TWiV 1375: One Virus is Not Enough

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 1375, recorded on November 13, 2026. I'm Vincent Racaniello, and you're listening to the podcast, All About Viruses. Joining me today from Madison, New Jersey, Breanne Barker. Hi, it's great to be here. It is very rainy in Madison. Not as rainy as it was yesterday, but still raining quite a bit, and it is 41 degrees Fahrenheit. So, you know, a good day to be indoors. Yeah, it rained all day yesterday. Last night, it's raining now. Here, it's six degrees and cloudy. It's chilly. But I had my umbrella. So at least that works. Now, I realize you should carry an umbrella all the time.

1:04And even though it's heavy, I put it in my backpack and now I benefit from that. Yes, I paid for not carrying an umbrella with me when I had to run a couple of errands after leaving my office yesterday. Because it was a bit rainy when I left my office. And by the time I got to my car and got to the errand, it was extremely rainy and unpleasant. Also joining me today from College Station, Texas, Jolene Ramsey. Hello. Well, it's a little bit warmer here. 76 degrees Fahrenheit and 24 Celsius. It's chilly, a little bit beginnings of chilly here, but not really cold. and not raining yet. It's supposed to rain this weekend.

1:54So what I want to know is what kind of umbrella do you carry? You know, you carry the like one that's going to flip inside out when it's actually raining and you need it or just like you carry a good one. Is that why it's heavy? I'll show you. Can I show you? Sure. I don't carry one, or most of the time, but when I do, I do have a heavy one. And I have one that has pretty designs on the inside, so it looks nice when I look up at it. It has space. So it looks like I have a beautiful night sky of space. But it folds up compact, I guess. Yes. Okay. I had originally bought a really small one. Like it folded to this big.

2:35It was$100 and it broke within months. $100? Yeah. Isn't that ridiculous? So I bought this, which is bigger and heavier, and it's substantial. And this has lasted a while. But what I like about it is the color. It's a rainbow. Ooh. See, he also brings nice colors with him in his umbrella. That's beautiful. I like it. I mean, everybody has a black umbrella, but this is, it's also, it's great colors, right? Yeah. It stands out. I told Jolene, my umbrella has space on the inside. So it looks like a black umbrella on the outside, but then it looks, but then when I look up, it looks like I have a nice, pretty.

3:13That's very cool. I like that. So this one is pretty, I have to admit, this is the second one. The first one broke. It stuck. The thing is, it's got a button. It has a button that opens it, right? It's great. So you get it out of someplace. Boom, it's up. But then you have to push it all the way down by hand to close it. It's really a hard spring. And one time, I opened my previous one, and it wouldn't close again. So I'm going to put this in my backpack. Otherwise, I'll forget it. I bought this one, and it's been fine so far. Yeah, but they all break. Absolutely. Even if you buy one that doesn't fold.

3:53Yeah. They break too. That's the kind I usually have. And somehow I always break the handles. The handle. Wow. Usually it's the top of the thing that goes.

4:06Anyway, that's the weather for today. So if you enjoy these science programs, which we do speak mostly about science, we'd love your support. You can go to microbe.tv slash contribute. American Society for Virology Applications are open for the ASV Global Scholars for the annual meeting next year. That's in Madison, Wisconsin, July 19th to 22. These awards are for applicants doing virology research in low - to middle-income countries. You can go to the ASV website. That's asv.org. You click on ASV 2027 in the upper right, and you can get the application there. They're due Monday, November 30th.

4:54Great opportunity to present your work and interact with others doing cutting-edge virology research. Yeah, I would definitely recommend it. Yesterday, we got an email from the president of ASV talking about the ASV 2027 meeting, and it listed some of the speakers. and I always love the ASB meetings, but I got even more excited when I was looking through the list of speakers in that email yesterday. So I definitely would recommend checking it out. You know who the keynote speaker is? I do now. Harold Marmis. So I would ask him for TWIV, but the problem is TWIV is on the end of the meeting at Thursday and guys like him will come and go, you know but we'll try because that happened originally with the original speaker for last year they said no i'm not staying yeah to the end well they said yes originally and then they canceled not too long before i was very lucky to get um the measles catanio and and his associate forgot her name do you remember her name work talk about sspe yeah i was really lucky because he canceled like two weeks ahead of time.

6:12But they were great, those two. They were. It was a really informative episode. I will talk to... I have his email, Varmus's email, and I will talk to him. Anyway, I hope there are more international people this year. Last year was hard because of the climate. And I hope it loosens up a bit under the oversight of Congress, which is how it should be, right? Yes, I hope so, too. Her name is Kalpana Yadav. Thank you, Kalpana Yadav. Great. She was excellent. Okay. I often talk about how I get a lot of AI-generated email to TWIV, like, you know, asking me if I want to have someone on the program. They're all from PR people, but they're clearly AI.

7:01And I got one today. The other thing is they offer services. Okay, this is totally AI generated. Listen to this. Your November 7th episode, TWIV 1373, packed a dengue human challenge study and a flu immune evasion paper into one sitting. The kind of dense two-paper episode where the sharpest 60 seconds are easy to miss if nobody is cutting clips. So why is this classic AI? Packed? Nobody says packed. Okay. Dense two paper episode. No. And the sharpest 60 seconds. Nope. Also the M dash. The M dash too. Right. That's between sitting and the kind of dense. And then finally, nobody is cutting clips.

7:49I never talk like that. Nobody is coming. I'm actually, does that mean like you need, they're asking if you want someone to make clips for you? Yeah. The rest of the email was, I can cut clips for you really fast because they do it by, Hey, Riverside that we're recording on now automatically makes clips for us by AI. I don't need to pay anybody. I don't use them. I actually use a human to make clips. Why? Because I think they do a great job. And maybe AI would do a great job, but I'm not going to switch when I already have a great job, right? Plus, I'm supporting a human. That's cool. Anyway, that's an example of AI.

8:30I get so many of them. And the thing is, you read the first one, your November 7th episode, I'm thinking, oh, somebody who listened. Nah, there's nobody who listened. Anyway, there's a real news item here. This is an interesting, it's just a press release from Moderna. But they have developed an mRNA vaccine against Bundibugio virus, right? and that's going into a phase 2-3 efficacy study in the DRC because they've already done preclinical and phase 1 data. And so Bundibujo, of course, is the virus causing the outbreak. Right now they're using Hervebo, which is not a matched strain. So this will be presumably better, but they have to check it first before they just give it to everyone.

9:19Okay, so that's why they're going to do this. So that's really good. That's great news. I mean, the outbreak only came to our attention in May, right? So this is not bad that we already have a vaccine. That's great. That's mRNA technology. It's mRNA technology and cooperation between Moderna, CEPI, Gavi, the Vaccine Alliance, and other global health partners, right? It's not... Yeah, sure. It's a lot of people and infrastructure that's already in place to do the testing. Yes. And bring it into people. And a lot of past basic research to understand things about Bundibugio, to understand things about these vaccines, etc.

10:03I like in the last sentence, there's no vaccine for prevention of Bundibugio virus disease. Thank you, Moderna. Okay, that's pretty neat. There's a call from some group yesterday. Daniel mentioned this. So right now the Ervibo, which is the Zaire Ebola virus vaccine, is not being given to a lot of people. It's being tested, right, to make sure it confers some protection. I think they give it to so many people and then X weeks later they give it to other people to make kind of like a control without being a control. But no, I don't think it's appropriate to expand it until we know if it works, right?

10:51Right. Even though you would love to. You would love to save people's lives, and I understand that. I think doctors, Medecins Sans Frontières wanted to do that, yeah. And I understand that, right? Because that's what they do, right? Anyway, got to test it. Because if you don't test it, then people will say later, you used an untested vaccine. And that's why we have this and this and this. Or people will say, hey, you already gave me a vaccine. Why are you trying to give me another one? If we later find out that it doesn't work. That too. Okay. So before we get to our paper, I have one thing I want to tell you guys.

11:29So I had a good question on live stream. They said if there was a Mount Rushmore for a virologist, who would be there? And I said, I had to think about that. It's an interesting question, isn't it? So we should do an episode sometime where we talk about that. Maybe the year-end wrap-up. That would be good. Are you going to be on that, you think? Probably this time, yeah. Okay. Okay, now we have a paper from Jolene where I learned more words than I ever had before. Great. Oh my gosh, Jolene. I think both of these papers today have a term in the title. Exactly. That's what I was thinking. That we had to put some thought into.

12:14I was very glad that I knew someone who worked on Merrimyckdick Lakes when I saw Jolene's paper. Oh, okay. Okay. Yeah, well, the paper that I picked today, I picked it because I saw a talk by the first author at a local branch meeting of the American Society for Microbiology, and it was an awesome talk. And I was like, this is so cool, and I want to talk about this in my class, so I should read this paper. I was like, what better way to make myself read the paper than to talk about it on TWIV? So this is a paper from the ISME journal that came out in January of this year called Abundance Activity Decoupling in Sulphur Cycling Bacteria Reflects Viral Infection Types in Meromyctic Lakes.

12:59So it's talking about how the viruses that infect bacteria that can change sulfur into different forms are influencing that sulfur cycling. And they're studying these bacteria in meromyctic lakes. So meromyctic lakes are the kinds of lakes where the whole thing doesn't mix at least once a year. So they have layers of water in the lake that are essentially stratified from the other layers. And that usually means an oxygen-free or anoxic lower layer, and then maybe a middle layer that's partly mixed, and an upper layer. So that kind of is my attempt to deconvolute the title a little bit. And this is a paper by Jordan Walker, first author and co-corresponding authors Alice Bosco Santos and Cynthia Silvera.

14:04I was excited about this because I like to tell people that viruses are important for everything, you know, and that they drive global biogeochemistry. And it's true that they do. And this kind of talks a little bit about that. To give a very brief overview, they were studying this lake that has purple sulfur and green sulfur bacteria. Actually, there were three lakes. Three lakes where they had these layers of bacteria. They pulled out samples of everything that was living in those areas of the lake. They extracted DNA and RNA to figure out who was there, who was being infected by what viruses, and how sulfur was being cycled in those areas because that is the way that they recycle their sulfur to do photosynthesis.

15:02And they found out that the virus relationship with their host bacteria was not reflective of the abundance of the host bacteria. Um, sometimes you might think that if a bacterium is a certain level, then the amount of viral activity will be proportional, uh, or that the, uh, the contribution it has to the level of a particular critical nutrient in the environment will also be proportional, but that was not what they found here. And some of the reasons this is important are because sulfur cycling is linked to carbon cycling. And it's something that you can see in the geologic record as an indicator of when ancient Earth wasn't just oxygen driven.

15:56So when were we primarily pre-oxygen and when were we moving post-oxygen and what parts of the Earth were this way versus another way? It also is really important when we're studying non-Earth environments where oxygen isn't present. It gives us ideas of who might be there and what the chemistry is in those places. and then the one other thing that I remember that they were talking about is kind of a reason why this is interesting. Maybe I'll bring that back up later. I can't remember what the other thing was. Can I ask you something? So they mentioned that. So at some point in Earth's history, there were a lot of anoxygenic photosynthetic bacteria, right?

16:44And so they, I didn't even know this, they do photosynthesis, but they don't make oxygen. Yep. Yeah. Right. That's right. And so these bacteria are the modern day equivalents. Is that the idea? These purple and green sulfur bacteria are, they call them photooxetrophs. So I presume they are. Yeah, that's right. So they're living in the sediment. And I looked it up today. You can find these in muddy. So how do they get light in the sediment? Well, that's actually part of the first part of what they do here is they go to these lakes and they are measuring how much light that can be photosynthesized gets down into that part of the water column.

17:24And you see a sharp decrease in these bacteria right past where the light can get. Okay. Yeah. Okay. So the bacteria do not exist below the area where there's no light. These bacteria don't. Other bacteria do. These particular Yeah, because if you're photosynthetic, obviously you need light. You need the light. Yes. Okay. So I'm going to go through this, but chemistry was never my strong suit right next to math. So I'm going to say some things the best that I understand them. Correct me if you know that I'm wrong. And check me if you're listening when it comes to the chemistry side of things. So they tell us here at the beginning that in the pre-Cambrian era, prior to Earth's atmosphere being oxygenated, which is estimated to be greater than 2 billion years ago, these photosynthetic and oxygenic bacteria were the ones that did most of the primary production on the Earth.

18:28And as Earth became more oxygenated, they became less of the primary producers during the Paleozoic. And that depended on oxygen limitation in different areas of Earth. And that was more around 540 to 470 million years ago. And then also into what is called the Devonian period, 420 to 360 million years ago. But today we do still have these kinds of bacteria. Like I said, they're called purple and green sulfur bacteria. Here's two words for you, Cholene. Eugenic and ferruginous. Yes. I guess ferruginous has something to do with iron. Correct. What's eugenic? I looked that one up before. But it has to do with water bodies where there is not a lot of oxygen and there is a lot of sulfur.

19:30Yes. And the other one for your genus is a lot of iron and not a lot of oxygen. Definitely the iron part. Not sure about the oxygen part. Let me finish my Googling. Okay. It's really amazing. the, maybe virology is the same for an outsider, but the words here are totally different. Yeah, it just says contains iron. Contains iron, okay. So these are examples of places where these purple and green sulfur bacteria can predominate over others. And they produce certain kinds of pigments that help them harvest light and do photosynthesis. And these are the presence of those kinds of pigments and other special metabolites that they make are known as diagnostic for their presence, and we call those biosignatures.

20:18So we can find those and use those as indicators of where in the geologic record or where in a water column these bacteria are growing. And in the kind of lake that, or the three lakes that they studied, these meromyctic lakes, like I said, there is a surface layer that is more mixed and oxygenated. Then there is an intermediate layer where there is some mixing with the oxygenated layer and some mixing with the anoxic layer below it. And this has significant changes in concentrations of different chemicals depending on the lake itself. And then the bottom layer, which is anoxic. And the middle layer, which they call the chemocline, that is where there's enough light that the sulfur bacteria can grow, and it forms what they call a microbial plate.

21:17And I think that this plate is an indicator of like if you took a cross-section view of the lake, it would literally be a layer that looks like a plate of the microbes growing. And if you go to the website of the last primary author here, the Silveira website, lab website, you can see a video of them taking samples of the water in the lake. And it's really cool because they go down a certain depth and then they put the tube into the bottle. And as they get to this area of the chemocline where these bacteria are predominating, the water changes color because of how dense it is with the microbes.

21:58So you can get purple water or green water. Are they in a boat and they're putting tubes in? They are. Okay. Yes. So years ago, I was sitting in Montana with Dixon as the sun went down next to a lake and he was explaining all this to me. Yeah, he knew this stuff. I'm sorry he had to miss the discussion of this paper because I would appreciate his commentary. He would love it. Yeah. So like Vincent said, these bacteria are oxidizing sulfur compounds instead of oxygen to fuel their photosynthesis. And it's not just the green and purple sulfur bacteria. There are also sulfate-reducing bacteria that are present in these environments.

22:45And so kind of one of the big questions that they had going into this study was about some observations that had been made where they were looking at these biosignatures and seeing the abundance of these sulfur bacteria, but then they were noticing that it wasn't matching what they would have expected in terms of sulfur compounds in that area. Because if they were growing there, they should have had a certain ratio of sulfur types, and they weren't seeing that. And they were wondering, could viruses be contributing to this decoupling between abundance and actual sulfur that is present? By the way, you said they don't use oxygen for photosynthesis, but it's actually they don't use water, which makes oxygen.

23:32Thank you. Okay. Yeah. And so, yeah, so in micro, sometimes I talk about, you know, phototrophy and photosynthesis, and photosynthesis being the oxygen one and phototrophy being broad to the non-oxygen ones, too. Okay. Okay. So when I say sulfur ratios and sulfur cycling, we're talking about going between hydrogen sulfide or sulfide compounds and sulfate compounds. So when you oxidize sulfide and you can get sulfate, and then that can be converted into organic forms of sulfur that living organisms can use, or the sulfate can be reduced back to sulfide. And so that is the cycle that I'm talking about here.

24:25And then in terms of ratios, there are different isotopes of sulfur. The super common type of sulfur is sulfur-32, and then there's sulfur-34, and these are present in a certain ratio. It's like 95 and 5 % in most environments. But in specific environments where you have more microbial activity, then you'll get a shift in the ratio. Okay, so to figure out if viruses might be involved in the decoupling between abundance of certain bacteria and the kind of sulfur compounds that they were finding. They went to three of these meromyctic lakes that don't completely mix. They were present, one is Mahoney Lake in British Columbia, Canada, and then two were in Washington.

25:18One is called Poison Lake and the other one is called Lime Blue Lake. They went and they collected samples and they measured many physical characteristics of the water, and they also filtered a certain amount of water, and they collected all of the microbes that were present in that water, and then they froze the filters, and they could then extract from those frozen samples DNA or RNA to see who was there, and the RNA would tell them who was active in that environment, who was transcribing RNA. And then they also did a last really cool set of experiments called Hi-C, which is where you cross-link the nucleic acids together in your sample.

26:07And then you are able to see who was next to who. So this kind of helped them say which viruses were associated with which hosts, because when the nucleic acid of the virus is inside the host bacterium, they will cross-link together and come out together. So they did these three things to collect their information. And then they, okay, so let me tell you about the kind of just the basic characteristics of the lake. In all of the lakes, they were able to clearly detect a region of the lake that was the top layer that was oxygenic, the middle layer, which they call the microbial plate, and then the bottom layer that was anoxic.

26:52But the exact depth at which that occurred varied from lake to lake. And the specific concentrations of hydrogen, sorry, of sulfide was different from lake to lake as well. It varied actually quite a bit across several orders of magnitude. And what they did was they plotted which of these sulfur bacteria were present and which ones were most active in these different lakes. And they saw different patterns for the different lakes. I have a hard time keeping them straight, but I'll mention the names just as a way to separate between them. So Lime Blue Lake, for example, had not very many purple sulfur bacteria, but it had a lot of purple sulfur bacteria activity.

27:41And then the other two lakes, Mahoney and Poison, had a lot of purple sulfur bacteria and a lot of purple sulfur bacteria activity. The Lime Blue Lake had lots of green sulfur bacteria and they were active, but the other two lakes didn't have very much at all. So that was kind of some differences between them. And then they also, okay, yeah, so that's kind of the basic characterization of the lakes and who was there. After they looked at which bacteria were present, they then looked at the viruses. And they sequenced these viruses and they classified them according to whether they were the kind of virus that will always go into the lytic cycle and immediately replicate, make progeny viruses and release into the environment.

28:35They call those lytic, although I don't like that terminology. I think it's very ambiguous. I would call those virulent viruses. I was just going to ask you that. And then, so there's those. And then anything that wasn't a virulent or what they call lytic virus was a temperate virus. And these are the kinds that can enter into the host cell and immediately make progeny and release by lysis. Or they can enter the host cell and either integrate or go quiescent for a while and replicate with the host without making progeny. So actually, they first classified which ones were temperate. And anything that wasn't temperate, they called what I'm going to call virulent.

29:23So when they looked at the abundance of these different viruses with different lifestyles, and these are hugely broad categories, by the way. I'm sure that individuals who study sulfur bacteria might say the same thing about purple sulfur bacteria actually encompasses many different groups. I'm not as familiar with those, though. So for the viruses, they saw lytic viruses in all of the lakes. They also saw temperate viruses in all of the lakes, but the distributions were different. So there were fewer temperate viruses in the lime blue lake where there were lots of green sulfur bacteria, and there were more temperate viruses in both the Mahoney and the Poison lakes, and they were also more active in those lakes compared to lytic viruses.

30:15And they then did some interesting analysis where they were looking at which kinds of bacteria or which kinds of microbes had defenses against these types of viruses. So they made an argument that there are multiple kinds of defenses that bacteria have against viruses, and some of those can be broadly classified into these two categories. One category are restriction modification systems. These are proteins that can recognize DNA that is modified in a different way than the host DNA and immediately cleave it and prevent the virus from replicating. That's the restriction modification system. They said that these restriction modification systems can be active against both the temperate and the virulent kinds of viruses.

31:11And then they also checked for defenses specifically that are CRISPR-Cas type of defenses. These are the kind that when a viral nucleic acid comes into the cell and the system has a match to that specific nucleic acid, then it will cut it. And these ones are definitely effective against virulent viruses, but when the host cell has to have that matching nucleic acid to the incoming virus nucleic acid, there's a risk that it could accidentally target itself because the temperate virus could integrate into the host chromosome. And so there's a maybe there is a stratification between which types of defense systems you would find based on which types of viruses are more active in a particular microbial population.

32:05How did you feel about that, Jolene? Because this was something that sort of, I was like, huh, I never thought about that. That's really interesting. Yeah. And so I was intrigued and I'd love to hear more about how you felt about that discrimination. Mm-hmm. I think that I was like, I've never heard that before. Does that really make sense? I think logically it makes sense on like a really broad scale. I could say that the pattern is probably there. But it's definitely not true that you won't have a CRISPR-Cas system against a temperate phage or maybe, yeah, temperate phage. so restriction modification systems it makes more sense they're active against everything I think the problem is with the the CRISPR-Cas it depends on its specificity is it RNA DNA and how active is it all the time how specific is it to having the to having the PAM sequence next to it.

33:09And I don't know, I'm not, I'm not as convinced, but there are some, I could see why we would call that to be the trend. Yeah, cool. Yeah. So what they noticed with the CRISPR-Cas systems is they saw there were more of the genomes that they had with CRISPR-Cas systems in the Lyme Blue Lake, which was where they had had green sulfur bacteria more than the purple sulfur bacteria. They also had restriction modification systems there. And then they had not very many of the CRISPR-Cas systems in the Poison and Mahoney Lakes where the purple sulfur bacteria were more abundant. And they were also less active in those systems where they were present.

33:58Okay. So the next thing that they did was they then classified each of the viruses that they had found according to which host they were infecting. And there are not just green and purple sulfur bacteria in these environments. There are many other kinds of bacteria and archaea and probably other microbes. And so this was really helpful for starting to really hone in on which of the viruses that were present were infecting green sulfur bacteria and purple sulfur bacteria that would affect the sulfide and sulfate sulfur cycling. So they saw that in the lime blue lake, there were all kinds of hosts that had virulent viruses and temperate viruses, but there was a huge bias of green sulfur bacteria that have virulent phages infecting them.

34:55And they didn't really have any temperate phages that could infect green sulfur bacteria present in their samples. Then they saw the opposite for the Mahoney and Poison Lakes. They saw that there were mostly temperate phages infecting the purple sulfur bacteria, and they didn't really detect much or there was negligible amounts in the virulent category. That's kind of surprising, really interesting. There are other bacteria that people study that seem to have a bias towards one kind of phage that will infect them. For example, the Clostridia, everyone's always looking for a virulent phage, but they pretty much only observe temperate phages, and we don't entirely know why.

35:45So this is a pretty clear signature that they have in these samples that they took in the summer of 2023. So then they did some, they tried to figure out, okay, so is there a pattern between the abundance of temperate phages with purple sulfur bacteria and the virulent phages with green sulfur bacteria, we would expect or we would predict that if they're there in the green sulfur bacteria, and these are virulent, so they should be active. So we should see lots of activity, transcriptional activity, RNA making by these viruses. And so they kind of plotted abundance of the viruses and their hosts in a ratio per each environment and by virus type.

36:38And they see that they have, for purple sulfur bacteria, they have very similar ratios that you would expect. When there's temperate, you see lower activity. And this is expected because temperate viruses tend to be integrated and not active, although there are cases where they are activated into the lytic cycle, and so you will see their activity. For the green sulfur bacteria, they saw really a very different pattern. It was spread out. They had some cases where there was low numbers of green sulfur bacteria to virus ratios and low activity. And then they saw another set where they saw a high ratio, so high activity and high abundance.

37:30And these are probably, let's see, this is here. So these were probably going to be telling us that they said the most dominant ones had the lowest abundance in activity that could result in a lower frequency of infection given the large host population. Because green sulfur bacteria were super abundant there. And so that might partly explain it. They also saw other sulfur-reducing bacteria in these areas, and those had no patterns. They were all over the place, high and low activity across all of the different kinds of sulfur-reducing bacteria that were not the purples or the green ones. Okay, so now after they had seen these patterns, they wanted to look at the effect of the viruses on sulfur cycling activity specifically.

38:36So they started to look at the virus genomes for what kinds of metabolic genes they encode. That could give them a clue as to what pathways the virus might be affecting directly. Of course, when the virus infects the host, once the host is killed, then that is no longer going to be changing any of the kinds of sulfur. But while a virus is infecting or when a temperate virus becomes a lysogen, gets integrated, then that can dramatically impact the kind of metabolism that a host is doing. So they were looking for things that were involved in all kinds of pathways, not just sulfur metabolism. They saw genes involved in carbon fixation, glycolysis and gluconeogenesis, photosynthesis, production of specialized compounds called porphyrins, and other parts of the sulfur relay system that are not the specific metabolism that they were looking at.

39:40They plotted this across the different lakes, and they saw that there were all of these kinds of genes. Some were more abundant than others in the virus genomes. And they also saw that the genes that could be involved in these different pathways were active in most cases. They were active across the different lakes. Maybe in some lakes they were more active than in others, but they definitely saw that the viruses, both lytic and temperate, could impact these metabolic pathways by the activity that they were observing. And then they honed in even further onto a few specific examples of enzymes that are involved in sulfur reduction.

40:38So they picked a gene called CYSC, or CIS-C that makes something called adenyl sulfate kinase. This converts one form of sulfur, adenyl sulfate, to 3-prime phosphoadenyl sulfate, which, like I said, is an intermediate in reducing sulfate to sulfite. And they looked at all of the CIS-C genes from the hosts in that environment and the cis-C genes from the viruses in that environment. And they took the protein sequences and predicted the structures, and then they compared them to see how related they might be. And when they looked at the classification of those predicted structures and at how closely they matched each other, They saw that there were viral genes, virally encoded kinases that looked very similar to the bacterial ones.

41:42And they saw that not just for the CIS-C gene, but for a couple of others as well that were part of this pathway. They saw it for one called CIS-K. and then they looked at several others that are involved in processing of compounds that have sulfur groups in them. So the overall pattern that they saw was that the viruses did have genes that were related to the sulfur metabolism pathways and that they were very similar to the bacterial ones, and they then hypothesize that these could be active in shifting the balance of the abundance of these sulfur compounds. Okay, so overall, kind of some conclusions that we can draw is that there were a lot of purple sulfur bacteria activity is correlated with the presence of temperate phages.

42:56And that suggested that perhaps the temperate phages could have an impact on their ability to thrive in that environment. And these, they said, would contribute to shifting the population from having lots of sulfates to a high sulfide concentration. That is for the purple sulfur bacteria. But then when there were the lakes that had high green sulfur bacteria, where they saw more of the virulent phages. The high phage activity would contribute to the green sulfur bacteria being turned over, as in the phage infects, then the bacterium lysis and releases all of its compounds that are inside the cell for other bacteria to consume and use.

43:53And that would result in maybe more of a balance, but less of the sulfide accumulating. And so that's the way that they describe the difference between where green sulfur bacteria predominate and where purple sulfur bacteria predominate and how the viruses might be impacting. And so one thing I didn't say that I think would make a lot of sense to me is that where purple sulfur bacteria have a higher predominance of temperate phage. When the temperate phage are integrated, not all of the virus genes are being expressed, but some of them can be expressed. And those can be either directly involved in sulfur cycling, or they could be influencing the bacterial genes that are doing sulfur cycling.

44:39It could confer to them a fitness advantage compared to some of the other microbes in that environment. And then some other things that they suggested this would mean would be that the, let's see, the one that I really was super interested in is the idea that when you see a certain amount of the biosignatures of purple sulfur bacteria, They originally, these had been used as diagnostics of a particular era. So the Proterozoic, or what is 1.6 billion years ago in those sedimentary rocks. And so if we want to be able to use those markers to date, either to date or to know what was happening at a particular time in our past or what might be happening in another environment where it's anoxic and we're trying to figure out if we want to go to this planet or asteroid or something, then it would be helpful for us to understand what kinds of growth conditions and what kinds of viral factors would influence the abundance of a biosignature for purple sulfur bacteria or other sulfur bacteria.

45:59And it seems like the viruses do impact the ratios of sulfur-34 to sulfur-34 sulfate and sulfur-34 sulfide. Um, it's, I was really surprised, I don't know, because the chemistry for me is a little bit hard that, um, we have preferences for which we, as in living organisms have preferences for which kind of sulfur isotope we use in our metabolism. I, I did not know that about our sulfur metabolism, But I did know that about carbon. And so I guess it doesn't shock me. And I guess it has to do with how the enzyme works. But I don't know. I just know there's a difference in carbon. Yep, both. And some of the open questions that are left is like, how are the viruses actually having this effect?

47:01It would be helpful to have some maybe either more controlled pairs where they could look at a known interaction between a temperate phage and its host or a virulent phage and its host. Or even more interesting would be to use stable radioisotope labeling where you could add a sulfide compound that was labeled into an environment and then watch how it moves through the population and see it get cycled. Another important caveat, or not literally the caveat, but a thing to think about is that when they were talking about when you measure the ratios of either sulfide compounds or sulfate compounds and you don't see them changing, it doesn't mean it's because there's less activity.

47:49It could just be that there's more cycling in both directions. We don't always think about it, or you can't always see that when you just take one kind of measurement. So adding in the activity measurements for viral genes and bacterial genes was useful for helping tease that out here. They call that cryptic cycling. And I think those are kind of the major things I really wanted to bring up. I thought this was a good way to look at how viruses actually are influencing this major biogeochemical cycle. And we don't maybe think about sulfur as much because it's not really a limiting nutrient in any of the environments that we grow in.

48:32But it is cycling everywhere around us. And it is linked to other major biogeochemical cycles. And it's, you know, in places where we don't necessarily frequent all the time. Maybe because, you know, kind of sometimes it makes it smell like a rotten egg. But it's everywhere on the planet. So, yeah, that's what I've got for this paper. It's cool. It's very cool. I like that. So this also tells you a little bit about what might have happened many billions of years ago on Earth, right? Yeah, I sort of wonder, you know, we could make interesting, I don't even know if they qualify as hypotheses, interesting conjectures about, you know, changes in viral dynamics or viral diversity or changes in viruses and how that might have impacted biogeochemical cycles or sort of history of life or geochemistry of Earth.

49:30Yeah, I think I hadn't thought about it a lot because I don't think too much about the metabolic genes that viruses have. But, you know, they pick up the ones that are going to give them an advantage. And the ones that are going to give them an advantage are the ones that matter to the host. so it makes sense that they would really be having the kinds of genes that were prevalent for a particular host in that environment. So a long time ago, it would make sense that the viruses would have what was necessary for their host to grow. And then we can see that today in the environments where that is still the geochemistry, or I guess just the chemistry of the environment.

50:13All right. Thank you. Jolene, meromyctic, and eugenic, and ferrogenous. The other two was myxolimnion and monomolimnion. I did not talk about those ones, but they mean the top and the bottom layers. Then they have lacustrine systems. Lake systems. Lake systems. That's interesting. But, you know, I like chemocline. I like that word because it summarizes a lot of words into one. You can imagine that, you know, you don't have to say all the things. You just say chemocline. That's perfect. I mean, we use words for a reason, right? Yes. Communication. That's cool. All right. Now, on to eukaryotic cells.

51:05This is a cell paper. the title is innate sensor responses set a supra unitary threshold or viral replication it's possible that this could happen in prokaryotic cells too they just don't look at it here yeah it is possible yes because they have their own they have their own kinds of defenses but this one is this study is eukaryotic cells yeah now i was thinking of that at the end of the paper I was going to ask Jolene, do you think this could happen in bacteria? And, yeah, I don't see why, because bacteria have defenses against viruses, and that's what this is all about, as you can see. Oh, yeah.

51:45The first two authors are Kohiro Mukai and Dongya Shia, and then the corresponding author is Nihal Alton-Bone, and they're all at NIH, the Academia Sinica in Taiwan, University of Maryland and the Middle East Technical University in Turkey. For some time now, all right, so, you know, in virology, we talk about plaque-forming units. A single virus is all you need to make a plaque. So it's all about, so they call it standalone infectious units, right? freely moving independent virus particles. That's what does everything. They infect cells. You only need one to infect a cell. And that's it. But what they and others have found previously is that a bunch of RNA and DNA viruses transmit unblock, okay?

52:45They're inside extracellular vesicles. There are bunches of virus particles inside these extracellular vesicles. Extracellular vesicles are released from cells. They're membrane encased. compartments. And if the cell is making viruses, these EVs will have viruses in them. And the arguments are that these EVs are the main mechanism for transmitting viruses to other cells. So there has been already quite a bit of evidence supporting that. And this paper tries to understand why or what's the function of that. What's the function of having so many viruses be required for efficient infection? Which is not to say that a single virus particle will not infect the cell.

53:33It's just that if you have more, it's more efficient. And they do a really nice job because I was sort of worried about this the first time I read the paper in distinguishing between mean, is it a issue of the extracellular vesicle or is this really about multiple virions? And so for many of the experiments, they're really using multiple virions. And so while they got into this because of this extracellular vesicle release of multiple virions, this does not require those extracellular vesicles. And so for many of their experiments, they actually use many virions. And so they're thinking kind of about things like multiplicity of infection and how many viruses are going to each cell, not about the extracellular vesicle thing, but you could imagine the extracellular vesicle thing is definitely a way that this could be sort of made almost even more efficient or something like that.

54:45Oh, that the vesicle itself was contributing to the infection? Yeah. Well, or just that then it becomes a more directed way of getting multiple virions to one cell. Right. But in fact, the observation has been that cells release EVs with these particles in it. Yeah. A lot of the experiments are simply done adding more viruses. I really liked that because there were a couple of places where I wondered how much of this is the EV and how much of it is the multiple viruses. And so I liked that they did it as multiple viruses and could really focus on that issue. So previously, a number of other viruses, enteroviruses like polio and Coxsackie, rioviruses, calissi noroviruses, volioma adenoviruses have been shown to be transported in extracellular vesicles.

55:35And here's what they call a counterintuitive pattern. If you have viruses in EVs, far fewer cells may receive input compared to free because there are fewer vesicles compared to cells as compared to free viruses. But overall, the virus replication efficiency is much higher when you deliver them within an extracellular vesicle. And so why is that? And there's two ideas. So one is that because, you know, viruses are quasi-species with lots of mutations in the population. Maybe they have to come in in bags because it complements the defects, right? Or second, it may allow counteracting innate defenses, right?

56:31That's the second theory. And related to all of this, because I'm sure this will come up at the end as well, is there is this whole literature about defective viral genomes and defective viral particles. And you can sort of think about the fact of how defective viral genomes or defective viral particles might also be able to do some of these things to help replication. So I have a question, though. So I noticed that all of the viruses listed as having been seen to move through EVs are non-enveloped. Is that right? Yes. Is that a real correlation? Yeah. I don't. Well, they say this has been seen with SARS-CoV-2 in this paper.

57:22That's true. That's true. But that is a question, right? Yeah. The other thing that I thought was really interesting, going to the exact same sentence, I looked at that sentence and I said, so you've got, they are listing some RNA viruses and some DNA viruses there. But elsewhere throughout the paper, they're really focusing on RNA sensing and immune responses to RNA. And they are not considering DNA sensing and immune responses to DNA in this paper whatsoever. And so I think that that's an additional complication or an additional piece that I think I might spend some more time thinking about after this paper in terms of how DNA adds into some of this.

58:10I mean, those experiments can be done, right? Right. Similar to what's been done here. Oh, exactly. So if you listened to TWIV last week, you'll be familiar with RNA sensing because we talked about the influenza virus paper. And so as you may remember, there are cytoplasmic sensors of nucleic acids, right? They abbreviate them CYVSP. Okay. I don't really like that. Too many abbreviations. There are a lot of abbreviations in this paper. I am an immunologist. I love abbreviations. Immunologists are known for how much they love abbreviations. And this paper was over the top with abbreviations, even for me.

58:45So, cytoplasmic receptors like RIG-I and MDA5 and PKR, which distinguish viral and host DNA. And if you remember last time, this is actually important. We learned in the paper last time that RIG-I actually binds to host RNAs, but they don't have features that are marking the RNAs as foreign, so they're not immunogenic. And one of them is like a five prime triphosphate and double stranded regions and so forth. And so that's what they're going to look at in this paper. So here's the way the idea is that the poliovirus comes out of a cell, not as single particles. You know, in the old days, we used to say the cells lice and out comes poliovirus, right?

59:38Well, that still happens, right? But before the cell's lice, extracellular vesicles are budding off with polioviruses in them, right? And so it's going to be a mix of free virus particles and EVs. And what they argue is that these EVs, as part of their normal trafficking, they bind to a cell and they fuse at the plasma membrane, right? Or they're endocytosed. So let's say poliovirus-containing EVs are endocytosed. within the endosome, the membrane gets disrupted. And then you have virus particles, which then can bind to the poliovirus receptor. Yeah, that was crazy to me. Endosome. But the thing is, right away here, people have done experiments showing when poliovirus binds, pre-poliovirus binds cells, it binds the receptor, it's taken up by endocytosis, and then it exits very soon after leaving the plasma memory.

1:00:34So there's a pathway there already. So we can't negate that because that exists. And in fact, we don't know how much one contributes versus the other in an infected host. These are all mostly cell culture experiments except for one. But that's the idea, all right? So what they do in this paper, they isolate extra EVs from poliovirus-infected cells, right? And then they break them open. They then take the virus particles. Well, then they take those.

1:01:13They keep one fraction intact. They lyse the other and they measure the amount of RNA in it. And then they add the other to cells and they only leave them on cells for 30 minutes. So the RNA gets in and it doesn't start making proteins. All right. And so the first set of experiments are in primary human macrophages. I don't know why they use those because it's not a site of polio replication in people. people, but okay. Because it's a site of, my guess is because it's a cell type that makes a lot of innate immune responses and does a lot of innate immune sensing. Right. Anyway, we also use HeLa cells here, which are similarly not relevant, but okay.

1:01:54You need to use model cells. I understand that. So then they can use RNA and C2 hybridization to see what cells got RNA, right? And what they find is that when you add in an extracellular vesicle, very few cells get polio RNA. Some do, but most of them don't. But if you add free polio, every cell gets RNA within 30 minutes. But if you added the extracellular vesicle, very few cells get RNA, but those cells get a lot. Those cells get a lot. And when you add free particles, every cell gets RNA, but each cell gets a little. So here's the neat thing. Then if you let the cells go through the replication cycle, who wins in terms of more genomes, the extracellular vesicle infected cells?

1:02:45So they're measuring genomes by PCR here. Okay. So they say 40 % more, which is not really a lot, right? 40 % is not. And it's PCR. So who knows what fraction is infectious? So these, I'm just pointing out caveats here. Because as someone who was brought up on a plaque assay, I need to be really convinced, right? And I think this is very interesting. The whole paper is very interesting. They've got me wrong, but it needs a lot of support. So what they conclude is that viruses require a super, supraunitary input, which means more than one genome to, they say actually to replicate, but that's not correct.

1:03:25To efficiently replicate should be in there, right? To replicate best. Yeah, something like that. I'm sorry, Jillian. Yes, it was okay. So a question about these extracellular vesicles. Is there any kind of consistency in how many poliovirus genomes will be present in each vesicle? I think so. Is there a range, I believe? So obviously it's not. It's like a tight range? I don't know what the range is offhand. I didn't look it up. That's okay. Sorry. So when they look later, they're able to see that there has been replication and that they also look at the release of viruses and they see the yield.

1:04:12Is that right? I think they did that. Yes, that's right. Yeah. So the idea, but do they know at the later times if this is from the original single infection or if there have been multiple infection cycles after the initial one? So they only allow three and a half hours. Or the five and a half, right? Yeah. In this one experiment, they do three and a half hours. So I think you could have more than one cycle there, right? That's the issue. So you don't know how that second and third cycle is happening, right? But what you do know is there's a difference between free particle and EV. But that's a good point, yeah.

1:04:57All right, so then they inoculate cells with just different concentrations of free poliovirus, no vesicles, right? So they look at how much gets in and how much is made. They use this 30-minute inoculation period. and they do quantitative imaging to measure the number of RNAs. And then they do another experiment where they let it go longer so that they can measure infectious virus, three and a half and five and a half hours. Oh, they say it's one round of replication. So my previous words. I'm not as sure about that. So that's what I was asking. Yeah. And when they do this, they're using free virus, but they're using different amounts of free virus.

1:05:42And so they're basically either using about enough free virus that cells tend to be getting one virion or amounts where the cells are going to be getting multiple virions. And so this is where they're trying to sort of force the super unit, super whatever. Yeah, they have one X, 10 X, and 100 X, where X is 10 to the 4. So these are multiples of 10 to the 4. All right. And so what they find is that at one exinoculum, 25 % of HeLa cells replicate. But 97 % had the genome to begin with. They had at least one genome. So I have a question about this, though. Yeah. The way that they did it, can we say for sure that that one genome that they're seeing by microscope was inside the cell in the cytoplasm?

1:06:37Yeah. I mean, they would argue that because of the wash they did at 30 minutes, that they got rid of extracellular. But it's hard to... These are macrophage cultures, right, Brianne? Right. So do they pile up on each other? No. So they're monolayer. They're suspension. They're suspension. But I guess for the imaging, they're sitting down. So probably you can say that they're in the cell. But what about saying that they're in the cytoplasm versus in a lysosome or something like that? Yeah, I don't think they can say that. Well, I think all you could do would be cytoplasm versus nucleus. These viruses stay in the cytoplasms.

1:07:21But I don't know. Those other compartments, no, we couldn't tell. All right, so the results are 25 % of HeLa is at 1x. At 10 to the 2x, 95 % of the cells replicate with 100 % starting, okay? In the macrophages, the proportions were 59%, 94%, and 100%. So as you go up, you get more production, right? A greater percentage of cells making, replicating the genome. Yeah. Even though, even at the low numbers, cells have the genome, have the first one copy of the genome. That's it, yes. They don't replicate the genome until you get to cells that had received multiple copies. And I mean, if I were going to give them the benefit of the doubt, their images are showing the genome and they're showing DAPI.

1:08:23And the DAPI might be looking at nucleus. So you can kind of see that at least in some, in say the AN5PV, the HeLa cells, the green HeLa cells with the polio, most of that green staining looks outside the nucleus. um i don't and so in some cases i would say sure that's outside the nucleus but well i mean i can't guarantee it you're right about on the entry side um yeah here they're looking at production of 2b which is poliovirus protein so clearly it got to where it needed to be but the early on ones if the virus if polio got in uh as a free virus but then it didn't get out of the cytoplasm i mean out into the cytoplasm right then it was like a failed entry uh yeah no Oh, that's a great point.

1:09:12So they conclude that the discrepancy between cells that contain viral genomes and those that are replicating the virus highlights that the entry of a single genome is insufficient to guarantee replication and you need more than one to enter a cell. And I do like that they use the word guarantee there. It could still happen. It's just not guaranteed. Yeah. I mean, I don't think old experiments have very low MOIs address this because that first round, the viruses could be released mainly in EVs. I just don't know. Anyway, so they make a model of these data, of these single-cell data. And they come up with an idea of Virion Input Threshold for Replication, VITREP.

1:10:03Okay, another acronym. It's not a nice acronym. vitrep is not just doesn't flow off your thumb it's kind of like moi but moi that make that make sure replication happens that's right that's right so for uh for heli cells it's larger than one but varies um sorry so it varies from cell to cell it's 14 in heli cells so you need 14 particles to get good replication but in macrophages it's 190 and these are averages Or this is, yeah. They are, plus or minus. So it's 14 plus or minus 1, 190 plus or minus 40. Yeah. Okay. They also looked at other viruses and other cell types. They looked at polio and pluripotent stem cell-derived neurons, where it's 9 plus or minus 1.

1:10:56They have Coxsackievirus in HeLa and A549 cells, where it's 37, et cetera. And here is SARS-CoV-2 in HeLa cells, 86 plus or minus 9. So let me just pause and just say something about SARS-CoV-2. So we know that SARS-CoV-2 in lung cells binds to the surface and fuses at the surface. The viral and cell membranes fuse and the RNA comes out into the cytosol, right? So here they're saying that you need a vesicle to fuse, but then the vesicle is going in an endosome, and then the virus is now entering from the endosome. So it's really different. So I tried to look at the supplemental figures for this paper.

1:11:52Yes. And I had all sorts of technical issues and could never actually open them. My computer – actually, I used multiple computers, and the supplemental figures just were not seeming to happen. So did they actually have vesicles with SARS-CoV-2, or did they do the same thing where they purified virions out of vesicles? Yeah, they purified virions. But they're making a case for the vesicles based on these experiments, right? But my point is, if SARS-CoV-2 is coming in through an endosome, it is now going to be sensitive to camostat, that inhibitor of endosomal proteases. And it's not. In lung cells, it is not sensitive to that protease.

1:12:36That's why we got all screwed up with kidney cells, where it does go through endosomes. So I'm saying that in vivo data where, and people used, what was the inhibitor that, hydroxychloroquine, right? That was the inhibitor that was actually used in people because of that. Oh, yeah. And so in people, it didn't work because in lungs, SARS-CoV-2 enters at the plasma membrane. Right. So to me, that's not compatible with the EV delivered virus. Yeah, I don't know how much their mechanism really depends on the EV versus just multiple viruses. Oh, you're saying if there are a lot of viruses around, you need a lot of them to write.

1:13:16Yeah, and it doesn't matter if there was an EV or not. It's how I'm taking this paper, but they wrote it at the beginning that it was all about EVs, and then they kind of forgot about EVs, and we're just talking about multiple viruses. That was the observation that led them to think multiple viruses entering at the same time into the same cell. And I just looked. They're not using vesiculated or vesicle-bound SARS-CoV-2 in the supplement. Yeah, they're just using freeze. They're just adding more. Yeah, I do. I know, but they present their model in the very first paragraph as saying the EV is endocytosed and the virus gets – So implying that in vivo, that would...

1:13:57So I'm just saying it's... I don't think EVs are involved, but maybe... Okay, maybe many particles are better. That's possible. But not every in vivo infection is going to be a high multiplicity either. Depends where you are, right? Oh, yeah, absolutely. So anyway, so that's my beef with SARS-CoV-2. So let's move on here. So multiple viral genomes must enter cells for efficient replication. They always leave out efficient or good as if it's all or none, but it's not, right? Right. And Vitrep varies for different cells and viruses. OK. So now we're going to look at the mechanism. What cellular barriers are working to block low virus input?

1:14:38Right. And somehow you let you add a lot of virus in it. It's it's overcoming something. And so they look at the profile transcriptional inflammatory responses And they see that vesicle-cloaked poliovirus induces weaker inflammatory responses than free polio, you know, like downstream of RIG-I and MDA5 and so forth. Okay. So, okay, that's now part of their hypothesis. So what they do is they look at a chemokine CXCL10. And they look at single cells here. They find that cells replicating poliovirus don't make CXCL10, and cells that have CXCL10 don't make poliovirus. Okay? And increasing virus, what they call intake, shifts the cellular response from inflammation, making CXCL10, to replication, which is not making CXCL10.

1:15:47So somehow having multiple viral genomes inhibits immunity. What is CXCL10, Brianne? It's just a chemokine. It's just a marker of inflammation. I don't know where CXCL10 has you do chemotaxis, but it's a chemokine. Okay. This is cool. I'm sure this was not necessary, or maybe this is new, but probably not. If you look at all the cells in the population and see the distribution of where virus is replicating, and then the immune response is not on high. This is what we would expect, but it's just really cool that they saw such a clear. It is. I don't know how often it's been done in sort of a single cell setting where you can totally get this bimodal distribution as clearly.

1:16:34Yeah, well, that's the key, I think, here, looking at single cell. So, separately, they find bulk CXCL-TAN and interferon beta is higher with low virus input and lower with high virus input in macrophages, healer cells, A549. They can detect suppression of these innate responses within an hour post-infection in macrophages. And they can look at phosphorylated IRF3, which is a downstream consequence of innate sensing, right? They can find phosphorylated IRF3 at low inoculum poliovirus. And then as you add more and more poliovirus, it decreases. So what they say is this is a surprising non-monotonic phenomenon.

1:17:23More is less. It's a good way to put it. Viral input levels above a threshold result in diminished innate immune responses. Yeah. So it seems like, as I often talk about in immunology, this is a Goldilocks kind of thing. If you have too little virus, then you're not going to get virus replication. You have kind of a lowish amount, then you've got the virus, but you're also triggering immune responses. And so that's bad for the virus. And then you have too much. And again, now the virus is going to be able to overcome those immune responses. So sort of too low, medium, and too high are different things.

1:18:05So let me throw out this. So in the old days when I had a lab, we used to take a plate of cells and add poliovirus at an MOI of 10, 10 PFU per cell. So every cell gets at least one particle in a Poisson distribution. Not every cell gets many particles. But those cells would be wiped out in six hours. Completely overcome any innate defenses. And if you put on one MOI, right, or 0.1, you just gave it enough time, eventually it would wipe them out as well. Which we would argue that at the low MOI, you should get overwhelming innate responses, but not in… What kind of cells were you using? Yeah, HeLa cells, right?

1:18:49Yeah, HeLa cells don't make very good innate immune responses. We studied the antagonism of it in HeLa cells, right? Rig-I is cleaved. MDA5 was cleaved. Anyway, I'm just saying that if you looked at the old results, you wouldn't come to this. All right. So they say next, a conventional explanation would be, oh, if you put a lot of viral genomes in, you got a lot of viral proteins made, and this is suppressing the immune response. Right? That would be the idea. So what they say, let's use RNA that doesn't code for anything. Poly-IC, a synthetic double-stranded RNA. And low levels put into cells by transfection triggers high CXCL, talent interferon beta expression, right?

1:19:43And if you put a high amount of poly-IC in, it suppresses the production. Do you know this? People must have figured this out previously. No, Brianne. Um, I, so I know that titration of poly IC is really important. Um, I don't know of a ton of experiments where people have done, um, synthetic molecules plus viruses. Though, as I look at this, um, I actually think we didn't use poly IC, we didn't use polio virus, but we did combine some other stuff. And I think I do have similar data. So when we looked at polio-induced cleavage of RIG-I and MDA5, we used to pre-treat the cells with poly-IC to get nice levels of those proteins for the Western blot, right?

1:20:32All the time. And it really induced them. I'm sure we added a ton on. Yeah. Well, the other thing that is sort of nice about this is it tells you that it's about the amount of nucleic acid. It's not viral translation or replication. Yes. But it also tells you it's not about complementation. So it's not about having two different defective genomes complementing each other because poly IC can't complement a defective viral genome. So they go through the pathway of sensing IRF3 phosphorylation. They even look at kinase TBK1 that's between. And they show that low and high amounts of poly IC have the predicted effects on their activation.

1:21:15Okay, so that's rig I, basically, or MDA5. I don't know. What is detecting poly-IC? MDA5? Poly-IC, I think, could be either, actually. Depends on the length of your poly-IC. Okay. So then next, they look at PKR, which is another sensor of double-stranded RNA that gets activated. It phosphorylates the translation factor, and that shuts down translation. So they do the same experiment, small amounts of RNA, high amounts. of RNA, and you get the same result. At high amounts, you get inhibition of phosphorylation of PKR. So basically, a cytoplasmic non-self RNA, either viral or synthetic, when you give it above a certain amount, suppresses activation of these cytosolic sensors.

1:22:10And it happens within 30 minutes of adding these RNAs on. Okay, so these... Again, it seems like having... That it's really about having excess RNA is suppressive. Right. It's not about you need to get enough to turn on the response. It's about having too much is suppressive. So they develop a model for trying to... Sorry, did you want to say something? Well, I think we'll come back to it later, but I'm like trying to think. So from the virus perspective, it's helpful to get, you get better replication because you've suppressed the immune system by coming in with more genomes. But from the cells perspective, which they do talk about later, this is also potentially beneficial because at that point, like you're going to turn on too much of a response and not be able to control it.

1:23:09Is that the idea here? potentially you'd get some immunopathology from a really big response. Well, they also say that it's designed to prevent the host from sensing itself. Yeah. Which I wouldn't agree with that. And that goes into last week's paper. It's really the phosphate or the double-stranded quality that's the distinguishing factor. Yeah. Because they talk about, this is the next part that they're going into, where they're working on this model that I had to read. way too many times, where they talk about the fact that there seems to be something different about self-RNA and non-self-RNA, and there's a difference in binding strength.

1:23:54And so it's sort of like you get sort of over the threshold when you have lots of foreign RNA that's a strong binder. So there's this other binding strength thing going on. Okay. But from like a multicellular organism perspective, it's helpful to respond to a foreign RNA that comes into the cell and potentially protect that cell and also the neighboring cells. But if you're being overwhelmed by the amount of RNA, then your response to an overwhelming number of foreign RNAs would be detrimental to the whole organism and not protective for that cell to respond that way. But maybe there'd be neighboring cells that would have lower exposure and you could still get that response that would be protective.

1:24:41Well, I think the thing that's useful in a multicellular organism is that there are multiple types of immune responses. So that cell that's overwhelmed with virus, we don't necessarily want to make a detrimental amount of interferon. We don't want to make so much interferon that we're going to get pathology. And we know that that cell that has a huge amount of virus is also probably going to present a lot of it on MHC. And CD8 T cells are going to take care of it. So you don't want to sort of have too much interferon causing you pathology in other ways when you do have that other type of response that can take care of that cell.

1:25:25Does that make sense? Yeah. Yeah, actually. It's a whole system. But we're just focusing on the one cell right now. Yeah, exactly. All right. So they make a model for how this might be happening. And basically, they think, they postulate that there are negative feedback regulators of these sensors, right? They're called NFRs. And these somehow are modulating the sensors in response to RNA. So for the cytoplasmic sensors. For MDA5 and RIG-I, there have been two kinases that have been found that interact and are inhibitors of those sensors. So they do some experiments to see if those inhibitors of the sensors make a difference.

1:26:12So they can knock down the RNAs encoding those. And in fact, when you do that, then you get higher levels of phosphorylation in response to RNA. So it overcomes that RNA dependent. Yeah. So the idea is it seems like the high level of RNA is turning on these negative regulators. And if you don't have the negative regulator, now the high level of RNA does induce a high level of interferon. Exactly. Exactly right. So that's part of what's going on here. So at high levels of RNA, these negative regulators are regulating sensing, and so you're not getting – I mean, that's counterintuitive evolutionarily to me, right?

1:27:11Unless the host activation of itself is really key there, and I'm not sure that that is right. Anyway, they show that one of these increases in response to non-self RNAs is consistent with that model. So what about for PKR? They actually look for some interactors. They find one by mass. It's a lot of work. They do mass spec. It was really cool. They're like, okay, we think this should be there, and then they found it. Yeah. So they found another protein, a phosphatase-2 regulatory subunit, which binds PKR, and that acts as an NFR also, as did the two RIG-I and MDA binders. And so there's a consistency across in that these NFRs are involved in this mechanism.

1:27:57So basically, low levels of viral inputs trigger innate responses, block replication. Higher inputs activate these inhibitors of the sensing, and that's why you can get—and you shut down inflammation, and you can get replication. Think about how that would be evolutionarily selective, or it doesn't—it's not clear. Okay. They do some experiments where they modulate the level of these MFRs, and they confirm the impact on replication, as you might expect. I'm not going to go through them because I don't have a lot of time here. But then the final, well, another experiment they did, they wanted to do this in mice, which is great, but they can't do polio in mice.

1:28:46It's too many loopholes to do that. So they do norovirus instead, and they inhibit one of these negative regulators in the mouse, and that gives them, let's see, they give them this DAPK6 as a negative regulator, so you're blocking sensing. And so you get lower intestinal viral loads because now you're making interferon. and getting rid of the virally infected cells. Yes. So the high amount of RNA is not countering it. Right. And what they, I mean, what you would be interested in, and I don't know enough about how you would do this in a mouse model, is to look at, say, over time, if those mice got some kind of interferonopathy or some kind of inflammatory disease because of the amount of interferon they're making.

1:29:44Yeah. I mean, if you did a knockout, that would be interesting, right? That's what you said. So this just says that if this DAPK protein has got the predicted effect, but it says nothing about multiplicity and number of particles and so forth. And then they ask if two viruses get into a cell. Sounds like a joke. It does. If two viruses get into a cell. Each at levels below their vitrep, would they help each other? And yeah, they do. Coxsackie and poliovirus. I like that experiment. And yes, they help each other. So they position this as a result that fundamentally challenges our concept of virus replication, that you need a lot of particles in a single cell to get efficient replication.

1:30:35and they think that this is because cells have evolved this. This has evolved in cells to prevent cells from recognizing their self-RNAs, which are there at high concentrations, right? Orders of magnitudes above virus RNAs when they initially come in. But I'm not sure that that's correct. That's their hypothesis, right?

1:30:59So we'll see. I mean, I think we need to do more experiments. I mean, I'd like to know, I'd like to see this in organoids maybe to get a little closer to natural infection. I would like you to see how cells get so many virus particles besides EVs, right? Because I think there are limitations with that idea. But yeah, it could be seminal. Let's put it that way. It could be seminal. I mean, I think the idea that the cells that get multiple virus particles are contributing a large fraction of the virus replication because viruses are replicating there more efficiently. So even if it's 50 % of the cells get a lot of virus, they're going to make way more than 50 % of the output virus.

1:31:51They're going to make like 90 % of the output virus. I think that that is a good take-home message, but I don't think you can say if a cell only gets one virus, it's a definite no for replication. Well, imagine if someone sneezes in your face and you get a few hundred virus particles. There have to be singly infected cells that go on. So to initiate it, but maybe then after that you get multiply infected cells. in an epithelial sheet, you can imagine that cells next to each other release a lot of virus, so there's a high multiple. Right. Okay. And so, yeah, the ones who got one virus contribute, but they don't contribute as much as some of the others.

1:32:37But they also, we can't say that they're like zero. It makes me think a couple of things. The first thing related to what you guys are talking about is, you know, when you see pathology in a tissue, you see the most, it's all, it's not, it's clustered in the tissue. and it spreads and then you get less and less pathology. And so that's consistent with this idea of more virus getting into the neighbor and less virus getting into the distant neighbors. And that could have a lot to do with the activation of the innate system. There are viruses that spread in the blood and that's pretty dilute and it delivers them to tissues where probably the multiplicity is not high.

1:33:18And so they're not saying that you can't get replication. Well, sometimes they're pretty definitive. They say it's... I didn't interpret that way. Maybe because I was thinking of the concept of permissivity. All these cells are permissive, but it's putting a heavier emphasis on the environment in the cell being conducive to virus replication. Yeah. I think some places, you're right, Vincent, they do phrase it as like it's all or none. And I don't think it's all or none. I think it's more likely and less likely or something like that and not all or none. I think it's mixed. I think there are situations where single particles work and there are situations where you need more.

1:34:06And that may be one of the key experiments to figure out when each of them is happening in an animal. I would like to see at least, right? but also in cells that are perhaps closer to where the virus is reproducing, right? Because these cells used here for poliovirus are not relevant for... What's that? Sorry, sorry. I was just trying to tell you that my friend Rocky, who's right here, he just went down. My friend Rocky is visiting on the window. Is that a squirrel? Yeah. Oh, how cool. Nice. I just saw it move. He's back here. Hello, Rocky. All right. Rocky said it's enough discussion on this paper.

1:34:54Okay. Anyway, that's quite interesting, provocative. There are still things, but that's how science goes, right? Mm-hmm. You do something that's different, and initially people don't believe it, and then if it's true, you're going to find it out, right? Anyway, so it's a cell paper. So reviewers thought it was high impact, right? Okay, let's do some picks of the week. Breanne, what do you have for us? So I have a New York Times article about a story that I've been following a bunch recently, which has to do with the U.S. having measles elimination status that we achieved as of the year 2000. And this week, Tahoe, the Pan-American Health Organization, has started reviewing data on whether or not the U.S.

1:35:56will lose its measles elimination status. And the way that that is determined is that they have seen, they started to see a number of measles cases about a year ago. And now the question is, has measles been continuously circulating in the United States for a year or were there separate introductions? And so we have to use sort of genomic sequencing to see have all of these different measles outbreaks been related to each other or are they coming from separate introductions? And that's really what's going to make the difference here. And so that's a sad idea that the U.S. will lose its measles elimination status.

1:36:44But it's also kind of interesting that we are currently able to address that question in much higher resolution in terms of thinking about sequencing than we've really ever been able to do before in terms of how we're able to see if this virus has been circulating continuously or not. And so this article talks about the fact that this is being examined right now. It also throws some shade on members of the current administration and their role in this. But the thing that I really like is that this kind of explains the big picture problem, but then also links to the next strain data so that you can actually look at the sequences yourself.

1:37:29And they ask different scientists to give them their take on the sequences and whether the sequences mean there's been continuous circulation in the US. And so I like this article because it gets into the process part of how scientists actually look at this and decide and links into the data so that you can start to look at it yourself as well. Yeah. In 2000, we didn't have this, these kind of data. Right. You had to do it all with epidemiology. Right. And that's hard. But yeah, it's clearly been circulating for a year and we're going to lose the status. but it doesn't mean you can't get it back.

1:38:10Right. You can get it back by resuming vaccination like we were doing before certain people were discouraging it. It's a good thing that there are scientists outside the U.S. doing this analysis, not just ones inside, right? They have no... Yes. That's true. Yes. Yes. No immediate consequences to themselves. I mean, there are over... How many cases so far in the U.S.? It's over 5 ,000, right? And there have been 10 deaths, which were all preventable by immunization. So this is not just a mild disease. Yeah, I like that article very much. By Apoorva, right? Yes. Jolene, what do you have for us?

1:38:56I picked a book. This is a book by Robin Wall Kimmerer called Gathering Moss. It's a natural and cultural history of mosses. So Robin is a great author. I love the way that she writes. It's kind of a, it's a mix of personal essays. It has a lot of personal reflection and scientific history of mosses, a little bit of life cycle, a lot of ecology. It goes, you know, all the scales up and down. I love hiking and I see moss all the time. And this book just made me like so excited about seeing different kinds of moss and where they like to live, where some thrive better in very humid environments in the shade.

1:39:38Others need some sun. Others are like almost all dark. They don't really have common names. They only have their scientific names. And that's why most of us couldn't really name any certain kind of moss. But she goes through the differences and interesting things between them. And she has this kind of connects to this bird arc that's been going on, maybe just like the last couple of email sets. She says that words and names are the ways we humans build relationship, not only with each other, but also with plants. So if anybody knows of a really good moss identification book, I would love to have that because I can't I can't say I can identify very many of them at this point.

1:40:17She also has this other short quote I'll read about how, you know, moss people usually ignore or pass it over. But I think it says a lot about who we are when we pay attention to those kinds of things. So she says, infrared satellite imagery, optical telescopes, and the Hubble Space Telescope bring vastness within our visual sphere. Electron microscopes let us wander the remote universe of our own cells. But at the middle scale, that of the unaided eye, our senses seem to be strangely dulled. With sophisticated technology, we strive to see what is beyond us, but are often blind to the myriad sparkling facets that lie so close at hand.

1:40:57We think we're seeing what when we've only scratched the surface. Our acuity at this middle scale seems diminished, not by any failing of the eyes, but by the willingness of the mind. As the power of our devices led us to distrust our unaided eyes, or have we become dismissive of what takes no technology but only time and patience to perceive. Attentiveness alone can rival the most powerful magnifying lens. I think that captures kind of some of the ways that this whole exploration of moss and the uses of it and its role in the environment with us as scientists and how we choose to observe things.

1:41:35And we learn interesting things by looking at things other people might not pay so much attention to. Cool. I just bought it. I like that quote. I just bought it. It really sounds great. I want to read it.

1:41:49particularly because they say the writing is so good, right? It's a great book. All right. My pick is also a book. I have this book called Neuropsychology. Okay. This is, it's a summary. It's not really a big narrative. It's a summary of everything. You can read this in a day. It's really cool. It's, I think this is written by AI, frankly, but the author is Devin Cade. I might be wrong. It just reads like AI. However, so what this is, is it tells you about all the different parts of the brain. First of all, in the beginning, there's a glossary, which they call jargon, okay, the language of the brain.

1:42:35They tell you what everything is. And then they go through each of the parts of the brain. Start with the neuron, right? And the chapters are very short. The paragraphs are very short, right? Think of dendrites as the antennae of the neuron. So we've got neurons, and then we go through synaptic transmission. They go through different parts of the brain and what each part does, how it's evolved over time. Here's a section called biohacking the vagus nerve. So the vagus nerve is important. let's see what they say about the vagus. Have you ever walked into the room and felt off about that vibe? That's your vagus nerve apparently doing it and other things connected.

1:43:26And you can do biohacking of the vagus nerve. If you're feeling stuck in a sympathetic, anxious, or dorsal shutdown state, can you force your system back up the ladder? The answer is yes, Deep, slow breathing. This is the most direct switch. The exhale is the trigger for the vagus nerve. By extending your exhale so it's longer than your inhale by about four seconds, you physically force the heart to slow down. You are sending a safety signal from the lungs to the brain. Cold exposure, humming and chanting also will calm you down. And social connections. So they go through every part of the brain and tell you that kind of stuff.

1:44:09They also talk about diseases of different parts of the brain, like Parkinson's. The neurons in the substantia nigra die, and these are the ones that make dopamine, which is the reward neurotransmitter. Dopamine is the grease that keeps the gearbox running. And so people with Parkinson's, it's a heartbreaking disease because the person is trapped in a body that refuses to obey their will. Their frontal lobe is shouting, walk, but the basal ganglia cannot lift the gait. They go through all the different diseases. They talk about language and writing as well and how that evolved. And then they talk at the end about a lot of the dementias, what's causing them, and also some technologies for studying diseases that we didn't used to have.

1:45:02So a lot of this we didn't know until someone died, and now we can do imaging and see it. So it's a very, very short book. I learned so much, but now I just bought Principles of Neuroscience, which is like 10 ,000 pages, and it weighs 20 pounds by Eric Kandel et al. And I want to read that because neuroscience is incredible. Are you kidding? It tells you what happens. It tells you what happens when you get a concussion. It tells you the Glasgow coma scale, why you need myelination, why kids are out of control in particular, why babies are flailing all the time because their neurons haven't been paired.

1:45:38They're just firing left and right. Oh, it's so interesting. If I had to do it again, I'd be a neuroscientist. Neuroscience is amazing. It is. But only with current technology, you know, because in the old days, all you could do would be to talk to people. And there was this, you know, the story of Phineas Gage, right? I do. The guy who lost his. Oh, my gosh. It's also interesting. So anyway, neuropsychology summarized by Devin Cade. If you are ever in Boston in the Harvard Medical School library, they actually have Phineas Gage's skull as well as the iron. Wow. And I remember seeing it multiple times in graduate school.

1:46:23And it doesn't look like that iron can go through that hole. Um, but you can see the actual skull and the actual iron, um, from Phineas Gage. That's amazing. That's intense. The other thing is they have examples of people that have, have defects in different parts of the brain and the things that, you know, where you can, you know what you want to say, but you can't say it or you can't put the sentence in. It's fascinating how it all works. It's just so cool. And memory too. That's another one. How memories are, are written. You get your memories and you sleep at night and you consolidate your memories into the, Cortex.

1:46:57Really cool book. Anyway, I'm sure there are better ones. And he's got a good biography at the end, actually, which I've started with. But that and physics, Brianne, that's what I'm reading. And I have a cool physics book next that I'm also done with. Oh, I have so many physics books. You should pick some of them. I want to hear them. I should, yes. All right, that's TWIV 1375. You can find the show notes at Apple. We're getting into years now. We're going to get into 2026 soon. Well, maybe not so soon. You can find the TWIV at microbe.tv slash TWIV. The show notes, you can send questions, comments, picks of the week to TWIV at microbe.tv.

1:47:45And if you enjoy our programs, we'd love your support. Microbe.tv slash contribute. Breanne Barker is at Drew University Bioprof Barker on Blue Sky. Thank you, Breanne. Thanks. I learned a lot. Jolene Ramsey is at Texas A &M University, ramseylab.versell.app. Thank you, Breanne. Thank you, Jolene. You're welcome. Thanks. This was fun. I'm Vincent Racaniello. You can find me at microbe.tv. 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.

1:48:32Thanks for joining us. We'll be back next week. Another TWIV is viral.