Unlocking New Frontiers in Mosquito Virus Control: Antiviral Approaches via Vectors & Skin Microbes

Hinzugefügt: 2026-05-22

[00:02:05][music] >> Everything.

[00:03:46]Welcome in our institute.

[00:03:52]Fernando, muito obrigada pela sua pelo very happy to host this session today.

[00:04:19]I'm very grateful to the institute to Marli and to staff for helping us setting up this presentation.

[00:04:33]Okay. Okay. So, I want to introduce Professor Linda Lang to everybody. I'm very happy to have Ling with us here today.

[00:04:43]Uh Linda Lang Uh she has an very interesting research group in Leuven in the I will not say that in Dutch. That is impossible, but it's the Catholic University of Leuven.

[00:04:59]And she has dozens of outstanding contributions in vector and virology.

[00:05:08]Um and we met I think a couple years ago and we started collaboration each other and it's been a great pleasure to get in touch with her research which is outstanding. You will see that in certainly in the next minutes.

[00:05:24]And so, I'm very grateful for having you here and I will just let's go straight to the point. Thank you very much, Ling.

[00:05:33]Thank you very much, Fernando.

[00:05:36]I'm very happy to be here and thank you for the invitation to come to Rio and to give this presentation. So, yeah, if my slides can appear.

[00:05:50]>> Okay. So, I will only start with a disclaimer. I'm a virologist and not an entomologist, so just you know, but we do work with mosquitoes. So, okay, there it's coming.

[00:06:08]Okay, so like you can see, I will talk about mosquito-borne virus control and I will try to show you some new strategies that we are currently trying to study, but I will start with the more traditional approach of the antiviral drugs like you will see my talk. So, I want to start briefly with introducing my team.

[00:06:28]Because yeah, I will show data today and I'm they are of course the people who do all the hard work in in the lab. So, this is my my current team in the in the Rega Institute and the Rega Institute is a part of the University of Leuven.

[00:06:45]So, KU Leuven.

[00:06:56]So, I want to introduce Professor Linda Lang.

[00:07:07]Linda Lang She has a very interesting research Linda Lang She has Linda Lang for everybody. I'm very Okay, okay, okay. So, I want to introduce Professor for everybody. I'm very happy to She closed her Okay, sorry. That's okay. That's okay.

[00:07:45]Okay, so I want to start with quickly introducing my team like I said and we are located in Leuven and Leuven is in Belgium. So we are here next to Brussels in the Flemish speaking part of of the country and this is our city town hall. So it's one of the squares. It's really pretty.

[00:08:03]We are not as big as Rio, of course. We are more like a small city, but it's still nice to visit if you ever come to Belgium. Please visit us as well. And what's nice about the Rega Institute is actually that we do We have several groups there that combine different expertises. So you see we have virologists, but also bacteriologists, immunologists and medicinal chemists and that's quite I think something that's quite special that these expertises are are together.

[00:08:28]And we are very the Rega Institute is very famous for antiviral drug discovery because also tenofovir was actually discovered there, which is still the backbone of HIV treatment.

[00:08:39]So let's dive into the topic of today.

[00:08:41]So I will talk about mosquitoes and yeah, you all are very familiar with mosquitoes and I don't have to tell you that a small bite of a mosquito can actually be a big threat. Not really because of the bite itself and of the itchiness that you get afterwards, but of course of what can be transmitted during this bite in your skin and your body.

[00:09:01]So yeah, mosquito-borne viruses they are worldwide problems. So vector-borne diseases are thought to be more than 17% of all infectious diseases and here on the maps you see the the current occurrence of dengue virus and chikungunya virus in green.

[00:09:16]So you see it's present on almost every continent and they're also overlapping and you can see of course Brazil is also having quite a lot of occurrence of dengue and and chikungunya virus. And so we study these viruses because they are present worldwide, but also of course because they are a health problem. So it's started every year more than 700,000 people die of vector-borne disease, and this also includes malaria, which is of course not a virus. Uh but also if if you don't die, there's also this high disease burden. So, people can become really sick, and yeah, we all know um the babies that were born from um women that were pregnant and infected with the Zika virus uh have a babies born with microcephaly, uh skin rash.

[00:09:58]This is a woman infected with a chikungunya virus, and then this is a person infected with yellow fever. So, it's it's having a lot of impact on the health of of many people.

[00:10:09]And so, yeah, dengue virus, I will keep this short because I I'm I'm sure you're all familiar uh with the virus. It's the most widespread uh arbovirus at the moment. And it it's thought that half of the world population is actually at risk. So, that's really a big group of people, and it is expanding uh every year to to more and more uh new places.

[00:10:28]Um so, of course, transmitted by Aedes mosquitoes, and when you get infected, yeah, you can become really sick. Uh not everybody gets symptoms, but uh if you have like a secondary infection, you can develop the severe dengue. And these are pictures I show uh to my students because it shows the impact of a dengue virus outbreak. It's uh in a hospital in Bangladesh, where you see uh lots of children are being affected, and it really overwhelms the the health system.

[00:10:53]So, it also affects actually people that are sick of other diseases because when they come to the hospital is there's no place for them, there's no health care anymore, and you see people have to be on the corridors, the parents have to be there to take care of the children. So, it's really when there's a big outbreak, it's really really uh yeah, a big problem. And yeah, luckily, we have some uh vaccines now, the live attenuated vaccines, but not all of them are perfect, as you know, but we still don't have any drugs that are specific for uh dengue virus. So, if you are having the virus infection, there's nothing that we can do besides treating the symptoms.

[00:11:29]Another virus we work on a lot in my lab is a chikungunya virus, so that's an alpha virus. It's a different virus from the dengue virus, but it's also the most widespread alpha virus. It's transmitted by the same mosquitoes as dengue virus, so that's why it's present in the same regions. And with when you're infected with chikungunya, you get an acute infection with high fever and joint pains that are apparently very severe.

[00:11:53]It's a very painful disease.

[00:11:55]And what's also peculiar about this virus is that you can develop this chronic arthritis. So, even months or years after this initial mosquito bite and infection, you can still have this arthritis that can come up in episodes, and people are really having difficulties of just opening a bottle of water and going down the stairs. So, it's affecting your daily life a lot.

[00:12:17]And that happens in a lot of people. In some studies, it's even 60% of the patients. So, that's really big group of people. Now, recently we got two vaccines approved. So, that's a really nice advancement in in in chikungunya virus control, but also for this mosquito-borne virus, we don't have any antiviral drugs. And actually, we can It's not only for dengue and chikungunya, it's like for all arboviruses, we don't have any specific drugs.

[00:12:44]And maybe something that you are less familiar that you know less about is that we had a big chikungunya outbreak in Europe last summer. So, that was really special, let's say, because that's not happening a lot. We have a lot of dengue now in a lot if you compare it to South America, you would probably say it's not that many cases, but for European standards, let's say, we have quite a lot of dengue cases now every year in like in the south of France and in in Italy, in Spain. But like in 2025, we had a lot of chikungunya virus. So, by November, we had more than 1,000 cases of local infections. So, people not people that were traveling, but got infected in France, in Italy. And the the infections were still ongoing in November because it's a it was a warm start of the winter. There were lots of tiger mosquitoes and people could become infected. And what was also special was that actually it was almost all over the country. There was even an infection in Paris. So, which is quite north in in Europe, let's say, for having a chikungunya virus infection.

[00:13:46]So, how can we control these mosquito-borne viruses? Yeah, you're familiar with the vector control. Yeah, we have insecticides. We can try to reduce mosquito habitats. We can do personal protection have spraying some repellents and so on. But, of course, this is not perfect. So, we have to try to find other ways. There are some vaccines for a certain mosquito-borne viruses like for dengue and chikungunya now, but not for all mosquito-borne viruses. And like I already said, there are no antiviral drugs for any of these viruses.

[00:14:18]So, this is something in the Rega Institute that we are looking into. We have a this history of searching for antivirals. And also the mosquito-borne viruses are now, since many years, on on the list of viruses that we want to try to find antivirals for.

[00:14:35]So, an antiviral drug that was discovered in in our institute by my colleague Johan Neyts and Suzanne Kaptein is this JNJ inhibitor. It's the NS4B inhibitor from that was developed in Leuven and then yeah, like licensed to Janssen Pharmaceutica, JNJ. And this compound yeah, it looks like this. It's very potent. This is in a in a cell culture system in liver cells um against dengue virus 2 and it has a really low EC50 value. An EC50 is the concentration that is needed to inhibit 50% of the virus. So, it's a nanomolar compound. So, it means it's very potent.

[00:15:15]And the CC50 is the the effect on the the toxicity on the cells and that's in the micromolar range. So, yeah, you really have a very big window between antiviral effect and toxic effect.

[00:15:26]And what's also important is that you have a pan serotype activity. Of course, we don't want a molecule that only works against one of the serotypes, only against dengue 2 or dengue 3. That would not make much sense, so we need compounds, antivirals, that work against all serotypes. So luckily this compound is very active. And so it was further studied in in vivo models by my colleagues in in in a mouse model. And here you see the viremia, so this is how much viral RNA there is in in in the blood. So this is the untreated mice and this is different doses of the JNJ-07 compound going from 30 mg per kilogram up to 1 mg. So you see a dose-dependent reduction in the viremia of the virus.

[00:16:09]This is a survival study, so you see the percentage survival over here and you see when they are not treated, they start to succumb to the infection starting from day five, I think, post infection. And when you treat them for five days, you see that they almost all stay alive and only with the lowest doses you see that some of the mice will not survive, but if you compare to the vehicle, it's like a very big uh um difference.

[00:16:37]So an analog of this JNJ-07, mosnodenvir, was then taken into clinical trials. And then last year in November, there was the paper in the New England Journal of Medicine where Janssen showed the antiviral activity against dengue in a human challenge model. So they infected humans with an attenuated dengue strain and they used it as a the antiviral as a prophylaxis and they saw really nice results.

[00:17:01]Now unfortunately, J&J decided to stop the development of this compound. So it's back at KU Leuven now and we are trying and my colleagues are trying to find another company that wants to further develop it, but it's the first dengue antiviral that shows really promising data activity in in humans.

[00:17:21]So, we really hope that it will be further developed um in the future.

[00:17:27]Only downside of this molecule maybe is that only active against dengue. So, it's active against all the serotypes, but it's not active against Zika virus or yellow fever virus. So, it's very specific for dengue.

[00:17:39]Then for chikungunya virus, this is work I did a lot during my postdoc. We also tried to find antivirals and we found that this NSP1 protein, which is the capping protein of the virus, um is actually a nice antiviral target for chikungunya virus. So, it's a protein that is responsible for this capping and it uses an other capping pathway than that what is used in the cell. So, that makes it a good target because you will not inhibit um the capping of your mRNAs in the cell because that would of course not be a good thing. So, here you see how the capping is working and it's actually a really nice protein because it forms a ring-like structure with 12 of these NSP1 proteins. And I I we have people in Marseille and in Singapore who are working on how these replication complexes are formed of chikungunya virus and it's it's really intriguing to see this field develop. So, what we found in in my postdoc was this molecule that we called MADTP. It was a collaboration with the chemist in Madrid and we found that there was antiviral activity against chikungunya in cell cultures and then in enzyme assays we found that it was actually inhibiting this guanylyl transferase step that is um one of the things that NSP1 is doing.

[00:18:55]A problem with the compound was that there was rapid resistance development.

[00:18:59]So, if you put the virus together with the compound at the suboptimal concentration, the virus just needed one mutation and then it was completely resistant to the molecule. So, that's not something that we like to see as antiviral drug researchers.

[00:19:13]So, we searched further for other molecules and then in our screenings this molecule popped up. It's a a molecule we call CHVB. It's a very different chemical structure from this one. And again, there we found that this molecule was also inhibiting the guanylyl transferase activity.

[00:19:31]What was nice about this molecule that it we needed four mutations now to get full resistance. So, we were very happy with that, but there was still some cross resistance with this other molecule. But again, so by screening we found another NSP1 inhibitor.

[00:19:45]And then our colleagues in the Netherlands have found yet another NSP1 inhibitor that now inhibited the methyltransferase activity. It It also was They They were able to get some resistant virus, but the mutations were different and there was no cross resistance. So, now even other NSP1 inhibitors have been found. So, it shows that this is probably an antiviral hotspot for chikungunya virus or maybe for all alpha viruses.

[00:20:10]But again, one of the Yeah, not so good things about these compounds is that they're very specific for chikungunya virus. So, even though the capping enzymes are very conserved, it's still we cannot use the molecules to inhibit other alpha viruses and we don't we are not sure yet why that is. But it's not a such a favorable profile for a compound if it only works against one virus because Yeah, companies are not really interested to develop something that's not broad spectrum. So, we are trying to find compounds that are actually being broad spectrum. So, because then we can use one stone to hit several birds, which would be much more favorable, let's say, than having all one stone per bird, let's say.

[00:20:53]So, one of the classes of antiviral drugs that are very broad spectrum usually are the nucleoside analogs. So, they these are molecules that look like our normal nucleosides, but there is a couple of changes there so that they cause, for example, chain termination when they're inserted in the viral RNA.

[00:21:11]And nucleoside analogs, yeah, they target the the active site of the polymerase, and that's a very conserved site because it's it has enzyme activity. If you make one change there, the enzyme will not work anymore, and your virus is dead. So, the virus has very has difficulties to get resistant to these nucleoside analogs.

[00:21:29]And what's also nice about these viral polymerases is actually that this 3D conformation is very conserved. So, if you look at a polymerase, you can actually imagine it as a right hand, and in the palm is a active site, and then you also have a thumb domain and a fingers domain. And so, these nucleoside analogs, they will act in this palm site, which is very conserved. So, that's why lots of these nucleoside analogs have quite a broad-spectrum activity against many different virus families.

[00:21:58]So, we have also in the lab tried to look at the efficacy of these nucleoside analogs against chikungunya virus. And for example, a couple of years ago, we found that favipiravir, which is actually a drug approved for influenza virus in Japan, um that this drug is also active against chikungunya. So, this is a data from a mouse model. This is the viremia. So, again, the the viral RNA in the serum, and you see at day three, there's a nice dose-dependent reduction of the virus.

[00:22:25]And in this chikungunya model, we also see the swelling of the foot pad. So, it mimics more or less what's happening in humans when they get infected. So, I would not call it arthritis, but there's inflammation in this infected foot pad. You can measure it with a caliper, and then you see that the highest concentration of favipiravir was able to reduce the swelling as well.

[00:22:47]Also, molnupiravir has activity against chikungunya, and molnupiravir was approved in a couple of countries, like in the United Kingdom, for SARS-CoV-2 at the time. Um also, this molecule is very broad-spectrum. It works against different viruses and also against chikungunya in our mouse model.

[00:23:06]So, yeah, maybe it seems like our it's all solved, right? We have two molecules that are broad spectrum. Uh not really.

[00:23:12]The problem with these molecules is that they act by little metagenesis. So, they will start to implement mutations in the viral RNA. So, it will not be implemented in the cells, but it's it's only in the virus. But, of course, it's something that's sounds a bit tricky, yeah? When you are putting in mutations and you make actually the virus to evolve more quickly than what it would do normally. And so, there's a couple of papers showing that the people that were treated with molnupiravir when they were infected with SARS-CoV-2, that they were having many more mutations in the viral RNA genome than what people that were not treated with molnupiravir. So, not everybody is in favor, let's say, of having these lethal mutagens as uh antivirals that we we use by many people because we cannot really predict what would happen with our viral genomes.

[00:24:01]And then there's sofosbuvir. Sofosbuvir is actually a molecule that is used for hepatitis C virus. It's part of the um the like the standard of care of this virus. And this virus this antiviral is used as as an antiviral chain terminator. So, when it's incorporated in the viral RNA, it will stop. You cannot add another nucleoside. So, you will have shorter viral RNA genomes and of course then the virus will not survive.

[00:24:28]Now, sofosbuvir also works in cell culture and then there was a paper from a Brazilian group in 2019 that showed there was also some effect in the chikungunya mouse model. You see, it's not very spectacular. It's like a one log reduction, but something was happening. But, probably this is not potent enough.

[00:24:47]So, we have three approved drugs for other viruses with some activity against chikungunya or a lot of activity with other problems. So, yeah, it's not ideal, let's say. So, that's why we thought maybe we can combine these nucleoside analogs and use lower doses of these two, have less mutations, but still have a potent effect uh against chikungunya.

[00:25:12]So, we started with analyzing the antiviral effect in human cell lines because that's not always done so much.

[00:25:18]We use lots of Vero cells, which are green monkey kidney cells, and yeah, chikungunya is not causing kidney uh disease, so it's not really a a great uh cell culture model, let's say, for the virus. So, we wanted to first see if these nucleoside analogs also work in in cell lines that are of human origin and more, yeah, let's say, relevant for the disease uh of chikungunya. And the skin is of course a site where the infection happens after the mosquito bite. The liver cells, liver is also infected by uh chikungunya virus. And so, this is a busy uh slide, but uh this is the active metabolite of molnupiravir, and then you have sofosbuvir and favipiravir. And you can see the molnupiravir derivative is acting actually against all the alpha viruses that we tried. Uh you can see maybe over here. Here is a different a panel of all alpha viruses that we tested in the lab. So, that one worked against everything.

[00:26:11]For sofosbuvir and favipiravir, it depended a bit. Like, for example, here the favipiravir worked really well against chikungunya, and I think this one is the Ross River virus, but not against the other viruses in this cell line. And here was uh there was some activity, and then for sofosbuvir, it was not very active against the alpha viruses we tested on H 187. So, we had a bit more of a mixed uh a picture there.

[00:26:35]So, then we went on to try to combine them. So, why drug combinations? It's something that has is being done for other virus infections, especially for chronic virus infections, because there's lots of uh advantages. Uh you can have an enhanced antiviral potency of your drugs. Uh sometimes they can act even synergistically, so 1 + 1 is 3. It will have a better effect than just the sum.

[00:26:58]And then when you have this enhanced potency, you can use lower doses. You can have a reduction of side effects.

[00:27:03]And also, when you combine drugs, the chance that the virus becomes resistant to two drugs at the same time is smaller. So, there's a decrease in a drug resistance.

[00:27:14]But so, how do you find meaningful synergy? Because it's to say that a compound two compounds are synergistic.

[00:27:20]Sometimes you read that very quickly, but how do you know it's really synergy and not an additive effect?

[00:27:26]So, for that we work together with people from the University of Helsinki.

[00:27:30]And so, what they did, they looked into anti-cancer drugs. Because in cancer, lots of drug combinations have been tested already and there's a lot of information. It's in in this database and they developed a tool, a software you can use on the internet, SynergyFinder. And they used the SynergyFinder to analyze all these drug data. And then they looked at the top 5% of the most synergistic drug combinations. And they looked at they calculated the synergy scores with their software.

[00:28:02]And what they found was that these 5% of the most synergistic combinations, they had a score above 12.

[00:28:09]So, showing that if you have a score above 12, that probably means something.

[00:28:13]That's probably synergistic. So, for our studies and many other people, they put their put their threshold for synergy at a score of 10 for these synergy scores.

[00:28:24]So, this will become a bit more clear in this slide. So, here we did this checkerboard assay. So, meaning that we have dilutions of the drugs on one side of our plate and on the other side of the plate. And then we Yeah, so we can test the antiviral effect of the compound alone, but also when they are combined in different drug concentrations. And then we use this data to with the SynergyFinder to calculate our synergy scores and you can also get these really nice 3D plots showing your additive or synergistic or maybe antagonistic effects of the combinations. And what you see here, the more upwards the mountain is, let's say, the more red it is, it it means that it's more synergistic in these uh combinations of concentrations.

[00:29:08]Uh when it's um just like at zero, it means it's additive and when there would be a peak downwards, it would mean that the compounds are actually working against each other, but that we didn't see here, luckily.

[00:29:20]So, we did we tried these three um nucleosides in combinations, like you can see here. This was for chikungunya on the skin fibroblasts. We calculated the synergy scores and like for two out of the three combinations, we actually had scores around 10 or higher. So, these actually showed uh synergy.

[00:29:37]Then we also calculate a mul- uh the most synergistic area, the MSA, and this is the the combination uh the combinations that actually have the most synergistic effect and they were uh so, we had for the three combinations, we had yeah, doses that were actually giving a high synergistic effect.

[00:29:55]So, we showed that if we combine these nucleoside analogs, we actually can have a synergy uh which is really nice.

[00:30:02]But yeah, this is of course in cell culture. Um that's not really what we want to show. We want to go in vivo, of course. And so, we first started off with testing sofosbuvir because we haven't done that yet.

[00:30:14]And yeah, you can see we tried two um concentrations that we found in the literature, but yeah, we didn't see much in our hands, which doesn't need to be a problem because maybe if we combine them, it would still have an effect.

[00:30:27]So, we picked the highest concentration, 80 mg per kilogram, and then for molnupiravir, we choose uh a dose that showed a bit of antiviral effect, but not a lot because of course, if we use an optimal concentration of molnupiravir, we would just like kill the virus. we cannot see the effect of the combination.

[00:30:45]So, either So, we had the the the single therapies and then the combination in blue.

[00:30:51]So, we tested this in our mouse model for chikungunya and here you see the swelling data. Um so, it's uh maybe a bit difficult to see, but here's the blue line, so that's the combination.

[00:31:00]And especially at day two, we actually see a better effect compared to the vehicle and then the monotherapies. So, this was uh significant statistically.

[00:31:10]On day three, we lost the effect. So, it's not optimal yet. We have to look into better drug dose combinations for these uh two drugs. And this is uh this is a the viremia, so in the serum, um you see at day three, we have a lot of virus in the vehicle group. There's not really a reduction that is significant for sofosbuvir. This is the molnupiravir group, but when we combine them, we actually have a a really nice effect.

[00:31:34]It's not completely inhibited, but at least there's the effect of the combination actually enhances the antiviral efficacy. So, we are now working on trying to predict better which doses would give this synergistic effect also in vivo because yeah, we cannot just test every combination in mice, that would not be very ethical.

[00:31:53]But uh so, we try now with computer modeling to see, okay, how can we improve how we pick the doses?

[00:32:00]And maybe what's also interesting is about the the mutations, eh? Because that was one of the reasons why we wanted to combine molnupiravir in a in a combination so that we have a lower dose with lower mutations. So, what we did was we took the viral RNA from the contralateral joints in the mouse model and we did uh deep sequencing uh on these uh viral RNAs. And here you see uh all the different mice, so every column is a mouse. Um and here are all the different mutations, the transitions, the transversions that you can have in the genome, also insertions and deletions.

[00:32:33]And so, for molnupiravir, the most common uh mutations are these two. Um will not go into how that's happening, but if you're interested I can can explain afterwards. And then these two mutations are a bit less common, but they also happen.

[00:32:48]And we see here that in our treated groups you we do see more of this type of mutations. And so the darker the blue color, the more this type of mutations we found.

[00:32:59]It's a bit difficult to interpret this data, so we also normalized it to the the viral RNA level because you can imagine in this group there was not much viral RNA left anymore. So you have to of course normalize them the mutations compared to the viral RNA genomes. And so that's what we did here.

[00:33:16]And so we showed actually this mutagenic effect also in vivo for the first time because you see the number of mutations go up if you have a higher dose of molnupiravir.

[00:33:27]And these are the data then of the combination. You see that the combination gives less mutations than if you would use higher doses of molnupiravir.

[00:33:36]Unfortunately, yeah, we didn't see really a difference with the 10 mg per kilogram alone. So this is the same dose as was used here. It was a bit more variable, let's say. But at least it shows that if you would combine the molnupiravir at the lower dose with another drug, you can actually have lower mutations than if you would use a higher dose of molnupiravir. So it's not perfect yet, but we are working further on this to improve the combinations.

[00:34:03]Now of course now thinking about the clinical course of an arbovirus infection, we know that it's a very rapid viremia. So we get after the incubation period there after the mosquito bite, you get the virus starts replicating, but then it will also go down again automatically because of it's an acute infection and your immune system will start to kick in and will fight the virus. So it's usually quite short. So if you want to use an antiviral, we of course want to lower this viral load in the organs and the serum so that we have less symptoms. And like for dengue virus it's very well described that the more virus you have in your body, the more severe your symptoms are. So if you can use an antiviral, you can lower this load and have less symptoms.

[00:34:48]And also if there's less virus in the blood, there will be less transmission because mosquitoes need a certain amount of virus in the blood to become infected.

[00:34:56]But so when would you then have to take this drug? Well, that's where the problem is. The treatment window is quite short. So it's when the virus virus starts to replicate so the viremia goes up and then here at the peak. But then when it's already going down, yeah, then coming with your antiviral will probably not do much anymore.

[00:35:15]The problem is that the symptoms they usually come at the the peak of the viremia. So people only start to know that they are sick and that they are maybe infected with something when they are at this point actually. So then you need to have really quick diagnosis to make sure that you know which virus it is they are infected with and then start up a treatment. So it's not very easy.

[00:35:37]It's not like with a chronic virus infection, yeah, people are infected for a long time and there's more time for a diagnosis, of course. So with arboviruses we would need this early diagnosis or maybe we can think of this pre-exposure prophylaxis like what we do for malaria travelers maybe that go to a region where dengue is endemic or another arbovirus.

[00:35:59]But then of course the drug needs to be really safe. It needs to be safe anyway, but if you give it to people that are not infected yet, yeah, you don't want to give them some side effects just to be on the safe side to not get infected with the virus.

[00:36:13]Of course in endemic regions we would yeah, also we also need a solution. So there we could think of household prophylaxis.

[00:36:20]So if one person in the household is infected and it diagnosed in time, then you could start treating the people in the house and hope they will not be infected. So, this is actually something that also Johnson & Johnson was trying to to test with this mos no denvir dengue drug that I talked about before.

[00:36:38]So, it is possible to find antivirals for arboviruses, but how will we use them in practice? We will still need to figure out. So, that's also why in in our lab we try to come up with some new strategies that we could maybe complement with typical antiviral therapy. And we try to work both with the host and the vector. So, I already talked a bit about our mouse models, but now I will come to our mosquito work.

[00:37:03]And so, of course we keep looking for new antiviral therapies and we are part now of a European consortium where we look at broad spectrum antivirals. Um we also do Yeah, uh since we are based in Belgium, we also work on Belgian mosquitoes and try to study with what type of viruses these Belgian mosquitoes can transmit because Yeah, we don't have Aedes aegypti, but we do have Culex pipiens. We start to get tiger mosquitoes as well. So, it's important to know what viruses can be transmitted by the Belgian mosquitoes. But for today, the rest of the talk I will focus on these two projects in the lab. So, talking about antiviral drugs and how they can be used as a transmission blocking agent in the mosquitoes.

[00:37:46]And I will also briefly talk about our work on human skin bacteria.

[00:37:52]So, one of the things we wanted to study is whether antivirals could have an impact on the the virus in the mosquito after the mosquito ingests it. And so, one of the first things we did was looking into blood meal ingestion. So, imagine that that we would have a drug on the market for dengue or another virus.

[00:38:11]Persons would hopefully take the drug, will be treated, but mosquitoes still are around and can bite humans that are treated with these drugs and the drug is in the blood in a certain form. It can be a metabolite, it can be the drug itself. So, the mosquito will ingest the drug and in the the mosquito, in the gut, but also in other organs, of course, the virus is replicating. So, we were wondering if these mosquitoes would ingest the drug during blood feeding, if this could have an effect on the virus replication in the mosquito and in the end, of course, also on transmission to new humans that are still naive for the virus.

[00:38:49]So, for this, of course, we need mosquitoes and this is how our insectary is looking like in in Belgium. So, it is a quarantine insectary because we work with Aedes aegypti and tiger mosquitoes and they're still invasive mosquitoes.

[00:39:01]So, we have all kinds of measures to make sure that they are not escaping, otherwise, I would be in big problems if we have an Aedes aegypti colony in the campus on Leuven. Yeah.

[00:39:13]So, here you see the are incubators for the mosquitoes. There's some trays there and cages. And then, of course, we Yeah, also in the summer, we do field collections. We have to We only have the summer for that and if it's a bad summer, which can happen in Belgium that it's cold and rainy, yeah, then there's not much mosquitoes and then we are a bit sad because we cannot do much research and we have to wait another year to collect more field mosquitoes.

[00:39:38]So, you see here, this is in the area of Leuven. This is my dad. He also has to help in the summer. He's retired and so, he Maybe he's watching. Sorry. So, he sometimes in the summer, he also does some mosquito collections for us.

[00:39:52]So, and then in the we need to infect these mosquitoes. We want to study the virus infection. So, this is our infection lab. It's in a biosafety level three. So, it's in Belgium, that's the highest biosafety level we have. And so, we Yeah, we have again climate chambers over here where we keep the mosquitoes. And then for the dissections, we use this mosquito cage where we first sedate the mosquitoes and then we take out the legs and the wings in this little area because if a mosquito would escape in here it would be quite easy to catch. If it would escape here and it goes behind the machines, yeah, you're lost. So that's not what should happen.

[00:40:30]So that's why we take off the legs and the wings in this um mosquito cage and then they cannot move anymore and you can take them out and do other like dissections and salivations with it.

[00:40:43]So yeah, and we have a hemolymphatic system that we use to infect the mosquitoes with blood and and and the virus. And then this is yeah, we have to since it's a biosafety level three, we have to dress up completely with a Tyvek suit. We have like our a masks and gloves double gloves and so on which makes it not so easy to manipulate mosquitoes, but it's working out quite well. And so we have done studies with flaviviruses like dengue, Zika, yellow fever, Usutu West Nile virus, chikungunya and now recently all also with the Oropouche virus um in the lab.

[00:41:19]So let's go back to the antiviral drugs that can maybe be used as transmission blocking agents. So I already talked a bit about favipiravir. So it works really nicely in cells, in mice for chikungunya. So when we started this project couple of years ago I thought like okay, let's try favipiravir that works so well. It's one of our favorite reference compounds I'd say in the lab.

[00:41:42]So we started in in mosquito cell lines and we were a bit sad because there was no antiviral effect at all of favipiravir and we were a bit surprised like why is this not working? And so we used another compound as a positive control the hydroxychloroquine which is um acidifies the endosomes and it's an entry inhibitor. Not a great antiviral, but it works well in cell culture. Also this one it was a bit active at the highest dose, not very active on the lower doses. But yeah, so we were a bit puzzled and then we looked into the literature and we found that actually insects don't have the enzyme that is needed to activate favipiravir because favipiravir is actually a very small drug. It needs to be activated. It needs to get a ribose and then the triphosphate to look like a nucleoside. And the enzyme that's responsible for this, apparently insects don't have it. And so we found some very old publications saying that insects don't have it and we were like, "Okay, this clarifies the lack of effect." So, we also showed that with with HPLC. So, this is in our Vero cells, eh, the monkey kidney cells. You see the monophosphate, the the diphosphate, and the triphosphate and they're all formed when you incubate the Vero cells with favipiravir.

[00:42:59]But, then we used two different cell lines of mosquitoes, eh, C6/36 from Aedes albopictus and Aag2 from Aedes aegypti. And then we we were not able to find any of those um phosphate analogs.

[00:43:11]So, showing in detail, okay, the cells the mosquitoes cannot activate favipiravir.

[00:43:17]Still we thought maybe we should check in the real mosquito because maybe there are some salvage pathways or other things happening that are not happening in cells. Um let's give it a try and then we also wanted to know is if this hydroxychloroquine at least could have an effect.

[00:43:32]And this is what we noticed. So, we infected Aedes aegypti with chikungunya and you can immediately see, there was no effect. Not of favipiravir, which was not unexpected, but also the hydroxychloroquine didn't do anything. So, we were disappointed because here it was quite active and here it didn't do anything.

[00:43:50]So, that's when we thought, "Okay, maybe we need a more relevant study model, something that can come in between our in vitro mosquito cells and in vivo infections." Because in vivo infections it takes a lot of time. It's very cumbersome because the biosafety levels, measures that you have to take into account, it's it's not very easy. So, that's why we wanted to have a new model that could actually be an in-between thing.

[00:44:16]So, that's when we started to focus on the mosquito gut. And yeah, you know, the mosquito gut is key for arbovirus transmission because it's the first site when the virus is ingested with the blood, where it will infect the different cells, and then it has to escape the escape barrier to get into the hemo lymph, and then infect the salivary glands and so on. So, we started to work more on the guts. And yeah, I will not focus on on this project because it just started, but I just wanted to show you one of our cryosections. My PhD student Cedric is working really hard on this.

[00:44:50]This is an Aedes aegypti. So, he does makes his cryosections of of the mosquitoes, and then he stains the nuclei with DAPI, and then the phalloidin for the actin. And the Prospero is an a marker for the entero the EC, so the enteroendocrine cells. So, it's not very clear here, but the reds is there, and we're still optimizing this also for Culex, which is for Culex it's again it's always a bit more difficult than Aedes, at least in our hands. And then we want to combine this with single-cell RNA sequencing to make a cell atlas of Culex pipiens, which is the most common mosquito in Belgium and in most of Europe because there's an there is a cell atlas for Aedes aegypti now, but for Culex we don't know much yet. So, we want to know which the different cell types, the different host factors, and study a bit more fundamentally how this infection is happening.

[00:45:43]But so, we also use the gut now as a this in-between model. And so, this is work of my PhD student Anna.

[00:45:50]So, she set up this ex vivo gut model.

[00:45:53]And that we were inspired during COVID by a seminar from people from the United States who have an ex vivo tick gut model. And so, they were showing that they were taking out the guts from ticks and keeping them alive and infecting them and we were like, this is so cool.

[00:46:07]We should try this as a Friday afternoon project, you know, see take out the guts of a mosquito and see what happens. And so Anna tried it. She anesthetized the mosquitoes, took out the gut, just put it in a plate with medium to try. And then she called me later saying like, "Lain, this is really cool. It keeps moving in the plate these guts and they stay alive." I was like, "Okay, this is really really nice." And you can see the peristalsis here of the hindgut of of of the mosquito. And so what we we so we tried lots of things and in the end we set up a protocol and we could also measure this peristalsis as a way of looking into the viability of our guts. And you can measure the peristaltic period so between every movement and you see it's quite short in the beginning. But then after 10 days in culture you see that these periods become longer so the guts start to move slower and slower. And in the end they will die but at least for 10 days we could keep them alive and we have collaborators now in the Netherlands that keep them alive for 14 days or longer so they're actually better at it than we are. But so I'm happy that this is a model that that can be used to study arboviruses.

[00:47:17]Of course we can also infect them so that makes it interesting for us as virologists. So like the Aedes aegypti guts we have tried with several viruses but this is for Ross River virus and chikungunya virus so two alpha viruses.

[00:47:29]So you see that the PFU's for the infectious particles they go up over time showing that there's active replication.

[00:47:37]We also did stainings on these guts so here in purple you see the E2 protein of chikungunya virus and we found it mostly in the posterior midgut which is also making sense because if the mosquito would ingest a blood meal it's also where it's digested let's say. And also here in the hindgut we saw some nice uh, staining of the, the protein.

[00:47:59]We also work on Culex pipiens, so we also tried the model with these Culex mosquitoes, and this virus is an alpha virus that can infect both Culex and Aedes aegypti. So, here you see that in both mosquito guts, um, there's active replication of the virus.

[00:48:14]And then, of course, we tried, uh, the antivirals in there because that was one of the reasons why we wanted to set up the model, huh, to have it like in between in vitro and in vivo. And so, we worked with this JNJ-07 compound, the dengue inhibitor. And if you use 2 micromolar, which is a concentration that is find found in the blood of, uh, mice that are treated with a dose of JNJ-07, yeah, there was complete inhibition of the dengue virus replication. And even if you only, uh, gave the drug 3 days after the infection, so when there was active replication of dengue, there was still, uh, significant reduction. Of course, not complete anymore because the virus has established, but this was for us, uh, a really nice, uh, results.

[00:48:55]And then, this is the metabolite of molnupiravir. Also there, we did see some effect, not as nice as the JNJ molecule, but at least there was also reduction in the ex vivo guts. So, for us, this was a sign, okay, we can continue with these two molecules and go in vivo.

[00:49:12]So, let's talk first about this metabolite, uh, of molnupiravir. So, we also, of course, checked in the cells.

[00:49:18]There was nice dose-dependent reduction.

[00:49:20]Yeah, this one you already noticed. Uh, so then we wanted to use a clinically relevant concentration because you can, of course, put so much compound in, uh, the blood meal that it's having an effect, but then, yeah, what's the chance that it's such a concentration will be present in patients? Yeah, that's not very relevant, we thought. So, we made up some calculations, and based on the PK data of humans treated with molnupiravir, this came, um, is, uh, similar to 25 micromolar of NHC.

[00:49:49]And you see already, this is not very effective in the gut model uh, while it's very effective in the cells but we gave it a try anyway.

[00:49:58]And yeah, we were disappointed again. So yeah, no effect on the bodies or the heads and we also tried 100 micromolar but also that one wasn't working although in our guts it was very effective.

[00:50:11]So then we worked with the pharmacy department in our institute and we asked can you do some PK on mosquitoes for us and they were like okay, that's special we've never done mosquitoes but yeah, we will we want to try. We gave them mosquitoes that were fed with NHC and then so they did this PK analysis and what we noticed was that actually 90% of the NHC was gone after 6 hours.

[00:50:35]So showing that it's so rapidly metabolized that there's no antiviral effect anymore.

[00:50:40]And so even with 100 micromolar this is also happening. So this is a molecule that will not work when it's ingested via the blood meal.

[00:50:48]So this shows that it's a nice model but we still need the in vivo to happen because the ingestion what's happening in the guts the blood meal digestion you cannot mimic completely of course in an ex vivo model.

[00:51:02]But we had some good results as well. So we worked with this J J L 7 and we started with giving the drug and the virus in the same blood meal.

[00:51:10]And then we analyzed at day 3 and 7 post infection different concentrations. So this is micromolar. And you can see that in the bodies so infection rate was very much reduced with the higher doses of J J L 7. This is then the dissemination infection rate so when the virus spreads to the the heads and the legs and the wings. Also there like at day 7 almost all the concentrations that we tested had complete inhibition of dissemination of the virus.

[00:51:39]And then most importantly the transmission so the virus in the in the saliva. So we see with this dengue model in Aedes aegypti only dissemination at day seven uh post infection. And you see that all the doses that we tried actually gave complete inhibition of transmission of dengue.

[00:51:57]So we were very happy with these results.

[00:52:00]And we also did some studies on mosquito survival because yeah, we don't want actually an antiviral to have a mosquito cidal effect because we don't want the mosquito to become resistant to the molecule. So here you see a survival curve with very high concentrations of J&J and yeah, there was no significant effect. We also looked at fecundity and you see that yeah, there was no effect on the eggs that were developed in the female. So that was nice.

[00:52:25]But of course yeah, the chance that the mosquito would feed on a patient that's having a lot of dengue in in the blood but also the compound yeah, the chance is really low. So we wanted to test some scenarios that would be more real life.

[00:52:39]So we started with giving a blood meal with the molecule, then waiting for the mosquito to lay the eggs to give a second blood meal with dengue virus. So mimicking that the mosquito would bite different people.

[00:52:51]And then seven days later we would harvest the mosquitoes. And here you see the infection rates and you see that even when the molecule was given six days before the dengue infection, there's still complete inhibition of the virus infection. And this is a dissemination also there complete inhibition of dissemination. So that was really unexpected actually. We were really happy.

[00:53:14]This is then the other way around. We first infect the mosquitoes with dengue, give them an established dengue infection and then expose them six days later to the compound and then again harvest samples. And of course yeah, there was no significant effect on the infection because the mosquito was already infection infected with dengue.

[00:53:33]But what was really nice to see is that the dissemination was quite heavily affected. So we went from 95% dissemination to 18. So that was having a really big effect.

[00:53:44]And even on the viral levels in the bodies, although the infection rate itself was not affected, we did see that in these bodies there was less virus present. So, molecule was still having effect on viral replication.

[00:53:58]And so this data, I forgot to add it also on the transmission, we did see a significant reduction of the the virus.

[00:54:05]So, then finally for this story, what we also we again did PK as studies and we noticed that this molecule can remain in the body for really a long time. We only went up to 7 days. We should have gone a bit longer, I think. But what was nice that yeah, you see of course that there's metabolization, but at this moment the JNJ or seven levels are still above this EC50 value. So, although it's lower, it's still enough to actively reduce the virus.

[00:54:33]And then we also work together with Alban Fontaine in a in France and he did a logistic model and an agent-based model which I cannot explain very well, but I will try. So, what they did was we used a systemic infection, the dissemination of different concentrations of our molecule. You see at two micromolar there's no dissemination. This is the control and then they used that in a model to mimic how many people would become infected in an outbreak, how many outbreaks there would be if humans would also be ex- the mosquitoes would also be exposed to the different concentrations of JNJ.

[00:55:10]And so what we noticed that that for example, if a mosquitoes would be exposed to this 0.05 micromolar, which is quite low, the number of dengue outbreaks would be much smaller than if they would be exposed to the vehicle, to the DMSO.

[00:55:25]So, this is just showing that yeah, these reductions in dengue kinetics would suggest that there would also be a decreased proportion of dengue outbreaks if mosquitoes would be ingesting this antiviral.

[00:55:38]So, we have been studying in quite a lot this ingestion via blood feeding, but this still requires that people take the drug, and so people need to be diagnosed or there should be some prophylaxis, which is maybe not going to be happening so soon. So, we were also looking into other ways, like for example, uptake via contact. If you have a lipophilic molecule, it could be taken up by contact of the the legs and then be taken up in the mosquito. And so, there were studies with a malarial drugs that showed that there could be an effect on malaria parasites, and we tried a small study with atovaquone, which has a bit of antiviral effect, not great, but it's very lipophilic and it's taking up by the legs of the mosquito, and we noticed that with chikungunya virus we actually had complete inhibition of transmission of chikungunya when mosquitoes were exposed for 1 hour with their legs to atovaquone.

[00:56:31]For Zika virus, we didn't see the same effect. So, I already say it's not a wonder drug. We would need something much more potent, but at least I think it could be a nice idea to think about for the future.

[00:56:43]Then I don't know how I am for time. Can I still have a small story or uh Yeah, I'm good. Okay. All right. So, then I want to go to our skin bacteria.

[00:56:52]You know there's people that are mosquito magnets. They get all the bites and then they can be another person in the room who doesn't get any mosquito bite at all. I have two kids, and one of them is like this and the other one is like this. So, if they're together in a tent, my youngest son will get all the mosquito bites. Um so, why is this? Why is a mosquito finding some some people more attractive than others? So, there's different cues that mosquitoes use to find us and to get a blood meal from us.

[00:57:18]And there's of course the carbon dioxide that they can already smell from a long distance and with the sensors in their antennae.

[00:57:26]But of course, everybody of us is exhaling carbon dioxide, so that's not what making people different. But there's this body odors, heat, and then the taste of our skin that is different between different people.

[00:57:38]And the the body odors are actually produced by bacteria on the skin. They produce these volatiles, and these volatiles can be different depending on the composition of your microbiome. And this is one of the main things that makes that some people are mosquito magnets and others are not.

[00:57:56]So, and of course mosquito-borne viruses, the skin is very important because it's the first site of infection. And here you see a female mosquito probing in the skin of a mouse.

[00:58:07]She's looking with her proboscis to find a blood vessel. And while she's doing that, she's of course spitting saliva and and she can deposit also a virus or a parasite. And so, you see here like she's probing around and then she will find the blood vessel to um drink blood from in a second. I think this will happen more or less now. Yeah.

[00:58:29]So, she finds the blood vessel and then she will drink up all all the blood.

[00:58:35]And of course in the skin, yeah, there's different types of cells. There's sweat glands and so on, but there's also bacteria in there. There's lots of bacteria on the surface, but more and more studies show that there's also bacteria in the dermis and especially in these glands actually we find bacteria as well. So, we were wondering, can these host skin bacteria also modulate the outcome of an arbovirus infection since they are present at the site of the infection?

[00:59:01]And there's some other studies that have shown that bacteria can affect the virus infection in the host. There's a lot of studies on the guts. There's lots of bacteria in in human guts and animal guts. And so, for enteric viruses it has been shown that bacteria actually can enhance infection of for example norovirus or poliovirus.

[00:59:21]There's also some studies on the respiratory microbiome and there influenza virus was studied and coronaviruses, but there it has a negative effect. The presence of some bacteria can reduce infection. But then for arboviruses and like chikungunya, dengue, how the skin microbiome might affect the virus, that's actually not being studied.

[00:59:41]So that's something that we then started to do and we first worked with cell wall components of the bacteria. So not with um complete bacteria, but with parts of their cell wall. And you see here the LPS, uh peptidoglycans and lipoteichoic acids from different bacteria that we could just buy commercially. So we started with um not all human like skin bacteria, but just the things we could get. And we incubated these cell wall components with viruses. And these are all alpha viruses. And then looked at infectivity. And we were surprised to see that some of these cell wall components could actually completely uh reduce virus infection. But it was not something that was happening all the time because first we thought it's something very uh specific. Uh um But then we tried different LPSs and you see that for example here the E. coli one was not having an effect while LPS of uh Pseudomonas and Serratia was having the antiviral effect. And then it also depended on the virus. So there is something happening. It seems to be quite specific. So we started to dig a bit more into the mechanism and we found that there was actually a virucidal effect. So these cell wall components were clinically relevant, yes or no. So that's for that we had to go in vivo. So for that we had to go in vivo and this is work of my PhD student Sam. So what she did, she used a Zika virus model that we had at that time. Um and she used a cream that contained uh four different antibiotics to have a broad spectrum uh antibacterial effect. And she treated first uh the the foot pads of the mice for 7 days with the cream two times a day. So she was rubbing in the cream on these foot pads. And then in this uh treated foot pad she injected Zika virus. And then we followed up uh how the virus was behaving in the mice.

[01:01:24]So here you see just as a control that we looked at the colony forming units of the bacteria on the skin. You see there was a yeah a significant reduction of the bacteria. They were not all gone but it was a really big reduction.

[01:01:37]And then of course the the viral data.

[01:01:39]So it's an it's a little model for Zika virus. So we looked at survival and you can see here in yellow that's the um the control and the vehicle cream treated mice. And then in light blue it's the the mice that were treated with the topical cream with the antibiotics. And in blue we we gave them oral antibiotics because there was one paper that showed that if you treated them orally with antibiotics there was an effect on flavivirus disease in the mice. So we used that as our positive control and also there we did see that actually the mice died sooner if they had less bacteria on their skin.

[01:02:15]And we also saw this effect on the infectious virus in the serum. So here you see in yellow the control mice. So they get a peak of the the virus infection on day five. And when they were treated with the antibiotics the peak actually went was two days earlier.

[01:02:30]So showing that there was some faster virus dissemination to the serum and to other organs.

[01:02:36]So we were a bit surprised by this but yeah we were happy that we could confirm the study with the oral antibiotics.

[01:02:43]So one of the things you wanted to know is this really because of the bacteria or are having the antibiotics maybe having some offside effects that are explaining this this phenotype.

[01:02:54]So what they did in the paper with the oral antibiotics they did a start stop treatment. So they treated the mice and then they stopped for three days to let the bacteria grow again and then infected with the virus. So we did a similar approach. So we had one group here in red that was treated for seven days but then we stopped for three days to let the bacteria grow again and then infected with the Zika virus. And you can see indeed there was like a recolonization with the bacteria and they even were more bacteria growing than on the control mice. So, there were some bacteria that were very active and like filling in this empty space, let's say, that we created by treating with antibiotics. So, these mice at the 3-day stop, they did have again bacteria on their skin.

[01:03:41]And so then going to the survival curve, you see that the actually the red curve is more or less the same as the yellow curve of the control. So, showing that when the bacteria are back on the skin, there was actually protection. So, the the mice were having the same survival rate as for the the untreated ones. So, we do think that the bacteria seem to protect to a certain extent the infection in these early stages. We don't know yet what's happening. We tried so many things. We did flow cytometry on skin, on lymph nodes, on all types of organs. We do see some little differences, but we cannot really explain it. We did bulk RNA sequencing on the skin, but also there nothing really popping out. So, it's still a bit of a mystery, but we hope we we can maybe find out what's happening there.

[01:04:31]But it just shows bacteria do seem to play a role in these arbovirus infections. And so, one of the thing we wanted to do is to get a better model because the Zika model we infect in the foot pad, but it's more subcutaneously.

[01:04:43]It's not in the skin and that's of course not what the mosquito is doing.

[01:04:47]So, we wanted to create an intradermal mouse model and we also wanted to be able to use immunocompetent mice because Zika virus cannot infect immunocompetent mice. So, we had to use immunodeficient mice and that's of course when you want to study immune system, yeah, that was not so smart, let's say, of us, but it was how we started. We didn't think it really through, I guess.

[01:05:08]So, for our next studies we wanted to have a model which is more clinically relevant and uh in immunocompetent mice. So, that's why we went to the Ross River virus, an alpha virus. They can This virus can infect immunocompetent mice, and we can use it on a basal two level, so making our life easier. And it also made that we could do a collaboration with Professor Pedro Marques in the our immunology department, who is a Brazilian professor who is now working in Leuven for a couple of years, and he's is a very good in intravital imaging. So, imaging life inside the mice. He has really nice papers on liver imaging and brain imaging. And we I asked him, "Can we also do the same thing in the skin?" And so, for that we used the ear because the ear is a very thin, and you can actually just put the ear under the microscope, and you don't have to do any surgeries or or things.

[01:06:00]So, and you can if you want to keep the mice then after the imaging. So, and this is what we did. So, we got a Ross River virus with a GFP, and then in blue you see the neutrophils and in red the monocytes. And so, we can actually make these movies. So, you see here the infected cells, and you see the neutrophils that are patrolling around these infected cells. We didn't see monocytes, which we were a bit surprised because this is 24 hours post infection.

[01:06:27]And then you can also make these like 3D reconstructions.

[01:06:31]So, the postdoc of Pedro made this for us, showing the infected cells and how these uh cells, the neutrophils, are around it. There's a couple of monocytes in this uh uh video. So, that was a nice, but yeah, it was nice to see this influx of the neutrophil. So, we're now using this model to study further how skin bacteria are yes or no important for arboviruses.

[01:06:54]And this is my final slide. So, we are in a consortium now where we try to modelate the skin microbiota of hosts. So, we do this in the mice, and for that we use lysins. These are um produced by bacteriophages and these lysins can um break down the the bacterial cell wall of a bacteria. And they can you can make this lysins in a very specific way for one bacterial species. So, our idea was can we use these lysins to modulate your skin microbiome so that we can reduce mosquito attraction uh because the bacteria attract the mosquitoes and maybe also arbovirus infection. So, we're focusing at the moment on Staphylococcus epidermidis, which is one of the bacterial species that we know is attracting mosquitoes. And we're trying to together with colleagues of the University of Ghent uh Professor Briers, she's a specialist in making these lysins of the bacteriophages. And we are trying to make one that's specific for Staphylococcus and then if we have it, we want to try to colonize the mice with it and then treat them with a lysin and see if there's an effect on mosquito attraction. Um this will happen in Zurich where people are having this video tracking to see mosquito landing and so on. And then we will do volatile analysis. So, we made this um custom homemade uh thing with a with a glass vials where we can put in a sedated mouse and we can extract the volatiles that are coming from the skin microbiome of the mouse and then we send it to Zurich where they do the the mass spec analysis uh for us. And then we of course will do the virus infections because uh that's that's our thing. So, it's a bit a complex project and it's advancing a bit slower than we hoped, but I think it's still something nice that we can try to see if we modulate the microbiome in a specific way, if this can help in the future.

[01:08:43]So, to conclude, yeah, there's no antiviral therapies for mosquito-borne viruses and but we can develop some if we want to if there's enough money and time and so on to to and effort that we put into, we can get these antivirals, but then we have to think of how to use these antivirals in a therapeutic way. And some of these antivirals can also inhibit virus replication of mosquitoes. Not all of them. You have to Yeah, even if they target the virus itself, there's like PK things dynamics you have to take into account. Uh and so we showed that change AO7 is very effective against dengue in Aedes aegypti. Tarsal exposure could be another way to um get antivirals into mosquitoes. And then we also want to uh study more the skin as maybe a a site of where you can apply things to reduce uh arbovirus infection and mosquito attraction.

[01:09:34]So with that I come to one of the most important slides are the acknowledgements uh thanking again people of my team and previous people, all our collaborators, Professor Jenta for inviting me here today of course, our funding. And then I want to take the opportunity to make some advertising for two um uh meetings that I'm co-organizing. This meeting is on hepatitis C virus and flaviviruses. It's in Copenhagen in Denmark in September.

[01:09:59]And they extended the uh the submission deadline for abstracts until the end of the month. So if you would still like to come, there's uh time. And then there's also this international meeting on arboviruses and their vectors. It's in the beginning of September in Liverpool.

[01:10:13]Um and that's also a very nice meeting if you study vectors or viruses in in in the host.

[01:10:20]So and with that I want to end. I thank you for your attention. If you have any questions, I would be happy to answer.

[01:10:32]Thank you Leen for marvelous presentation. Do we have questions?

[01:10:37]Okay.

[01:10:39]So thank you for presentation. It was great presentation. I have actually uh two questions actually.

[01:10:46]Uh the first one is about the combination, the synergistic idea of the project because uh it's got my attention that you were trying to mix two broad spectrum uh drugs.

[01:10:57]And I I I I I was thinking if you could try like a backbone treatment like with the broad spectrum and then mix with the more specific drug to treat the the virus because you can make like a toy. You can make like big spectrum thing and for different treatments you can implement a drug with more specific target. So, I think this is a curious thing to wait as well, but I am curious about what you think about it. And the second question is about the infection because you're uh uh uh, experiment this mice with the antibiotic treatment >> [clears throat] >> and you see the different, but I was curious about a second a second way like wait to the bacteria can recolonize the mice and then try again with the antibiotics see if this is a common way to develop the the disease the the the the the bacteria will influence always or not will change the depend on the way the the mice here with the bacteria or without the bacteria but in the series of treatments with antibiotics. This are my two questions.

[01:12:07]Okay, thank you. Um, so for the first question of the combination, I think that's a that's a really nice idea. So, we we used these three nucleosides that were broad spectrum because they are actually from if you look at the approved antiviral drugs, um, there's not many out there and from all the from these approved antiviral drugs we took the ones that are also active against alpha viruses and actually only three of them work well against alpha viruses and they they happen to be broad spectrum. But so, there's nothing out there yet that's specific of course like I said. So, in this study we really wanted to focus okay, what is there yet and if you would combine them could this maybe be used in in in in human patients. But I think it would indeed be a great idea to test that. So, we have some of these specific drugs. We haven't tried them in combination with uh with one of the broad spectrum ones, but it's I think it's a good idea.

[01:12:59]It is of course then you need to diagnose a patient and know specifically which virus they have. If you add like a more specific drug, you cannot say like okay, I have a combination that works against all alpha and flaviviruses which would be a dream I guess but then you really need to know okay, it's dengue so we add the NS4B inhibitor or it's chikungunya we add NSB1 or something. But it's a good idea to to test it. And then for the second question, I've never thought about it actually. I think it's a it's a good idea.

[01:13:27]I'm not sure if you my my student is defending her PhD next month.

[01:13:31]I'm not sure if she would be willing to do such a long study but yeah it is a good idea to look into if we then apply the antibiotics again how how it would work out. Yes, yeah, it's it's really great idea. Thank you.

[01:13:45]I I will No, okay. Congratulations. That's beautiful work, you know, really nice very nice work. And we are a little jealous of your insectary. I hope Finland does not take you to see ours, right Finland?

[01:14:07]A little different but anyhow. So I have actually many questions but if you have time, you know, we are up here maybe you can come and talk a little, okay?

[01:14:16]So you're actually thinking of using specific bacteria phage to target specific bacteria. That's so cool.

[01:14:24]That is very nice. Yeah, yeah, so you can actually control the microbiota biota on the on the skin. That's very nice. And um Maybe I missed it but I'm not sure you said what is the mechanism of action of the your compound at J J&J, right?

[01:14:43]And and also when you give it to the mosquito, you know, actually if I understood right, you you go and check the infection like 10 days later. I mean, where is this compound hiding, you know? How does it, you know, how is this working?

[01:14:58]I'll start with that and we can continue later.

[01:15:03]Yeah, that's some great questions. Yeah, I didn't show anything about the mechanism of action. It's of course well-studied. Um, so it's actually inhibiting an interaction between NS4B and NS3, so two viral proteins that are important to form the replication complexes. And actually for these NS4B inhibitors, the J&J ones, um it only works when the complexes have not formed yet. So at the beginning of the infection. So when the virus infection has been established, then these complexes cannot be disrupted anymore with the compound. There's other NS4B inhibitors because NS4B is like the hotspot for flaviviruses antivirals.

[01:15:38]There's one of Novartis and that one still works when the complex is already made and it can still disrupt the interaction. So that's how how it works.

[01:15:47]It's also very difficult to get resistance to. So I know my colleague, she passaged for 40 passages, so 40 weeks, the virus in the presence of the compound and then she slowly got some resistance and then you need four mutations to get a really drug-resistant dengue virus against this molecule.

[01:16:04]Um where the compound is hiding in the mosquito, we don't know and I would love to study. I was When when J&J was still working on the molecule, I asked if they didn't have like a tagged J&J molecule that would have similar properties, but they didn't really have that because then I wanted to see with with maybe some confocal imaging where it would hide.

[01:16:24]Another idea would be to dissect different organs and then do the PK analysis, but yeah, since they stopped every work on this molecule, we can also not work together with them anymore to do this type of of studies.

[01:16:35]But it was actually something we wanted to to do because it stays for such a long time. It's It's probably not only in the gut. It must I think it is it spreads but I yeah, it would be great to study that. Yeah.

[01:16:50]Hi.

[01:16:51]Uh about the the microbiota of the skin, do you do do you think the the virus modulation the the expression of anti-peptide microbian from the skin in the gut of the insect?

[01:17:09]Yes, yeah. So I I I didn't completely understand the question. So the the microbiota in the gut of the mosquito and the skin? Yes.

[01:17:24]Ah.

[01:17:25]Yes.

[01:17:29]Yeah.

[01:17:31]Yeah.

[01:17:39]Oh yeah, that's a that's a really great question. I actually don't know if the this anti-microbial peptides if they are being ingested and then do something in the mosquito gut would be really great to study.

[01:17:49]There are some people that have studied if if the virus infection is changing the skin microbiome of mice and it does.

[01:17:55]It's really cool study that shows that the skin microbiome is being adapted if if there's a virus infection and actually there's more volatiles produced by the bacteria so that there's more mosquito attraction to dengue infected mice than uninfected mice. So I think that's that's really great and they have the whole mechanism elucidated. I'm not sure if there if the anti-microbial peptides were involved. I I don't think so. It was more like this volatiles.

[01:18:20]But that the virus is changing the skin bacteria it does seem to be the case.

[01:18:24]How it's really happening that I don't think that has been um found out yet but it would be great to study. Yeah.

[01:18:33]I want to know if we have questions from the online transmission. Do we have questions?

[01:18:39]Okay. Okay.

[01:18:42]Oh.

[01:18:44]Is there Oh, we have something here. Okay.

[01:18:48]Oh, the questions from Hafiz from Pakistan.

[01:18:52]So, uh have you ever considered looking into the saliva of Aedes while working on the antivirals?

[01:19:00]And second, what's what was the impact of RRV on lymphocytes as well?

[01:19:08]Yeah, so we looked into the saliva when we infected with Yeah, with the virus and then JNJ 07. We didn't look whether the antiviral was in the saliva. If that's the question, we only looked at whether the virus was still present there or not.

[01:19:23]It would be very difficult to look into whether the drug is still present in the saliva because it's such a small sample, like probably most of you know, and we would have to get so many saliva samples. I'm not sure if you could ever really measure if the JNJ compound is in the saliva. Um yeah.

[01:19:40]It could be since I think it's probably spread around in in many organs of the mosquito, but whether it's secreted in the saliva that I don't dare to say.

[01:19:48]Second question was Sorry, the RRV and the lymphocytes. Yes.

[01:19:53]So, yeah, we only looked at the monocytes at the moment. We do want to look at other cell types. But yeah, we have to inject antibodies and we have to look at the spectrum of their of the fluorescence that we can measure couple of signals together. So, we cannot measure like 10 different cell types at the same time with this intravital imaging. So, it is something that we will do with flow cytometry. So, there We actually did that with Yeah, we looked at also B cells, T cells with the flow cytometry on the skin. I don't remember out of my head what the what the RRV infection was doing in the skin.

[01:20:27]I can look it up. You can send me an email. We can We can discuss further.

[01:20:34]Okay, I will make couple questions. So, if I if I'm allowed. So, I am first question is about are you looking into the mosquito microbiome?

[01:20:48]Because this is well established in parasite models that the parasites modulate the microbiome and the gut microbiome has a profound impact on transmission.

[01:21:00]This is one question. The second question, I I was very impressed with the effect of the antibiotics on on the viral infection in the skin. So, do you think what happens it's very common in Brazil. It's very difficult in in in how can I say the diagnostics point of care our point of care diagnostics is could be improved it a lot. But the the problem is that sometimes the the physicians just take kind of a broad spectrum strategy for fever, you know.

[01:21:37]And it's very easy to buy antibiotics in Brazil. So, people can it's very easy to get antibiotics here. So, it's very common.

[01:21:47]This is even a joke in the old times people the doctors just you go home and rest and drink milk. So, but now people are on ampicillin or stuff like that, you know. So, it's more expensive than milk, but it's it's quite popular. So, I was wondering if this cannot make everything worse, you know. So, I'm not talking about antibiotic resistance, which is obvious, but about getting dengue outbreak worse because there's many people taking antibiotics almost random, you know, and making them more attractive to mosquito, getting infections worse, and so on. Do you have any thoughts on that?

[01:22:35]Yes, so I will start with the first question about the mosquito microbiome.

[01:22:39]Yeah, of course also for the arboviruses it is well established that if you infect the mosquitoes with a virus that it's the transmission is affected and the infection of the virus and the transmission is affected by certain bacteria in the mosquito gut and also the gut microbiome is changed because of infection. So, many people are working on that. We have done a Yeah, one study when when we started when I started the lab Yeah, I was still like exploring a bit Okay, what are we going to do? And I was really interested also in this It was a sand fly paper showing that they found bacteria in the saliva of the sand flies. And I thought that was really cool and it was not described yet for mosquitoes. So, we started this little side project on for getting saliva by force salivation and plating it on different plates. And we also found fungi and and bacteria in the saliva.

[01:23:27]So, we did a little project there trying to do some sequencing on it and so on.

[01:23:32]But yeah, there's it's Yeah, we are not bacteriologists and we also not working with fungi so much. So, it was a bit difficult, let's say, for us as virologists. We had some collaborations, but and also combining viruses and bacteria in the lab actually biosafety-wise our biosafety managers didn't like that so much that we started to work with bacteria.

[01:23:51]Um So, yeah, we don't work so much on bacteria anymore. There's a couple of things that we do, but there's many people that work in the mosquito field with bacteria. So, that's why we decided Okay, let's focus on other things and let other people that are better in that do that.

[01:24:05]The second question Yeah, that's really something I wondered. So, there's this study with the oral antibiotics in mice.

[01:24:10]It's from Michael Diamond. So, they really tried different flaviviruses.

[01:24:15]Some of them immunodeficient mice, but also like West Nile virus in immunocompetent mice. And they did see every time if the mice are treated with oral antibiotics broad spectrum that the disease with flaviviruses got worse.

[01:24:27]Also for alphaviruses they found something similar. So it could be if it's really widely used and yeah you get a fever you take some antibiotics but it's maybe then dengue virus that people that had the antibiotics have a worse outcome. It would be a super interesting to study I think but I don't know if that's really what's happening. It's of course comparing mice with humans but it would be interesting to look into that yeah.

[01:24:56]We have time for Yara. Do you want to You can have a second round why not?

[01:25:05]Uh actually we work with sand flies and we've done some work with ex vivo you know guts but um we never tried that and that was very nice to know that you can keep it like for such a long time. It is just a very basic question has to do with microbiota. How do you control the microbiota? You just use pen strep or you have to do something different?

[01:25:29]Yes with this gut cultures we have to treat the mosquitoes for a couple of days before we dissect with pen strep in the sugar. We tried without but then like more than 50% of the guts got overgrown with the bacteria and we couldn't use them so it's a bit of a downside of the model but because of course we know these bacteria are important for lots of things that happen in the guts but with this if there's too much growth of the bacteria we cannot do anything and they also die the guts then so yeah. So we also in the medium when they we we have them in the plates we also add pen strep yeah.

[01:26:05]Just one last question. Do you have any idea about the mechanism of because one of your compounds was the one that was well more than one just vanished from the mosquito in 6 hours or something like that. Do you have any idea what the mechanism is if it's microbiota or the mosquito? Have you tried antibiotics to test that or something like that?

[01:26:34]No, actually we don't have any idea how why it's so metabolized so fast because in humans it's not like that.

[01:26:41]Of course there it's not being ingested with the blood meal so it's very different. But it's given orally so it must be but it's of course it's giving a small molnupiravir which is a prodrug which protects the active metabolite. We didn't use molnupiravir because that's not what will be be present in the blood when the mosquito will ingest it. So that's why we used the active metabolite which is in the blood and then >> [snorts] >> give it to the mosquitoes.

[01:27:06]If it's bacteria or not, yeah we we we didn't try that actually. Would be interesting to to give them antibiotics and then give them the blood meal and see if the metabolization is slower or not.

[01:27:16]Would be a nice side project to to look into. Yeah.

[01:27:20]Okay, so I think we we are good. Thank you Leen for your marvelous presentation and for your visit as well. Thank you for the audience for being here especially special thank you for people that came to the presentation and of course thanks to people that are online watching us.

[01:27:39]So merci and bye-bye. Goodbye.