49:31tax dollars with NIH and things like that. And so I always try to make sure that I'm helping out the taxpayer by doing that communication. I think about this all the time. I've been on the dole my whole life. I went to a state-funded university. I was on a training grant in both graduate school and for two postdocs. And then I've had grant funding at both my jobs, both at state universities. I've been on the dole my whole life. What does that mean, being an adult? Somebody else paying for my existence. I see. Okay, we have a listener pick from John. Dolly Parton probably did more to protect people against disease than the federal government.

1:50:20In her memory, I recommend this short video of her singing Jolene with the rewritten, reframed Vaccine Vaccine as she gets her COVID-19 shot. And John gives us a YouTube link. Thank you, John. That is TWIV1353. You can find the show notes at microbe.tv slash TWIV. You can send questions or comments, picks of the week to TWIV at microbe.tv. And if you enjoy these programs, we'd love your support, microbe.tv slash contribute. Jolene Ramsey is at Texas A &M University. Thank you, Jolene. Oh, sorry, ramseylab.bursell.app. That's right. Yeah. Thanks. I enjoyed our discussion today. Brianne Barker is at Drew University Bioprof Barker on Blue Sky.

1:51:13Thank you, Brianne. Thanks. I learned a lot. Rich Conda is an emeritus professor, University of Florida, Gainesville. He's currently in Austin, Texas. Thank you, Rich. Sure enough. Always a good time. I'm Vincent Racaniello. You can find me at microbe.tv, where I don't have to go, but I do every day because I love it. I'd like to thank the American Society for Virology and the American Society for Microbiology for their support of TWIV, Ronald Jenkins for the music, and Jolene Ramsey for the timestamps. You've been listening to This Week in Virology. Thanks for joining us. We'll be back next week.

1:51:52Another TWIV is viral.

1:51:59Thank you.