50 Years of Ebolaviruses: From Zaire ebolavirus to the Bundibugyo virus Emergency- Nadine Biedenkopf

We start with this now third webinar in our little panda theme. So that was an idea in the core group that because of the current ongoing yeah Ebola outbreak we could do something actual on learning a bit about Epolo virus its history because we now have 50 years of Ebola virus and luckily Nadine agreed to give this seminar and tell us about the history of Ebola virus outbreaks and from the beginning I guess then until the ongoing outbreak which I understand is now becoming or is at the moment the second largest so far and to introduce Nadine a bit she studied human biology at the Philips University in Marborg so that's actually something we have in common but you then went into infection biology I stated biochemistry then I understand you did your post your doctoral thesis and also your post-doal work at the Institute for Viology in Marborg where you're already early focused on the transcription and the replication of Ebola virus and then since 2019 you are a group leader at the institute also that your research then focus on transcription and replication of filo viruses and also corona viruses But I also understand you have a applied yeah applied subjects in your lab looking in how you can test vaccines and high throughput screening under these high ESL4 conditions which I guess it's quite a tricky task to do. So with that introduction, I think I give the word to you and we are looking forward to a very interesting update on what is ongoing with Ebola viruses. >> Yes, thank you very much for the kind introduction. Can you see my full screen? >> Yes, but uh still the >> not in the full screen. I am true now, right? >> Yes, perfect. >> Yeah. Thank you very much for the kind introduction and also for the invitation to give um a bit of an overview on the really currently concerning outbreak of bunduklu virus um disease um although it's not so you will learn in a few minutes. So um it's it's also a 15 years of anniversary of Ebola virus in general and as I mentioned I will give an overview on the history on these filo viruses and later on I will talk a little bit on what we do on research um of these highly pathogenic viruses in our institute so in Maruk and we'll also guide you a bit through a project which is based on maruk virus and um so maruk virus brings me already uh to the first introduction because before you're going to do to talk about Ebola virus you need to mention um mukbook virus as it's you know so to say older brother of Ebola viruses and this has a long tradition and uh you will learn from that here um also in Maruk because in 1967 so 59 years ago there was the first documented filo virus outbreak which was maruk virus it was uh in Maruk where members and lab workers of a company it's the bearing company got infected by so far unknown disease of unknown atology and um there were in total it was not only in Maruk but also in Frankfurt and in Belgrade and in total there were 31 cases and seven of them died and by then nobody did know Maruk birth and what was going on whether it was an infectious disease or poisoning so there this is um the Um now you can see my pointer. This is the old newspaper. So people were really um concerned about this mysterious disease of the employees of that company. Now what happened? Basically the company were engaged in uh in producing vaccines and they regularly imported African green monkeys from Uganda. And obviously these monkeys have been also been infected by these um infectious agent and then transmitted the virus to the people who care for them. And it was then in Maruk um where they isolated the virus which then caused the disease and named it after the city Maruk virus although it uh it has its um endemic in African region as you will learn in a bit of a second. So and this is um the castle of Maruk of Maruk of Maruk city now. Then 9 years later or and then um 50 years ago there was then uh the first documentation of Ebola virus. This was um after two simultaneous outbreaks in the Democratic Republic of the Congo which was named by then Zya and Sudan. And this is the really first um publication I found. And you can see here the two um the two cities that were affected. Maridi in Sudan and Yambbuku in um Zy back then and they are only um divided by roughly 800 kilometers and this is also people who visited this Yambuku village. Um it was a rather yeah middle size outbreak of 200 to 300 people but with a high mortality rate. Um and um so although it was closely related by um geographical means um the sequences of the virus were um were completely different. So it was then named one was then named um Ebola virus or back then Zya Ebola virus and the other one um Sudan virus and also of note most of them um have been in nocomal um infections and the virus has been transmitted here and the term Ebola virus I forgot to mention is um a name of a river Ebola river this is a picture by that and you can find that river here which uh is also um feeding into the river Congo and which is close by to this village in Zahir back then Zahir Yamukun. So that's um the reason why the virus gets got his name for. Now how do these viruses look like? They are then or or were then grouped into the family of fetto viruses because of their filamental shape as you can appreciate here. Maruk virus and Ebola virus they really have this long filamental shape roughly almost up to one micrometers in length with a diameter of yeah 80 nanometers and they contain a negative single stranded RNA genome as uh I I mentioned they are brother and sister so to say so we have the maruk virus and also the Ebola virus nowadays it's called or Ebola virus and back then then the Ebola virus and the Sudan virus but this is an is growing family. This is uh the the family tree of um the filo viruses in general. Um due to yeah advance advancements in sequencing analysis there have been also sequences found in in fish for example here in fish in in Switzerland leburg fish landfish but it's only sequences and for us for the humans mostly relevant here the green ones which is the maruk virus or marabuk viruses and the oro ebola virus among them also bunibukium virus. So it's also a member of Ebola virus but not the the original one which is the Ebola virus. Um so since its discovery 50 years ago in general filirus or maruk virus but also also Ebola virus have caused several outbreaks um in the central African region. you can appreciate it's always in this um it's sometimes also called Ebola spelled in this humid uh humid areas um central African regions and they have been characterized by rather small small outbreaks sometimes the virus run ran into a village and people people um got sick and have died but it was rather self self-limitating um in in the last outbreaks but frequently um and uh this changed um tremendously in 2014 2015 when there was um the first outbreak of Ebola virus um here in West Africa and it started here in the Geekadoo region in Guinea and then um spread into the mostly offended countries Sierra Leone and Liberia and you may also remember um all these pictures. So this was the so far f biggest or largest Ebola virus outbreak with more than 11,000 deaths and 28 cases and this um outbreak was roughly one and a half almost 2 years long and it had really huge um impacts also regarding socioeconomics. So the schools have been closed also. Public health care system was completely crashed because obviously normally the sick people um engage with the with the uh with the doctors and they get sick because until they know that they have been uh contracted Ebola virus they they already have been infected um leading also to decline in gross domestic products and also food production and also this crossber trade. for really huge social social um economic impacts. Since then you may appreciate also really almost every year we face and rather luckily small outbreaks of filo viruses among them maruk virus mostly Ebola virus here's a larger Sudan virus outbreak all in the central African African regions and um today um we are facing a really large outbreak of the bundy virus Ebola virus so again bundyu is a member of the author Ebola viruses. It has been known since or it was firstly discovered in 2007 after an outbreak of 140 49 cases um in Uganda and then 5 years later um there was also a smaller outbreak in the Democratic Republic of the Congo. So today it's the third outbreak of Bundi virus. um here in the Democratic Republic uh of the Congo affecting mostly these um orange provinces and the stars mark where um the previous outbreaks have occurred. So it's really close into this border region, Uganda and Democ Democratic Republic of the Congo. Um it was rather early notified by the WHO already um beginning of May um uh that there is a high mortality outbreak with a disease of at that time point unknown eiology in this Mong Mongalu um um health zone in this northern Italy province. Um and then in um yeah roughly 10 days later the the ARNB and Kenshaza confirmed that it's um bundukio virus disease. Already two days later um the WH declared this really important um public health emergency of international concern um because it already spread the virus already spreaded also to Uganda. So to the neighboring country two two confirmed cases and one death and had um this is a region which is rather mobile has crossber activities and by then there were already um 200 roughly 250 suspected cases. So this was rather early detected and rather rather early declared. The good news is that at least in Uganda where they had had been um in total now 200 uh uh sorry 20 confirmed cases and two death this outbreak is declared over after the double um incubation time of 21 days. So after 40 days uh 42 days this was end of July. So in Uganda uh this outbreak has ended but in the Democratic in the DRC the Democratic Republic of the Congo this is um a current situation of really much of concern um as data from um early this week um now it's five provinces 51 um health zones affected with more than um 4,000 cases and more than 2,000 deaths and giving it a case fatality rate of 46%. And again the most affected um regions is here this ituri region also north ku some cases have been in the south ku region and you can um see that there is really um yeah distribution and transmission ongoing in this northern region. Um so far this is um the largest Ebola or Ebola virus outbreak in DRC although it's it's 17th outbreak. So before there have been 15 outbreak of Ebola virus in the DRC and two bundukio virus um outbreaks but so far it's the largest one and it is the largest one because it's also a rather insecure region and has some more um more uh factors that contribute to to that to the transmission because it's already there in human humanitarian crisis as I mentioned insecure insecure region and high of criminalism in that region. Um you can find um mining also illegal mining for gold and manganesees. So there are really like um yeah criminalism and bands um ongoing. There's also large urban centers which means high population density and as I mentioned earlier also high mobility cross border to Uganda and also to the other countries and this is all complicates the outbreak or the containment of the outbreak um which is um can also be seen here in this high transmission rate already. This is calculations based on how many uh so um at which day um thousand people have been infected and we are currently here um this is the ongoing bundio virus outbreak and um where you can see so the first thousand cases after notification have been after um 45 days roughly and um oops and um during the the largest Ebola virus outbreak in West Africa this thousand cases was reached um after 235 days. So in total this means that there is an high transmission rate meaning um meaning that there will be likely many many people affected and um especially with the insec insecure um dynamics and regional politics um there this is really of concern. Also you can you have may might have heard in the in the news that they have burned um treatment centers um etc. So this is really really concerning. A bit more facts on felo viruses. As I mentioned the felo viruses um maruk and ebola virus they mainly occur here in the central African region. um um and this is because of its um reservoir and this has been shown and it's evidenced for maruk virus is um the af the African um rosettus Egyptian rosettus which lives in huge colonies um mostly overall um Africa and the reservoir for Ebola viruses is suspected um to these um three also fruit bats um that also live here in the region. um where the outbreaks occur. It's only suspected because the final proof of isolating life virus from these bats in nature um is still lacking. So you can infect that species, you find RNA, you find um um um antibodies against the virus, but so far um life virus hasn't been isolated. But it's very likely also to the close um relation to maropar that that's the reservoir maybe also other other of these fruit beds [clears throat] how then the virus transmitted from the reservoir to the human. Now um at least a good thing Ebola virus or feliruses are transmitted not via via airborne transmission but via infected body fluid. So this is then blood, urine, feces, also breast milk and um it's a classical zonotic virus meaning that the infected animals somehow get in touch um with the humans which is either by direct consumption of infected animals because these fruit vests are rather large and they are hunted for bush meat or by um by also other hosts like um chimpanzees or these dukers this small antelopes that are hunted hunted by the people and that um have been infected also um by the fruit beds by um for example co-feeding on the same um on the same trees [clears throat] and this is why in that region where this really rather frequently um happens the transmission it's also then um you should never touch or take uh dead animals that are found uh in the forest um because they could have been infected and died from um Ebola in that case from Ebola virus. Human to human transmission then works either by um also by direct contract of course with infected body fluids for unprotected health care workers or other people who are caring because um who are caring for the infected patients and also traditional funeral practices. This was at least um prior to the 2014 outbreak um a huge problem since then. There is more awareness um that traditional washing and um practices are rather restricted now because um the bodies are full of virus and and [clears throat] to prevent infection. Um the incubation time of Ebola virus is 2 to 21 days. This makes then so this is the latest time point which makes then um um a double incubation time to to have um to get an outbreak ended after 42 days. It's not very infectious and compared to other viruses that are also um droplet spread like measles virus. So um the the basic reproduction number is roughly three people. So one infected with Ebola virus infect three people. So compared to other viruses, it's rather okayish I would say. Now the symptoms are rather unspecific starting from unlike uh from from flu-l like symptoms, fever and also um headaches, tiredness, muscle pain. So rather unspectacular which can really be get um severe severe vomiting, diarrhea and that's then the problem of um dehydration, stomach pain and also hemorrhages. It's not so common as um previously thought. So the the the lethal cases they had this hemorrhages but it's not a classical um viral hemorrhagic fever virus Ebola virus. Um the virus enters the uh enters the body via as as mentioned infected body fluids and there infects um directly um macrofasages and dendritic cells and thereby distributes the whole body right and it it enters the lymph nodes and then enters also all organs and there leads to organ um or tissue damage and also to an overreactive immune response with the release of um inflammatory um cytoines and chemocines leading then to a cytoine storm which really harms the organs and um and the vessels and the tissue. Um also there is um impairment of adaptive immunity and this can then lead after roughly one 8 10 days um to multiorgan failure in total and then to death and the lethality rates um of um ao virus vary a bit. So for Ebola virus it's um estimated 55 to 88% for bundyu virus with the current outbreak it's 46%. Um but one needs also to consider that it's these numbers are sometimes really biased. So because we only normally see the people that are severely infected but not that are um have only um only weak symptoms and um yeah maybe the reason lies somewhere somewhere in between. There's also post Ebola symptoms like myalgia um impairments in vision and hearing hepatit hepatitis fatigue and also not to forget also stigmatization and social isolation in small villages when you are a Ebola virus um survivor because there's also like sometimes um yeah the the the religion or believing of witching and this is really also a rather um severe symptomatic with this um or with the stigmatization really huge problem there. Okay. What can you do against um Ebola virus? And the good news is there is two vaccines licensed um which were had a much of um improvement and much more format forward energy so to say after and during the um 201416 outbreak in West Africa and both are based on viral vectors that carry the surface protein of the Ebola virus. One is based on BSV and one is a two regimen um vaccine based on an adino virus and an MVA virus. Um and this is a life attenuated virus. There are also antibodies um that have promising um therapotics and and and protect against the disease. Ibanga in Mazab and also nucleioid analoges like the ram deseir and obel dese and also um what remains is then supportive supportive k care mainly um targeting dehydration. This is the good news but the bad news is this is only working for Ebola virus only. So for the Zaha Ebola virus train and so far for the bunibia virus train there's no licensed or approved um theoropix neither theraputics no vaccine available currently um so the WHO together with the CP um they have initiated um funding and trials for the most promising vaccine candidates. One is also again based on the VSSV. This is basically the licensed AVU which has here the Ebola virus GP um on its surface which is then changed against the bundukio virus GP. one is based on chadox um so also a chimp adenoirus um vaccine from Oxford University and one approached by Merna um is um prioritized which is based on mRNA so but these trials will take some time of course because you will you would they start with um GMP production and then pre-clinical trials um talking about um phase one trials first in men and um and so on. But last weekend um the WH suggested also um also to use these uh this AIBO vaccine candidate which carries the Ebola virus GP um due to um yeah hopefully promising cross protection against the Bundi virus GP at least to do something um until until bundi um specific vaccines are available. Currently they are also doing rem desave and orbital dese which are rather yeah unspecific nucleioide analog analoges that block the viral polymerase and there's also an antibbody which is crossprotective monoconal antibbody and this antibbody has been also successfully used for treatment of two American um citizens that have been hospitalized in Germany one in Frankfurt and one in Berlin um a few weeks ago and that worked rather successfully. um until then. So until waiting for a vaccine and clinical trials um it's of course um how can you otherwise contain such outbreaks and also this comes true for prevention of outbreaks of causes to raise public awareness also for for the symptoms. be aware and leading to early detection and also diagnosis which was also a bit of problem um during the early outbreak that there was not too much of diagnose um centers and also equipment and of course what always helps is contact tracing and isolation measures if the people um stick to that which is also kind of a problem because the the the political situation um is so insecure by there and also to implement um safe funerals. And with these vaccines, what um people then do there in in trials is to do this ring vaccinations and but also use um post-exposure prophylaxis. So likely with this antibbody especially for healthcare workers to be at risk and also with this ring ring vaccinations you then vaccinate um contact of contact so to hopefully somehow contain contain the virus. Yeah that's um for now for um the bonjibbuku ebola virus. Um now I want to um talk a bit on what we do on inab virus. um with a with a few pictures. So as I mentioned due to the to the original um um to the isolation of Maruk the first isolation of Maruk bars here in Maruk there's a high tradition of these highly pathogenic viruses this is the building of our whole you know institute so we are here in the institute and this is our BSF4 building and we work here with mukbook virus Ebula Sudan virus so the feta viruses but also with lassa virus paramixo virus like me virus cf F and also highly pathogenic corona viruses. And our um devotation to or addiction to Maruk virus is also seen on our outside BSF4 building because um because it carries EM pictures of the Maruk virus on its outside. This was um running operational in 2008. So it's not it's um not that old. I know first I want to show you how this works. So this is basically um a house in a house um system. So we work only in one of that floor and all the other floors are for technical reasons. So for um yeah for for maintain for maintainment and and everything and basically this is um we enter that via airlock systems and the actual work side where we work is um internal and this is um under or negative pressure. So everything is um is so so to say nothing is going out and everything is pulled pulled in. If you picture how this looks like, so we wear the suit with an um positive um with a positive pressure in the suit in a negative pressure surrounding and it looks pretty much like a normal lab I would say. Maybe a bit more uh cleaned and everything because we are not allowed to take too much things um inside the lab. Yeah, as I mentioned, the old building is is not so old, but during the pandemic with Corona virus, we recognize that we still need more um more place and more space. Also, when it comes to to experimentation um and that's why we currently are building a new BSL4 building. It's called the MSAP Mar Center for Epidemic Preparedness. So the big black brother of the smaller um red BSF4 lab and we will continue there our pre-clinical testing of vaccine candidates and antivirus which we do in in cells but also as I mentioned in in vivo in small animal models like mice or um ferrets or hamsters and of course we also do functional characterization of the of um these viruses also when it comes to virus host interactions and so on. And in total um we have then a lab space of 450 square meters which is I think three times more than we have in the old building. But again here we will work only in this uh at this floor and all the other floors are just to get our our lab running at that floor. Um I choose a project now where where I wanted to to talk to you a bit or explain a bit what we do uh which is also on my ab v virus and which is based on reverse genetics approaches. Yeah. To study the molecular biology of these highly pathogenic viruses and what we do rather frequently uh in our in our yeah daily life. just um again to keep you to keep everybody uh on on the terms. So what means reverse genetics? Reverse genetics means that we look from the gene towards the function. So otherwise that you would do like the plant is small and then you have um and you look what what has changed in the genome. We go from the genome to the to the function. And for the foirus or for Ebola virus or Maruk virus um this virus is a rather simple virus. As I mentioned before, it has a ne negative stranded um single stranded um RNA genome and has it has only seven viral proteins. So rather basic functions but also redundant functions um and they rely also on on rather important um interactions with the with the host cell with host factors, host proteins. They have they carry um post-transational modifications by the host proteins but the function and how how do these viruses replicate the genome and um and assemble together in the infected host cell it's not always clear and therefore we use here frequently this reverse genetics and where we also where we also use this reverse genetics is to denovo synthesize um a recominant virus which is of most importance when a virus isolate is not available which is also um is also um the reality um currently with bundyu because we don't have a current bundy virus isolate and I choose Marvok virus because this is a nice um the nice story where we succeeded to denovo uh synthesize um the genome and the the the history of that is rather similar so to say to the to the budooia virus outbreak, but it was a bit with a more luckily outcome which is 5 years ago. There was a note uh that there was one case of a Maruk virus diagnosed in Guinea um which is here in in West Africa and it was only one case and it was lethal um and um but there was because it was diagnosed postmortm mortum there haven't been a natural isolate of this marocus guinea and also only an inco incomplete sequence but still back then when we had that note it really concerning because that happened. It was the first time Maruk virus was detected in western in West Africa. Before it was more in Uganda and also in DRC. Here it was the first time and it was exactly in this geeku um region where the devastating 2014 2016 outbreak started. So also with this high mobility to towards Liberia and Sierra Leone and people were really or we were really concerned that it could also uh also um increase into an rather huge outbreak. But we had no virus isolate and we did not not know too much uh of that virus also whether like um antibodies for detection or um therapeutics would help. So we decided um we decided to generate this virus from denovo with the Maruk virus. Um it's not too um it's not uh it's not like that we have so many different um species but more isolates and this is where the Guinea outbreak the Guinea virus isolate mapped towards the Ghana and also towards an Angola virus isolate. So how how do you build and how do you construct a recumbent virus? So as I mentioned so the genome is RNA and we do this by reverse genetics on DNA on DNA plasmids and this is uh what Isabelle Fites um by then she was a doctoral student um had in her PhD thesis and then she started then to to synthesize um the genome um the the DNA fragments and to liate them together. there was a bit of a problem because the sequence um was not complete. So they were missing the three prime and the five prime ends of um the genome um which is rather often because um normally if you do just NGS sequences um you don't get uh when when you don't do raise PCRs and so on you don't get the absolute ends but they were missing. So by thinking of a of a cloning strategy, we we thought okay we can um resubstitute um the absolute genome ends by the closest relative which was which is Angola and also the genome ends are rather highly conserved. So we filled that up with the with the angola specific sequences to get uh at least in in in zilico uh complete re combinant marbuk virus guinea genome. But before trying to rescue that virus, we wanted to be sure whether the genome ends are somehow compatible. And here we used again these life cycle um modeling essays and also reverse genetics by using um or constructing a marquar skinny specific mini genome. So this then has instead of the proteins um a vanilla reporter. So in our case it's a vanilla luciferus a reporter but it has still contained the absolute genome ants that are required for efficient um replication of the genome and transcription of the genome and um with using um that essay we wanted to check whether the ends that we just filled up were rather com um um suitable for the for the viral polymerase before trying to to work that out all in BSF4 level1s because The benefit of this essays is that we can work in a regular BSL1 or BSL2 lab. Okay. So these are called mini genomes and we can work as I mentioned in a BS2 lab. How does that work? So we use um basically we need the the viral proteins that we need for efficient transcription and replication on DNA plasmat and transfect that and transfect that into cells. So let the let the cell uh synthesize the viral proteins. Also the mini genome which is also on a DNA plasmid is under control of a T7 promoter. So we also transact the T7 polymerase. T7 polymerase then uses um the DNA plasmid in order to synthesize an RNA mini genome. And this um RNA is then recognized by the viral proteins and then it's similar to an infection cycle. um the viral mini genome RNA is transcribed into mRNA and then translated into the vanilla or into the reporter gene and in that case it's the vanilla luciferase and it's also replicated and by using um um that assay we can then um measure the luciferous act activity as a mean um that reflects viral transcription and here this is minus the viral polymerase and this is with the viral polymerase. You can you can see that it basically when we add the viral polymerase that it works and that the viral um genome ants are compatible for the maruk virus specific um viral helper plasmids. So all that are all required for viral transcription and viral replication. Yeah. So these are are are all working and this um made us uh hopeful that we can also rescue this recombinant virus. So this is then a fulllength reccombinant Marvok virus that has all the information of um the guinea virus proteins together with the substituted angula virus sequences on an RNA. But again, this is then transferred or cloned into a DNA plasmid. And this is then rather similar to the essay that I showed you before uh where we had only a reported gene um included. Here we have now the whole information. So basically the whole genome and what happens is then again the T7 polymerase um synthesizes an RNA which is then the viral genome. We also need then the viral um proteins that initiate transcription and replication. We do that by plasma transfaction. So the proteins are here and similar to the to the mini genome essay I described before we have then a new round of viral transcription. So with the with the helper proteins we can have that all our new viral proteins um um produced but also replication the viral the virus reconstitutes assembles and bucks and since it carries the information now for all viral proteins we have a reconstituted recombinant virus which we can use for infection and infection if the virus successfully works and luckily it worked. So um marverse does not show a very pronounced cytoic effect. You need to believe me. So this is without the viral polymerase cells are rather dead. It takes se 7 to 10 days. So the cells are pretty much uh also not in a good shape so to say. But of course we sequenced everything and finally were successfully in rescuing um a re combinant um marbor guinea isolate which was also similar. In parallel we did also with the muzzuki. This is the traditional or the at least for us the transition traditional maroc virus that we use but it was no different here in this negative emaining of the recominant particle. Then um of course we we wanted to characterize um the recombinant um guinea virus in comparison to the other maroc virus isolates that we had um in human cells. And here you can see um the growth kinetics for the guinea virus um for the recominant guinea virus here rather similar to also to other viruses except of the recominant muzuki virus in human cells but also there was not not of a big uh difference in veros cells. This is monkey cells. So these are all relevant um host cells and we also tested in bat cell line. Um and also there were neither a neither a benefit or an disadvantage of the virus so that it could propagate in human cells, monkey cells and in um in bat cells. We also then tested whether we can inhibit it uh with remiss which is a nucleioite analog um I have already introduced and you can also see also in compar in comparison again to the recumbent mole virus that that works pretty much. So obviously they are not too much of a difference and they are both um or the guinea virus is also targetable by these um antiviral and we also check then for the available antibodies that we had in our in our lab whether they also recognize although they have been um normally targeting the muzzuki virus spec muzzuki virus muzzuki maruk virus whether they could also detect guinea virus and and this was true for for GP for the nuclear protein and for VP40 and interestingly another viral protein. This seemed to be um rather specific since the Marvok virus mazuki specific antibodies were not able to recognize the guinea specific um whipp and so for we do not know um for what kind of reasons. Okay. Um so in principle we we used this reverse genetics um approach for this denovo generation of at that time point new virus since then there haven't been any cases so this had a bit luckier than the bundia virus outbreaks so but in principle this works not always as fast as we um as we were hoping for because we are relying also on lia so yeah DNA synthesis and lation and this is a rather 19 KB is a rather huge genome but finally it worked also by compl by complementation of the genome and by conserved um by conserved um sequences worked out quite well so we don't need to wait for rays uh and specific PCRs which would have wouldn't have been uh possible at that uh with that case anyways because um because there was no more RNA information or sequence information available And then we characterized the virus and it replicated similar to other marble bar viruses in cells in bad cells in monkey cells and in um human cells. It has a had a rather dominant CPE also comparable to so rather more um cytoic effect more damage and um it could be inhibited by the rema as a nucleioite analog and there were cross reactive antibodies against um the viral proteins at least GP MP and VP4 meaning that also people that might have been infected with the Muzuki virus um they should have should be crossprotected by this virus. So this is also important when it comes to vaccine development which was not the case here but back then when we start that project we didn't know whether this would be an outbreak or only a single case. So we tested tested our our um capacity and also our um our expertise how how fast we how fast we can generate these kind of recominant viruses. So I hope I could convince you that this is was really or is in general a powerful tool with using this reverse genetic systems with a fulllength genome synthesis but also using this life cycle modeling essays which I haven't gone into uh detail too much today only um for checking the genome ends but this is rather a powerful tool to study this molecular viology even without a BSL 4 so with in with um BSL 2 conditions um for molecular studies for various host interaction. But here as I showed you also for translational approaches because if we are lacking a natural isolate um we are completely relying on the generation and and the expertise of generating these recombinant viruses to have a virus to um go for um animal models u which we also need then of course for vaccine preclinical analysis of vaccine candidates or other theorics or antivirals um because we we need of um a virus specific animal model. And with that um yes and with that I I would like uh to acknowledge all the people. So mostly at least the work that I showed you today was uh from uh during the PhD cases of Isabell and also um heavily involved always the VSSL4 team that we have here in Maruk and all funding partners and for you for your attention and I'm looking forward to your questions. Thank you.