TWiV 1353: Phage bet-hedging and EBV antibodies

Welcome and introductions

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 28th, 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.

0:40We 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. There you go. And I forgot to bring my computer glasses, so everything is fuzzy.

1:11Oh, 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. Yeah. I can read the dashboard. I can see the computer and see all sorts of other stuff at the same

1:45time. It takes a while to get used to it. The problem is that if you look at something close, you have to tilt your head. I don't like that. So I get computer glasses, but I didn't bring it on. 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. 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.

2:16Hello. 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. It's a pretty lovely 82 Fahrenheit sunny today, so not too bad. And I also vote for progressives.

2:46When I had to get glasses a couple years ago, because I couldn't read tubes in the lab anymore, I had to... 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. I 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.

3:22The 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.

Remembering Dolly Parton and Scientific American

3:35If 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. How about that? Happy birthday.

3:56I have almost everyone from TWIV's birthdays in my calendar to send out a message. Almost everyone, Jolene. That's proprietary information? Yeah, I don't have Jolene.

4:12Actually, 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,

4:44for marginalized communities. And of course, the first clone 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 didn't, she chose not to be a billionaire.

5:20Yeah, 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. But 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

5:52able 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, um, and how many people are easily

6:24able to recognize how good these things were. Yeah. Cool. I'm, uh, I'm really happy to, uh, see somebody on the radar who is, seems like nothing but good. Uh, and, and, and for everybody, uh, I don't see, you know, nobody has trouble with this. It's, uh, except the fact that she died, but cool. Yeah. Great stuff. She said, I didn't like her music, but I liked her.

6:56She, you know, she sang quite a range of different kinds of music over her lifetime. Uh, so yeah, I can't say that I always listened 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. Oh, I was, I had a, uh, we had a guest on twin a couple of weeks ago. Her name was Melissa. And I said, did you ever listen to the song Melissa by the Allman brothers? And she said,

7:32yeah, we played at my wedding when my daddy gave me away. It's so cute. Melissa, uh, who put in the Scientific American? Uh, me, uh, uh, so yeah, my wife, uh, she reads the paper to me every day. Uh, and she came across, she came across this on this day, August 28th, 1825, 181 years ago, Scientific American, uh, the first issue was published. So Scientific American is 181 years

8:05old. 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. Hmm. 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? Hmm. Right here in, in Washington on the reflecting pool before it, before it turned green, right? Right. Well, I mean, the reflecting pool has a history, apparently.

8:50Yeah, 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 want 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.

Phage-encoded contingency loci and bet hedging

9:12That'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

9:43really 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. And 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. So we know that the DNA encodes

10:24for 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

10:57of 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. But this is different than that. So that was 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,

11:29a simple sequence repeat. And so instead of having a translation frame shift, 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

12:01something that I hadn't heard of in terms of genetic heterogeneity. So 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.

12:34Yeah. 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. And importantly, you also still keep the same piece of DNA so that if, say, the product is non-functional after your frame shift,

13:06you 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, you know, towards populations versus

13:43individuals. 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. So 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

14:17occurs. 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. So 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

14:56replication 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. This group had previously reported the discovery of a new defense system

15:32that 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 bacteria, to defend against bacteriophages. In this case, bacteriophages with modified DNA.

16:02So 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-hydroxymethyl cytosine. This 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

16:32for 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 its cytosines? 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. And yeah, I'll leave it at that.

17:06So the phage has a method to evade this defense system. It 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

17:36is 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 trade-off that it was making by not modifying its DNA, just like Rich mentioned. So maybe you're going to go here, but the phage defense gene is in at least one of the

18:17enzymes responsible for that base modification, correct? Yes. So the alpha-glucosyltransferase gene, or AGT, is what helps add the modification to that DNA. Right. 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? Yeah. So when they lose the ability to modify the DNA, now it can survive on

18:57the type 4 restriction system defense, but not on the other one. And when they looked at the part of the genome that seemed to be responsible, the AGT gene, is specifically at the sequences, they noticed that there was a repeat of adenines. So there are seven, one, two, 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

19:29had, 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 adenines in this spot in AGT. And so those deletions or insertions were changing the ability to make the, the ability of the RNA to make the protein, changing the reading frame. And you can imagine if you look, think about those numbers, six versus seven or eight is pretty

19:59important because something that's seven or eight is going to be a different reading frame than something that's six. Exactly. Because of the triplet nature. So like three makes or codes for an amino acid. Okay. So they saw this, they saw this in multiple, multiply independently isolated mutants. They also saw a few mutants that were not, um, in this, uh, homopolymer of adenines and those actually changed the sequence of the protein. And so that what they did was they then compared

20:35the mutants that were in the, uh, homopolymer or the contingency locus as they call it. And then they, they compared that with a mutant that would change the, uh, the base to something else in a different spot in the gene. And they looked at how often did they get reversion and they could put the selection pressure for going back to the original sequence, uh, or the ability to make the AGT that could modify the DNA based on, uh, plating it on the bacteria where they otherwise

21:09couldn'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, um, compared to the frequency at which they got a change back in the regular mutation that changed the coding sequence. So they saw that, uh, the, the frequencies were quite different. I think they said it was 10,000

21:39fold different reversion frequency. Now that was interesting to them. And they, 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, they, so what they did was, um, they took their, they, they sequenced to get these original, um, frequencies. They took that information of the sequencing and they looked not just in the phage

22:15that they're working with. So I told you that, uh, 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, uh, uh, sometimes called set of seven dwarves of phage that were isolated and studied by a number of groups a long time ago. And so they compared these T2, T4, and T6 genomes.

22:46And, uh, they saw that the, this locus, um, had this contingency locus in the same, in the same place. And they then looked across the entirety of the genome and they compared how often did they get, uh, the difference in reversion, um, across them. And so when they, when they propagated these three different phage, uh, and then they isolated five of them from each of them that had been propagated

23:19and they sequenced them, they saw that, uh, sequenced to what, 2000 fold. So like every single spot they checked, 2000 times, um, they, 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, they saw a lot of them. Um, they, there were some that were encoded between genes, but they focused on the ones that were coded

23:52inside of genes because then you know that if you get a change, one, uh, nucleotide more or less, that you're going to affect the protein product that would be coming out. And they saw, um, the majority of them were about six. They had some that were seven and eight base pair long. Uh, and they also noticed that the majority of them were AT homopolymeric runs and not GC homopolymeric runs.

24:23And I think the significance of that, as far as I understand, is that AT base pairs, uh, only have two bonds that hold them together in their complementarity configuration and Gs and Cs have three. And so when the polymerase is going along, the DNA polymerase is going along and replicating the, it's easier to break AT bonds and slip than it is to break GC bonds.

24:51So as they were analyzing where all of these, uh, simple sequence repeats were found, they, they wanted to know how often were they not just in the T even phages, but, or were they found in other phage? Uh, is this something unique to these ones? So they took advantage of a set of phage called the, the Basel collection. This is something that was isolated, um, I believe in Alexander Harms's 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. Uh, they definitely found them, uh, across these genomes. They noticed that the amount that was in the different genomes varied. Uh, 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. Uh, and then

26:02again, again, they saw this same pattern that the GC SSRs were not nearly as prevalent, uh, as the AT SSRs. I'm, I'm really amused by the fact that T7 has none. T7 of course is my old buddy. I did my thesis on T7 and I have always, I've always, I think Bill Stadier even, uh, 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

26:34get out. Right. I mean, that's, that's really what it does. So maybe it's not an, 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, what they do. Uh, they noticed that they, they, um, in general, they were fewer than you would expect in the genes that were involved in

27:12replication. So polymerases, for example, were the least common to have an SSR. Those are essential. I mean, if you lose your polymerase, then you're, you're done for. Uh, so the, 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

27:46contingency locus, uh, observation that they had seen with the first, uh, 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, uh, in the entire field of genetics and molecular biology. And that's because this, this gene R2, uh, which is actually two genes, R2A and R2B were the ones that

28:19were used, uh, to determine that there is a triplet genetic code. Uh, and they were also used for demonstrating homologous recombination. So, um, they, they were, they have a lot of literature, a literature about them, even though we don't entirely understand how they work. Uh, and turns out that R2A had a simple sequence repeat of six adenines in it. And conveniently, um, R2A is also,

28:51has 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. Um, they didn't really see much of a, a statistically significant change, uh, compared to what they saw everywhere else in the genome. Um, so they took, they then took that information that's in

29:26the 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, uh, 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. So, but just a little bit. So R2 is known to be important for, uh, the plating of this phage onto a particular host. So in this E. coli host, uh, it has a lambda

30:05bacteriophage already in the genome and lambda encodes, uh, basically an antiphage defense system called Rex. So when Rex is there, then you need R2 to be able to plate, uh, to infect and make a plaque. But if Rex is, I'm sorry, if R2 is not there, then it won't make plaques on this E. coli. And so there, this is setting up a similar system to what we saw with the AGT gene, where if you have the gene,

30:36then you can plaque. If you don't have the gene, then you can't. Uh, and that, so they took advantage of that to look and see if they got a change in their, uh, frequency. And they did, uh, they did see that there was an elevated rate of reversion, um, in the mutants that were in the SSR. And there was not a nearly as much, uh, for the other change that they had made that was what I might call a normal

31:07mutation that changes the coding sequence. And, uh, that's not surprising, I think, based on the, the way that we think that the SSRs are working. Uh, but it was really cool that they could demonstrate it with their actual phenotype of can the phage plate or can the not phage not plate here. Uh, most of the time, accessory genes don't have a phenotype that's easily trackable. Uh, but the R2 genes do, if you plate them on the host that makes that Rex defense system.

31:39And their overall, um, conclusion is that this region of the R2 gene, as well as it seems many other genes may have these contingency loci. They are genetic, uh, 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 they, this is also what is known as the red queen co-evolution between bacteria and

32:14phage. So they're constantly having to go back and forth and just slipping the DNA polymerase here and there, uh, is way easier and faster than having to acquire a new gene or fix a gene that is broken. You, you just always have some that's not working and, and some that is. Uh, 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. Um, I know that viruses of eukaryotes do this as well. And, um, I think that there are also cellular genes

32:51that do this, but cells grow at not quite the same rapid rate that viruses do. Yeah. 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, uh, contingency loci were sort of understood in bacteria systems and eukaryotic systems. Um, and we're not really well known as much in phage and even we're not known quite as much in archaea. And so, um, I guess when I was reading and thinking about this contingency

33:26locus, 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 population, um, and not have to exactly, as you said, gain new genes or, you know, make nearly so many changes. It sort of fits with our understanding of phage, or at least my understanding of phage really nicely, um, and allows a population of phage in these cases to sort

34:02of 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 know, 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, uh, uh, I think to myself, this is really familiar. Um, and I sent,

34:38uh, 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. Uh, and it turns out that, yeah, I was able to dig this out of the archive and I sent it to, uh, sent it to Jolene. Uh, this, this is a really weird, uh, gene in vaccinia called J3, uh, that has, uh, in our hands, at least three different act, well, our hands and others, three different activities. It's a two prime O methyl transferase. Okay. So it methylates the

35:12ribose of the second base in an mRNA cap. Uh, and so that activity, uh, in fact makes vaccinia messages look like cellular messages to subvert, uh, the cell, some of the cellular mRNA based, uh, defense mechanisms, uh, in cell culture, uh, this, uh, so biochemically, this thing also has some sort of transcription elongation function that we never fully understood. Uh, and, uh, it also biochemically,

35:47uh, affects the length of poly A tails. It's an accessory, uh, 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, we found, uh, uh, the, the gene, because of the transcription elongation function, uh, if you don't have it, it doesn't grow on normal, the normal cells and culture we use, but there's a drug

36:18that enhances the transcription elongate, a elongation that has the effect of, uh, correcting that phenotype. So the mutants were drug dependent, but you know, who knows in other cellular environments, uh, whether this might have a contingency. 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, cause they're easy to make, uh, for, for the virus. So there may be some circumstance in vivo

36:52where this is more a contingency thing, uh, 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. Are they, has 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. Uh, yeah, the, yeah, it's, it was not really described in depth here, but I did a little bit of other reading. Um, and there's,

37:26there are some who are very, people who are very interested in these in pathogens. Um, and I was looking at it because, uh, there's a phage that we study that has one of these. It's not in a coding sequence or maybe it is. Uh, 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, uh, sequence repeats. Yeah. Um, I'm not sure which genes in eukaryotic viruses have them though. Okay. So apparently eukaryotic viruses do not have these.

37:59They don't have any? Contingency sequences, but they achieve the same effect in other ways. Okay. Basically. Like high mutation rates. It, it feels familiar though. Yeah.

38:15Do, but Jolene, this is probably present in other phages too, right? They just chose to look at these, the set. Probably. Yeah. I think this seems to have been a follow-up from, uh, their type four restriction system work. I'd like to see, and maybe it's already been done, but I'd, I'd like to see, uh, these guys or some other eukaryotic virus person, uh, do this same kind of thing where they do extra deep sequencing. All right. To see if there are sequences in a population of viruses

38:48that show sort of hypermutation in one of these, uh, stretches of sequence. I thought that was a clever way to approach this. I also am blown away, uh, still by the way genetic mapping is done nowadays. It's just sequence stuff. Sequence the genome. Okay. Unbelievable. I didn't, I didn't, uh, 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

39:22well. 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 mutable, right? Exactly. It's polymerase-based or there are so many of them. Hmm. 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, um, having our professor

40:00actually read from Alice in Wonderland, the section leading up to that, uh, quote. Um, and I, I imagine it every time I think about red queen evolutionary dynamics. I imagine that same passage over and over. Hmm. All right. Now we will move to eukaryotes. And this is a paper in Cell Reports Medicine

Human monoclonal antibodies targeting EBV

40:25entitled, Transgenic Mouse-Derived Human Monoclonal Antibodies Targeting EBV, Epstein-Barr Virus, GP350, and GP42, Provide Basis for Therapeutic Development. Yep. So, uh, the, the authors are, um, 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

40:56in Seattle, uh, several different departments there. Every time I see a paper from the Hutch or UW, Vincent, I think about our visit there. Yeah. Uh, that was just awesome. I came away with such a wonderful impression of the place as, you know, uh, just a wonderful place to be. The, the, the, the either institution on its own, uh, is amazing. Uh, and the overlap and collaboration between the two is amazing.

41:26Yep. So I wanted to just give a super brief overview of this and Vincent will give the details. So 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, they did a mouse study where they made it make human-like antibodies, and then they tested whether they would protect

41:56from EBV. And they were honed in on the right kind of antibodies to protect from the disease that they were testing. Uh, and they found a couple. Um, 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. Uh, 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. We've talked

42:30about that work, uh, 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 cancer, new cases of cancers and 209,000 deaths globally. So not insignificant. Now this virus

43:01infects primarily epithelial cells and B cells. And so those cancers, uh, are typically originating from those cell types, um, uh, in people. We'll talk about that a bit later. So the virus is a herpes virus, right? 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, uh, B cells and epithelial cells. And what's interesting is that

43:34you have two different kinds of cells. And so the, the, the glycoproteins have different functions. There's a fusion machinery. So this envelope is going to fuse with that of the, 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, uh, membrane fusion. Uh, and then, um, when the virus binds to self-surface receptors, which we will get

44:08into, this GHL, uh, relays a signal to the actual fusagen, the fusagenic protein, which is called GB, cause fusion of the virus with the cell. Now the virus infects B cells by attaching to complement receptors CD35, uh, and or CD21, complement receptors one and two. And the, the viral protein that does that, uh, is, uh, is, uh, GP350 or a splice variant of it called GP220. So you need, uh, GP350, but you also

44:46need an additional viral protein called GP42 that binds GH and GL, right? The fusion complex that I've just told you about, um, um, binding of GP42 to class two 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,

45:17um, EBV on, on these B cells, uh, class two, uh, HLA. There's also a hydrophobic pocket on GP42, uh, that's different from the HLA binding site. Uh, and this is the target of neutralizing antibodies as we're going to see. So when, when you are infected with the first time you get infected with, uh, EBV, you, you, you, the virus establishes a latent state in resting memory

45:47B cells. Uh, and these, uh, these cells can reactivate, but most of the time they're eliminated, uh, by cytotoxic, uh, lymphocytes. Uh, so you carry the, the virus asymptomatically for most of, most people 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, called post-transplant lymphoproliferative disease or PTLD. Uh, and this, this disease is higher in EBV, uh, seronegative

46:24people compared to seropositive people, which suggests that preexisting immunity plays some kind of role in preventing it. People have wondered whether passive transfer of antibodies could protect, uh, 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,

46:54GB, GP350, GP42, with all these proteins we've just talked about that are needed for entry, uh, into humanized mice before, uh, EBV challenge. And that, that giving those antibodies can prevent viremia. It can prevent infection of the spleen. Uh, it can prevent viral-driven, uh, lymphoda and lymphoma and deaths to various, uh, degrees. And also monoclonals against GP350 and GHL can protect against a challenge with a rhesus lymphocryptovirus, which is basically the EBV ortholog that infects

47:27non-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've tried some, uh, experiments in people. They're, they use a 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,

48:04at least in that case, uh, mouse antibodies. So what they do here is they use, uh, a mouse called ATXGK. There are a couple of different mice that do the same thing. Uh, they have, 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, uh, the, the constant region is still mouse,

48:38right? 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 the, those, uh, those antibodies, you can fully humanize them in vitro. Yes. That was my understanding. So, so I am not quite picturing it. You have a chimeric gene, uh, or you don't have a chimeric gene cause you can fully humanize it later. Uh, so you are only replacing the V, D and J portions

49:14in the mouse genome with the human versions. You're not replacing the mouse consonant regions. So you've, so your first, your first pass antibody for playing with is effectively a chimera of mouse in the variable region. And I'm sorry, uh, human in the variable region and mouse in the constant 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

49:45to splice in the human constant region. If I got that right, Brianne, and then, and then that would be, uh, your, your therapeutic thing for humans. Cause you're not going to make antibody. Well, not going to make as many antibodies to that. So the, 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.

50:17The part that's the problem for us, if we get a mouse antibody is, is the constant region, right? It could be any of them. Any of it. Yeah. I see. It's foreign, right? So you make immune responses to it. I mean, and people before they had these mice, they chimerized them, right? They could, they could drop the variable regions into a, um, into a human antibody, right, Brianne? But then there are conflicts between the variable and constant region still. Yes. Yeah. To get the sort of biochemistry to work correctly.

50:50And some people, so there's a guy who did that. I forgot his name. It's been years figuring out how to change the constant region 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, how do you do that? This is a method to do that. They speak in the introduction of, uh, very briefly of human monoclonal antibodies that have been isolated that do this. And I, uh, uh, where do those come from?

51:24Oh, so you can get people, right? And you can get B cells and sequence the heavy and light trains, right? Okay. And then express them. So if you, if you have a way of selecting, uh, 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 of, 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, we're not going to use these mice

51:58and make enough monoclonal to like 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 selling culture and make lots of antibody. That 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, the catch with human antibodies, it's not, you may not find the antibody that you want.

52:29Yeah. Right. Whereas in the mouse, you could screen a lot. Right. And the, and the, and the human, you could modify the, 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. Okay. 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. Yeah. Yeah. Yeah. It's the fact that you're getting a full sort of diversification process in the mouse. The whole, yeah, the whole antibody production thing is, is normal. And then you have to,

53:04you have to do Cesar Milstein again. You have to take the spleen out. You have to fuse it with cells that will make them, the spleen cells immortal, the B cells. And then you have to put them in 96 well plates and screen supernatants for the monoclonal you want. Right. That's the original hybridoma technology, which was subsequently bypassed like by phage display and so forth. But it gives you what you want. Right. So sometimes you have to do the harder work. I know. I think the immunology students now don't even have to memorize what's in hat media. Like,

53:38I don't understand.

53:41Yeah. So you fuse it with a myel, 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 the, any unfused cells. Yep. And that's, 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 that what they do is they immunize these mice with, they have produced recombinant GP350 and GP42.

54:16Right. And then they immunize the mice. They 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

54:53inhibit 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. So in the end, so they're making recombinant human IgG1. They have two GP350 binders and eight GP42 neutralizing antibodies.

55:29Right. 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

56:00a perfect fit antibody. And 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

56:31that'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. But 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.

57:05So these monoclones are called ATX42 or ATX350, depending on which antigen was used. And then 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'd make FAB fragments, just the Y part of the antibody that's binding

57:37the antigen. They 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. Okay. So then after affinity, they say, let's look at the neutralization

58:11of 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. And ATX350-1, which had a very low affinity in 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 RAGI was a B cell line. Then they

58:45look in epithelial cells, and you see some differences compared to the B cell line, which makes sense because they're 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, 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,

1:00:00whether 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, too, has the highest neutralizing potency. They 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

1:00:32going 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

1:01:05together. And 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.

1:01:35together 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 stearic occlusion of the hydrophobic patch. Remember, we talked about that before on GP42 by GHGL. So the idea is GHGL is occluding this hydrophobic patch on GP42.

1:02:13Then 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 DBV 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,

1:02:46these eight GP42 antibodies, they actually belong to two clonal lineages that bind GP42 with high affinity. They 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. So by that, I understand it to be that multiple individuals in the lineage of a clone would have had the same germline sequence, but taken a different

1:03:25maturation pathway. Does that make sense? Yeah. So I might say that they picked the same V, D, 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. Because remember, 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

1:04:02infectivity 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.

1:04:39And then they give these mice, once they have the human B cells, they give them an intraperitoneal injection of the antibodies. So 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.

1:05:10So 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 immunohistochemical 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

1:06:17lots of EBER foci. And in the mouse treated with ATX42-2, there are no EBER positive cells. And 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. So 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,

1:06:54they can prevent viral DNA, they can prevent splenomegaly, the enlargement of the spleen.

1:07:04And then they do a, they do a, 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.

1:07:30And now the nice thing is, unlike some of the antibodies that have been studied previously, these are ready to go into people. So 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?

1:08:02See 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, 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. Right. Plus these mice ain't cheap, the humanized mice.

1:08:35So 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. So one of the issues here is that in these mice, only the human lymphocytes are susceptible to

1:09:05infection, 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

1:09:40this antibody, right? 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.

1:10:05And... So these... Yeah, I was going to ask about that, actually. Because these are... Surface glycoproteins, right? That's right. And so when they're doing all these immunizations, they're probably not including the anchor that would put it in the membrane. And so 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. There is... But it does still work. Yeah. Well, you make antibodies, but not the ones that are

1:10:38not 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.

1:11:13That's good. That's pretty good. There's also another phase one that's recruiting, which is a GHGL-GP42 ferritin nanoparticle vaccine. It 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 conformation in order to make the antibodies. But what this paper does, it defines the epitopes,

1:11:47right? You now have a structural target and try and reverse engineer a vaccine. You know, you have the antigen there. 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? So, and this would be more than a post-transplant. If this vaccine worked, it could be an MS vaccine, right? That could be cool. Anyway, so that's why this paper

1:12:23was 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

1:12:56fine, but it didn't have to be that way. Some of these critters are really complicated and really difficult to make vaccines for. But, we're getting better at it now. Getting better at it. And you go back to our podcast, you and I did Rich with McClellan, right? Is it two years ago? Yeah. He's doing that, too. Yep. Okay, let's do some

Listener emails and discussions

1:13:23email. 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 20C, 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.

1:13:54A 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. That 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

1:14:26to add a miniature white poodle urinating on Trump, but it refused. And the picture is below.

1:14:35Dr. Reconyelo, 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. It's 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 About 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

1:15:11albums. 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. This is my favorite scene from the book set in Arches National Park in Utah.

1:15:45Quote, this would be a good country, a tourist says to me, if only you had some water.

1:15:51He'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, siree. And where then would people go when they wanted to see something besides people? I see what you mean. Still, I wouldn't want to live here. So dry and desolate. Nice for pictures,

1:16:26but 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. Red River Gorge in eastern Kentucky is arches with water, at least as close to it as you can get and still be

1:17:01in 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. Thanks, Charles. 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. Amazing. 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.

1:17:36You 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. Mm-hmm. 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

1:18:13Jolene 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. Could you do it for the other programs? And so he said, here you go. We have TWIV, TWIM, TWIP, TWIVO, QUIN, Immune, Infectious Disease,

1:18:44Puscast, Matters Microbial, Urban Ag, and one for all of them. So you can search all the Microbe TV podcasts. Very cool. Thank you, Scott. Thank you, Scott. Very kind. 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.

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, Khadija.

1:19:37I'm reaching out to share a perspective on long COVID reporting based as a parent of a child diagnosed with autoimmune encephalitis in 2019. I 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

1:20:14year that Mayo no longer even includes that on their AE panel. My son, who was seven at the time of the initial diagnosis, had the most severe case of, in terms of initial neuropsychiatric presentation. But ONSAT began with what looked like tonic, clonic, and absence seizures, and longest duration of perceived neuroinflammation. A, his clinicians and I agreed that his relatively fast speaking, speaking fast diagnosis was a result of the following factors. One, age. At the time,

1:20:48didn'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,

1:21:21and Brianne chime in that it will help being believed. Partly, 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, 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 IV-IG and IV steroids in

1:21:52accordance 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. I 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

1:22:26improvement. 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. I met Dr. Douglas Waite, the developmental

1:22:57pediatrician 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

1:23:29will 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. My 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

1:24:01and 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. After reaching his lifetime limit of Cytoxan, he was still experiencing symptoms. Tocilizumab targeting IL-6 from a very small

1:24:38trial 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. P.S. I think part of the conspiracy theorizing about lab leaks, et cetera, 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

1:25:10you 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 Kodija'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

1:25:52groups are important things for all of us to remember. To me, my, 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. I 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

1:26:24and, 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

1:27:14it. It's got a nice picture on top of an emergency, a surgical procedure with it. It says, Fishkin 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. Sort of meshes of one sort or another, I think. But there is evidence that a product made from minimally processed

1:27:44cod 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.

1:28:16There you go, Dixon. Yeah, Dixon would have loved that. Oh, Dixon's here. Look at him. He's up there in the corner. And 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

1:28:46only 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. I'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

1:29:18yesterday with a group that I just absolutely love. And the harmony just blows you away. It's amazing. Anyway, that's a digression.

1:29:28Throwing 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. There'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

1:30:00presence 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. End quote. 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,

1:30:32as well as parts of nature. A bobab tree and a bat, the virus itself. And 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

1:31:05let 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,

1:31:44the path of most resistance. A comic set in a future London facing a crisis of antimicrobial resistant infections. Notable 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'm a student of political

1:32:21science, an illustrator, and a writer. The scope of what I can write and draw is limited by my knowledge and experience. Everyone 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

1:32:53than 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. Write 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

1:33:27communication, 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. I have been making crocheted art for, what, three months now and I'm improving greatly, so

1:34:05I'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 and 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?

Picks of the week

1:34:30My pick is an article from Scientific American. So I was extra excited when Rich told us about 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 trying to look at the last universal common ancestor of life on Earth. And this paper suggests that, in fact, life on Earth emerged twice, once for bacteria and once for

1:35:07archaea. And that previous ancestors before those would not have been alive, where, 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,

1:35:38you 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 think is an indication that this sort of transition to life happened twice separately. And I just thought it was an interesting thought. I hadn't really, I guess I always, you know, imagine those trees that we see in a

1:36:13textbook 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.

1:36:44So my problem with this is that, I mean, archaea and bacteria have the same genetic code, right? And 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?

1:37:17So this is a astronomy picture of the day pick. 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 or 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. So this

1:37:54is 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

1:38:31was 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, um, 2017, took seven years to get to Saturn. Was it 2000? No, no, no, I'm sorry. 1997 took seven years to get to Saturn. Um, make sure I got this right. Launch date. Yeah. 1997 took seven years to get to Saturn, uh, and then, um, orbited it for a

1:39:01number of years and finally, uh, ran out of gas in, uh, 2017. So we're talking, what's that? 20 years, uh, part of that time to get there and a lot of time, uh, orbiting the planet. Uh, 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

1:39:35atmosphere where it surely melted, but I guess we don't know. They actually, they, they talk about in a expanded article on this, that they, uh, um, deliberately crash it into Saturn where they figured it would be, uh, consumed because they didn't want to take a risk of contaminating any of the moons of which there are 260, uh, uh, with any, uh, trash from the spacecraft. Right. And some of

1:40:08them are, you know, candidates for having potential, uh, 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? Uh, so today I, I switched my pick. Um, I am picking a, uh, CBS News evening story about Dolly Parton when she first got her COVID vaccine. Uh, so she thought that vaccines were very important

1:40:41and decided that she would do, 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, uh, 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, um, with the words, vaccine, vaccine, vaccine, vaccine, vaccine. I'm begging of you, please don't

1:41:17hesitate. And then she says, vaccine, vaccine, vaccine, vaccine. Cause once you're dead, then that's a bit too late. Oh yeah. And so, um, it's just a, a really nice way for us to kind of remember the contribution that she made. And, um, I think it's a good, a good rewrite of the song. It's a much more wholesome than the first one, actually. The, uh, uh, uh, that, uh, nature article that was

1:41:52picked earlier, uh, makes the point that, uh, uh, Dolly gave a million bucks to Moderna to assist in, uh, creating a vaccine. And, uh, uh, she, uh, through a foundation, I guess, that she created, which is, um, uh, 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, um, a big portion of her money she gave to

1:42:22Vanderbilt, Vanderbilt. That's right. They did the development. Yep. Yep. All right. My pick is, is apparently a pick I've done before, according to Jolene, but, um, this is the book by Daniel Kahneman called Thinking Fast and Slow. And the reason I wanted to pick it again is the following. I did. So 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, uh, Thomas Edison Museum in Edison,

1:42:59New 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 resort to avoid the real labor of thinking. And I was looking at it. I said, where does, 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. I just kept this little cardboard thing because it just impressed

1:43:34me so much. Right. Cause I think it's, I feel that 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. Um, and that conserves energy, but real thinking, real deep analytic thinking requires a lot of energy. It's exhausting and most

1:44:07people don't like to do it. So I just read about this, you 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 takes, 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

1:44:40background in science, it's going to be really hard. So they switched to type one thinking and gets us all in trouble. It's hard for me too. Yes. I think it's hard for all of us. And yes, it is. I think that, um, as I've been spending my time recently trying to get ready for the new semester and, you know, thinking about, um, 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

1:45:14sometimes you got to do the thing that's hard to help out practice your brain. Vincent, I feel like you've talked about this, this, um, 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. Yeah. Yeah. Because Thomas Edison had this in his lab to try and motivate people. Yes. Cause 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

1:45:50you'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 is, 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, I just, just had this idea that maybe it can explain a lot of, um, anti-science skepticism, right? Anyway. This looks very interesting.

1:46:21Anyway, 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, and reading it yourself. And people will say, well, I don't have time. That's just, that's laziness. It's, it's cognitive laziness. It makes me think, go ahead. I don't think you said what the book is, Vincent.

1:46:57Yeah. I said Thinking Fast and Slow by Daniel Kahneman. I think so. But anyway, yeah. Thinking Fast and Slow, uh, Daniel Kahneman. Um, I linked to the Wikipedia page about it, which, um, you can go check out. Yeah. I think about, uh, procrastinating a task. Okay. Like, you know, in my case, uh, actually I don't have to procrastinate, uh, uh, doing TWIF because I like that preparing for TWIF, but you

1:47:29know, I do other stuff like, you know, I do administrative stuff for this, uh, music chapter that I'm in. So, uh, creating an agenda for a meeting or something like that, uh, or doing some other task. And I will, uh, it, 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. And I don't want to do that. But usually I find when I get into it, it's okay.

1:48:02I think the things that you have to do are the ones you want to put off often. Right. Uh, yeah. But I like to do something like this. I love doing this. I, this, this is not a chore ever, ever, ever. Well, it's almost as if, uh, you know, uh, the, 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, um, you know, technically nobody has to do this. We, you know,

1:48:35Rhian has to go to her lab. Jolene has to go to her lab. So this is not like that sort of thing, but. There was a interesting article that I almost picked, um, that, I mean, these come out periodically, right. But just arguing for how it should be part of our job to communicate to the public, uh, the science that we're doing. And, uh, I mean, the teaching part is an easy thing for us to say like, yeah, this is my job. Um, 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

1:49:09communicate the information in a way that's relatable to particular audiences. No, we are the ones, we are the scientists. We know the science. We are the ones who have to communicate it. Yeah. I cannot speak for all of us, but I at least was very much funded in that training through the U S tech tax dollars with NIH, um, and things like that. And so, uh, I always try to make sure that I'm helping out the taxpayer, uh, by doing that communication. I think about this

1:49:42all the time. I've been on the dole my whole life. Okay. I went to a, a state funded, uh, university. I was on a training grant in both, uh, graduate school and for two postdocs. And then I've had, uh, grant funding at both my jobs, uh, both at, uh, state universities. I've been on the dole my whole life. What does that mean being, I've been on a dole? Uh, uh, uh, somebody else paying for my existence. I see. Okay. We have a listener pick from John. Dolly Parton probably did more to protect

1:50:17people against disease than the federal government. In her memory. I recommend this short video of her singing Jolene with the re rewritten, reframe vaccine vaccine as she gets her COVID-19 shot. And John gives us a YouTube link. Thank you, John. That is TWIV 1353. 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.

1:50:55Jolene Ramsey is at Texas A&M University. Thank you, Jolene. Oh, sorry. Ramseylab.bursell.app. That's right. Yeah. Thanks. Uh, I enjoyed our discussion today. Brian Barker is at Drew University, Bioprof Barker on Blue Sky. Thank you, Brian. 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,

1:51:29but 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. Another TWIV is viral.

1:51:59We'll be back next week.