 Today, we have the pleasure of Dr. Philippe Le Mercier, who will tell us about the SARS coronavirus 2 data and viral zone. Philippe, over to you. Okay, thank you. So do you see my screen? Yes. All right, so I will present you the SARS-CoV-2 in viral zone. So viral zone is about putting together the knowledge and textbook in PubMed and whatever source I find together with seconds data and to provide it with easy access for the users. And just I wanted to highlight a little bit of the scale of biology. So in biology, you have kind of three scales. You have the molecular biology, which is what viral zone is about. Which tells about molecules, the cell, the variant replication, everything that is inside the single cells. We have the clinical biology, which takes care of a human body or for veterinary biology for animals. And you have the epidemiology, which will take care of the scale of the world, how the virus gets back and forth and epidemic controls. So these three kind of scales are working together to prevent these infectious diseases. But you will find the implication for antiviral drugs, for example, which can be effective in molecular biology in cells, but not in clinical and so on. So we go back to viral zone. Okay. So in viral zone, we have created a resource dedicated to SARS-CoV-2, which can be accessed in the home page by this banner here. The resource comprises many two parts. The resource developed in viral zone, which are there, and the external resources. We put links to many other places, which provides a lot of useful information. So in the viral zone resource, the fact sheet is a classical fact sheet in viral zone about the beta-coronavirus genus. You have some more information on genome and expression because coronaviruses are very special ways of making messenger RNAs and translating them. You have a link to the proteome, the interactome, the curated interactome, the life cycle of the virus, and some data about drugs tested against COVID-19. So to start with the fact sheet, so in the viral zone, you have always this kind of fact sheet for all kinds of viruses, actually. So SARS-CoV-2 is a beta-coronavirus, which is a genus, is a family of coronaviruses and it's a positive trend RNA virus, which means the genome is composed entirely of RNA, which is positive trend. It means that the genome can be directly translated by riposa. So when the genome enters the cells, it will be translated right away. We have the virium structure here. You can see that we have also the marine hepatitis virus. So something you have to know about the beta coronaviruses and coronaviridia at the world is that in the first place, I mean, before the SARS came out in 2003, it was only pretty much animal viruses which were studied and you have the hallmark of the model organisms was a marine hepatitis virus where a lot of studies made at that time on this virus and it turns to be a virus making hepatitis in mice and it's when in fact a local breed in the lab of mice, it's very difficult to get rid of. It goes all over the place, you have to burn and disinfect everything. It's really a nightmare. It was studied because of that, but it's a little bit different. Symmetric. There's a part which is kind of budding scar there, I will show you a little bit more later. And there's a membrane. So this drawing has been made by using very nice studies of cryoEM and you can find in this paper that I link in Valozone, the study of the vion which suggests that it may be not completely wrong actually because when you look at cryoEM you have different kind of pictures can be a little bit elongated and if you take a cross from this part you have a round one but it's not necessarily the real shape of the vion and it's not also so symmetric as you see in the news on all their course. Now you have a part of the genome of the variant which we will be detailed later. I may just topology of the polyproteins, a little bit of gene expression, a sketch of replication which is entirely cytoplasmic which the virus doesn't need the nucleus to replicate. And on the left you have a lot of informations like links for databases, you can go into NCBI directly to find the attack on the virus taxonomy and linking to the sequences. You have also some dedicated resource, etymology, so different species officially recognize the part of beta coronaviruses. You see you have a lot of bats. You have also human coronaviruses, which is a mild disease, a human virus known for a long time. You have links to refined strains with a sequence proteome genome. You have some part of the host, so you see that it's mostly present in bats for SARS and MERS and human, cattle, pig, rat, moose are occasional also. These are RNA viruses. Those viruses are able to infect many more than one species usually and I expect SARS-CoV-2 to be able to infect many animals. Actually it has been shown that it infects cats very pretty well. It can go back in bat pretty sure as well. So this is not entirely human virus SARS-CoV-2 and this one also it can go back and forth from animals. The true piece means many epithelial cells for all the beta coronaviruses. Some of them are making neurological disease but not SARS-CoV-2 of course. You have different interactions for cell receptors. So you see the both SARS are using a CL2 but the humurine is using another enzyme and other including human coronaviruses, classical human coronaviruses using psilic acids. These are many respiratory humans being SARS, MERS, or even H-CoV-O-C-43. And I will go back to the drug later. So this is the classical fact sheet for Valso. Now in this resource we have added a lot more information. So I will try to start with the coronaviruses life cycle which I draw to present the whole molecular biology of this coronaviruses. So the virus starts over the infection by being outside of the cell. So the viral particle is completely inert. It just contains a genomic RNA and the information but it's pretty much you could make the analogy of a USB key. It's completely inert. It contains a program but it doesn't do anything. And it's one but when the variant enters the cell it will start to get translated to produce protein and to take over the cell. So it's pretty much living inside the cell but not living outside. So the variant contains this corona of spike protein and the spike protein are of two activities are mediating the attachment of the virus to the right cells. So the virus will enter the target cells and also the fusion of the membrane to release the genome into the cytoplasm. The fusion activity is not active when it's synthesized directly inside the cell. And it's the same for all envelope viruses. They got a fusion protein inactivated at the start because if the fusion was active it would make a lot of fusion directly before getting out and would completely prevent the virus from being expelled by exocytosis or whatever means. So the fusion protein is not active and it needs to be the spike needs to be cleaved at a special specific place for the fusion peptide to be active. And this in SARS-CoV-2 this is ensured by TMPR-SS2 which is a host a human protein which would cleave the spike protein outside. It's present in the lung and will activate the fusion protein which then in turn can make it function down. But SARS-CoV-2 have a difference with the SARS-CoV-1 which is has a poly poly arginine tract in the cleavage site which could allow fluorine like proteins to cleave it. So it would allow the variant to be active in many more tissues than respiratory tract for example. This is not yet well defined. So the first step of the virus is an entry into the cell which starts by attachment. So the spike protein binds to EC2 which is an enzyme present at the surface of the some of the lung cells. This is an entry receptor. The binding will trigger by the cell endocytosis of this complex. So when the virus is endocytose it enters into an early endosome which will mature into late endosome. And late endosome have a more acidic pH a lot of things change inside. And this will trigger the fusion activity of the spike. The spike then make fusion between variant and endosome membrane, release the genome into the cytoplasm which is encoded. And then you get the genome. So the genome is about 30 kilobase is long. It's the longest RNA genome for virology actually. And it's just a messenger RNA. Okay. Just a simple messenger RNA which will be translated first into two polyproteins actually. So polyproteins are way of viruses to create a lot of function by just having one opponent translated. So it's pretty efficient. You just translate once and you can have up to 16 proteins there. For this the virus need to include proteins to cleave this polyprotein into individual chains which have each one a different function. So the purpose of the polyprotein is to establish the first infection and to create double membrane vesicles. So these are the two part of the polyproteins which create that. So double membrane vesicles are special feature into the cell induced by the virus which actually is there to hide the replication intermediate. Because the virus is RNA when it replicates it will create a minor strong RNA which will anneal into a double-strand RNA. Okay. And double-strand RNA never happen into modern cells, be it in bacteria or in the ocarotide or whatever others. It's all mark of virus infection. So all these cells have strong anti-viral system which recognize double-strand RNA and will completely shut off any virus producing double-strand RNA. So the virus need to hide itself against these defenses. And with positive standard RNA viruses it's done by creating a lot of vesicular system which will actually hide the double-strand RNA intermediate from the detection system of the cell. So in these vesicles you will have replication of the positive genome and transcription of new messenger which in turn can produce new polyproteins on the cycle's goes on. Okay. So you got amplification of the genome. But also it will create subgenomic RNAs which are RNAs are kind of spliced RNA. We explain later how it works. But these RNA are then encoding for all the structural protein which will allow encapsulation of the genome budding through the Golgi membrane with all the protein and then getting out by exocytosis. Okay. Also the non-structural protein sorry translated from this as G RNAs which somehow disregulate host immune response. Okay. So this is for the life cycle. Now we see a bit more of how the coronaviruses express its genes. So the genome is about 30 kilobases and it's comprised in two main parts. The first part and cause of polyprotein which is 16 actually change. And the second part encodes structural protein and few host modulating protein which are all expressed by subgenomic RNA in the second phase of the infection actually. So the first phase will produce this once and the second phase will produce that once. Okay. So there are different ways of being translated. So when the virus enters the polyprotein is translated because it's the first opening infant there. You see that the protein have two ways to be done and it's different by ribosomal frame shift. So it's a special feature in which the ribosome on a special sequence will stall and sometimes go back or forth one nucleotide. Thereby it will start to translate in a different opening frame which is RF1B there and make this longer polyprotein. So we have the two kind of polyprotein produced the longer been less abundant. So the reason for that is that this RF1B encodes the polymerases and LEKs on a proofreading system everything for replication. And in pretty much all viruses the RNA polymerase are done regulated compared to the other proteins because you need a lot of these proteins to take care of the cell to really under the CFS system. This will produce the best on DNA. So you need a little bit just enough to not produce too much. If you over express these proteins into a cell it will go right away in a different state. So those are found a way using a very simple system to produce less polymerase but everything needs to take care of the cell. So the polyprotein topology is like here. You see it's you have three parts, three chains which are transplant brain. On these three ones we'll produce a double membrane vesicles interact together. So you have the small polyprotein on the biggest one with RTRP. The first part is the host modulation protein interact with host and this P1 is actually shutting off the translation system of the cell by inducing messenger RNA cellular messenger RNA degradation but not the viral one. So by doing so the virus take over the translation system for himself and the cell cannot translate anymore. It also shut off the cell defense because cell defense relies on creating new messenger RNA for anti-viral proteins but if these are not translated of course the cell cannot defend itself. In the second part of the infection the virus will produce subgenomic RNAs to actually translate all these open RNA frames. And this is done by a special feature which looks like splicing but it's called discontinuous transcription. Actually there are sequences which are called transcription regression sequence or TRS which are present pretty much at the start before the structural open RNA frame there. And when the RNA polymerase will replicate the minus trans it can stall on these sequences and directly jump to the leader TRS thereby producing a deletion and it's kind of splicing. So the more replication there are the more these subgenomic RNAs are created. Of course it's always just a possibility for the polymerase but it can go through so it will continue to make the full length genome as well. This smaller negative standard RNA will be replicated into positive strand, double strand RNA and then we produce messengers which will in turn allow translation of all these proteins. So you see in coronaviruses when you look at open RNA frame you can see a lot of open RNA frame presence but only some of them can be really translated by this system. If you don't have a TRS it's not likely that open RNA frame is translated. Also the virus relies presumably on list key scanning to produce sometimes two different peptides from the cell messenger. List key scanning happens when the ribosome doesn't start on the third AUG but we go find another one. So it's not very likely but it happens then you can produce the 7B or 9B proteins. So you have from what we know on SARS there are nine messenger RNAs so eight of them are subgenomics. Could be more it's not really well known so far but for this it's pretty sure. Now we have a part of the proteome. So viral zone is produced by the Swiss PROT group of Swiss Institute of Bioinformatics and our main activity is to annotate for Uniprot consortium the proteins and we did that for SARS but we have a problem is that the Uniprot release rate is about two months right now because it's very complex database a lot of computational have to be made for each release. So the virus we annotated in January will not be available until the 20th of April which is pretty long but it will be the first one of annotation of course a lot of annotations have been added since now. So Uniprot as a reacted by giving a pre-release entries so these are you see last update is 6th April so it can be updated very quickly and independently for using a classical release of the whole database of Uniprot but it gives access to this annotation to everyone so you have in this pre-release SARS-1 which is a CV HSA and SARS-2 there so if you click on that you will have the protein of SARS-2 SARS-1 who have also nine human proteins which are also dated for their interactions with SARS. Other than that it's classical classical Uniprot annotation that you can access to here you have rear with the different reactions and the different features I will not go into details for that. So in Valzone you can directly access by clicking also on this to the same site specific for SARS-CoV-2. You have some notes which gives you insight of the main purpose of these proteins. For example an interesting part is budding so viruses have pretty much two kinds of ways to bed out of the cell it's escort depanade escort is a system which allows the cell to bed out vesicles and many viruses are using that but some viruses like influenza coronavirus or flabby viruses are using a different system so SARS-CoV-2 using that here you see influenza so a mark of this budding and actually it's a budding that involves viral proteins it's just small pores that will by depolarization allow the pinch off of the budding virus and which is actually released from the membrane but you end up with a virus which is not completely symmetric to you kind of scar of budding there which is also a mark of corona viruses. Okay so I will go back to the interactome so the interactome represent there is what we have annotated in Uniprot which means it's an interactome which is created and for which there is a clear function in the interaction and whose interaction have been also experimentally well established okay because you can find much more interaction you have that in the external resources for example there is a large scale interactome bio archive uh Krogan paper which will add about 300 interaction you have virus host net also which um so we which shows a lot of interaction uh from SARS-CoV-600 it's for SARS-1 SARS-2003 but those are not interaction for which it's well established any function or anything so we don't annotate them yet we we wait for more experimental confirmation of this so for that we have just a few of them in light green you have those shown up to happen for SARS-CoV-2 and on one and for the others is for SARS-CoV-1 which by similarity we we think it happens also in the same SARS because the two viruses are very very close actually only on spike protein on some NSA it's different so okay um you can access to the protein also by clicking there in uniprot if you're interested and you have the paper which uh one of the main paper which tells you where come from that annotation from I see we see uh I see okay so this is for the interactome which will be uh dated as we have much more information and now we are going to speak about the drug so I have plugged the drugs on the replication cycle um but before that I just want to highlight things about antivirals in general okay so to treat the viruses we have pretty much vaccines on antiviral drugs so these are the treatment approved so far so you see for human viruses we have 17 viruses for which we have vaccine which are approved on using human okay for antivirals we have only nine viruses so it's lower because it's actually much more difficult to find antivirals to make a vaccine um to make a vaccine takes about two years maybe less if you push things up but uh for antivirals for hepatitis c it was about 20 years of research for HIV like 10 years and it has been making much better right now and uh and it's very difficult the reason that the virus actually is alive inside yourselves and it's using your cell system to replicate to translate and so on so it's quite difficult to find a molecule which specifically will attack the virus each virus is completely different so you need a specific molecule against HIV which will not work against hepatitis c and so on so you get different set of antivirals again each one when for vaccine you can use the same kind of carrier for different vaccines it's not the same for antivirals and also um you have in this scale of allergy you have a lot of ways to make antivirals in the cell culture at this scale it works very well for example i told you that double strong RNA is strong and user of antiviral system if you put double strong RNA on cells it will go into antivirals you will prevent cell from growing but this can not be used in clinical in clinic if you make this it's toxic so you have a lot of ways to have antiviral working in cell culture but will not be effective in clinic you need something to be not toxic in human or organism you want to to cure and also to work at the low dose because you in cell culture you can put a high dose of drugs but it can be the same in all organisms so this being said we go back to the anti-covid drugs so before COVID-19 came out we had no drugs which are really working against against coronaviruses coronaviruses are still much studied for veterinary veterinary diseases but so far we have only random severe which gave some good things in the experimentals with the COVID-19 a whole community of research go wild by finding antivirals and put that on cells and it ends up with a lot of trials which I will describe the main ones there but they are still working in in in cell culture but in clinics it's not yet very well established okay so in anti-covid system you have neutralizing antibodies which can be monoclonal or covalentine plasma this will neutralize the virus we call that neutralizing because antibodies can bite the virus but not prevented from entering the cells you need a specific antibody which is called neutralized to do that okay then you have camostat mesilate which is a drug which inhibits TMP RSS2 so this drug could prevent this activity in the lung so no cleavage of the fusion protein so no fusion activity okay you have also the chloroquine which is a reproposed drug it gets malaria and chloroquine activities may need to focus into the endosome and prevent the maturation of the endosome somehow so the virus cannot then make fusion because if the endosome is not mature the fusion protein cannot activate itself to make the fusion you have also you mean if you mean finovir which is a recent drug against influenza it's not pretty much recognized in Europe and in the US as active against influenza and it will prevent fusion that we tried as well but it does not work so chloroquine is still an investigation you have some drugs which act on the the protease to cleave the polyprotein so if you stop the protease from doing these jobs of course the virus will be completely stopped and there is a polyprotein as well in HIV and these drugs are reproposed drugs from HIV anti-protein okay the problem is that HIV protease is an ASP protease but in the coronavirus it's actual protease which means there are completely different ways of doing the cleavage it's not the same active site at all it's not the same reaction it ends up with the same but it's not the same reaction so these drugs will not likely be effective and actually the first two ones have been shown not to really work so they do their job in cell culture but for many reasons maybe side effects but they are not working to prevent the protease active of coronaviruses seems not to be happening now you have at the level of transcription replication you have nucleotide analogue which can stop the polymerase for walking so we have remden severe which drug came from from cancer research seems to be very active in in cell culture but the drug is not yet ready to be used in clinic seems to be so there are new new formula of remden severe coming out that could be promising but it's not yet there you have a favipavir which is an antiviral against influenza which was actually tested in influenza not my good results was tested against Ebola who's no good results and now is tested against the coronaviruses as well and at the end you have antibodies Tocillizumab which are against IL-6 which have some effectiveness to treat the disease because COVID-19 created a dysregulated host immune response which can leave you to death by own immune system so this protein this antibody will prevent IL-6 which is inflammatory cytokine to act and will make you so it's not really antiviral it's anti-COVID okay sister there are many others like Aparin which in some case can be effective also so here you have the main drugs are listed there you can link to Wikipedia which would have some general insight of the drug you also link to Kembo which gives you a lot of details like the max phase of use clinical research and the formula everything you need to know and a reference as well which gives you a paper to relate to from this antiviral here and external results have put a treatment of vaccine vaccine tracker which is made by Mika's sister as to keep up to date so it shows you what's happening against COVID-19 so you see here you have 79 vaccine vaccines tested you have 18 antivirals antibodies you can access to more details there then you get a list these are all the antibodies up to antivirals so this is Favipavir for example so they tell you a lot of details the clinical trials use some published results and so on so it's interesting to get these details okay at the end of the day antivirals are not pretty much really it takes really years and years to find good antiviral good formulation of molecules which are not toxic which are still working in a human body and I personally expect vaccine to be the first one to come out many after antivirals but I think we will have to wait for the vaccine to be there to solve the COVID-19 of course antivirals race is on but don't expect to find antiviral like that in two or three months it's going to be hard in this site an internet book of clinical care you have a lot of data on clinical aspects of COVID and on antiviral therapies okay a very nice picture is there which shows up at the stage of illness so you see in the time course you have kind of three stages of COVID-19 with host inflammatory response that's where on TIL-6 therapy can be or TIL-2 can be effective to prevent the death actually so this there is an inflammation phase the phase in which you have something out of breath so they give you symptoms, signs and the potential therapies there okay well actually this site provides a lot of information on clinic with for example you see how you can look at the disease directly so okay this is it for resource in valve zone now for external resources you have the unipot which has what we showed up there is a very nice video made by a pretty close singer University of California which explained the molecular biology of coronaviruses you have a site for epidemiology very well done which is made by people part of Swiss Institute of Bioinformatics it's next train so they show you up all the the sequence on a phylogenetic three of the SARS-CoV-2 show you the geography as well and the diversity I mean the different mutation you can find in these in these viruses here you have the opening frame of the virus you see it's not much diversity actually for RNA virus it's not like influenza mutating all over the place the reason is that there is a proofreading activity associated with the polymerase it's only the coronaviruses of that all the RNA virus are producing a lot of mutants but coronaviruses are the only one which have a proofreading proofreading means that when the polymers make a mistake it can be corrected okay it happens in all the polymers of the cell but not in classical viruses coronaviruses have that okay you have a site for PDB structures so PDB make a special resource for COVID-19 in which you can access to all the structure they have already and you see pretty much a lot of 110 structure for SARS-CoV-2 she's really amazing so you can access this by this link from PDB Swiss model is also part of Swiss Institute of Bioinformatics and it offers a special site for SARS-CoV-2 in which you have models of all the other proteins which are not crystallized yet so for example you have msp2 papain like potatoes and so on so it can be also very interesting to get this is a world matter so it's more epidemiology resource which gives you in all the words all the number of cases you see well about two million cases diagnosed number of deaths also which are diagnosed as deaths produced by SARS-CoV-2 and you have details by all countries so it's live data so you can see from yesterday how many deaths happen in the USA and all the countries how many in the world 5000 deaths it's really crazy and you can look at the country with all the data and you can see it's very new case in Switzerland you see it's really going down which is pretty nice to see active case going down as well but well the deaths are still stable it's normal because there's a lag between the deaths and actually the onset of this is okay and cellosaurus is a website which also from cibus free sensitive by informatics it tells about uh cell culture and every data on cell culture and it gives a site for SARS-CoV-2 cells cell lines that have been used to to grow and those that are are not suitable to grow okay so it gives you a lot of details if you want to grow SARS-CoV-2 in whatever cell system you can have a lot of information there and then medical data COVID-19 which I already presented previously okay I think I've went to everything there so I will stop and uh if you have any question thank you