 Thank you to to will for extending the invitation. I have to say there's a degree of irony in the invitation and that I'm probably the one in this virtual room who knows the least about genomics and bioinformatics, but and indeed a very heavy user. So I took this course. I don't want to say how many years ago certainly before the pandemic. I realized how much I didn't know. And then, you know, reached out and started collaborating with a number of you a little bit before the pandemic but definitely a lot during the pandemic. So, always like to start with a little bit of of gratitude and I want to to call out the people that have really been doing all the work over the last few years. So, so many people. This is just a group in in our lab. You know, I have to say, if it wasn't for these individuals, I don't know where my sanity would be. I've just been so impressed. And it gives me great hope in terms of the future, both in terms of technical leadership, but also just in terms of our ability as a country to respond to the threats in future. So these are a few people and, you know, I'm just maybe I'll put some of them on the spot and just ask those that are in this meeting to put their cameras on if they're not already just to say hello so people know who they are. John is holding this bat so you don't see his face but but he's in this call as well so yeah very grateful to all of these individuals and all the hard work they've done. I'm going to put some other people on the spot because I'm incredibly grateful to them. These are the people who came through. I think in the darkest of times. I'd say probably Steve share was the first person to knock on our door and say do you need money do you need help, you know, completely unsolicited. You know with with funds from the McLaughlin Center, Andrew MacArthur we've been working with you a little bit already but this is when you really swung into action same thing you know how can, how can we actually help and I don't. I'm not an endorsing Lumina in any way whatsoever from a commercial perspective but I have to say on a personal level. Peter graph and him making sure that we had instruments and reagents in place in fact he he was already working really hard late 2019 to get us a mini seek on premise and that made a huge, huge difference this is before the pandemic. But then ensure that things were up and running and that we had reagents in hand it was really a genuine effort on on a luminous part to respond to the pandemic early. I think it was during the darkest days of the VOC so many very early morning late night. Slack messages, usually when Finn was knocking it was sort of bad news. Come through our pipeline so we're very much at the of the cold face at the clinical field in the lab. We generate data but we really can't transform it into health intelligence without these people and many others that I'll mention as we go along. So this talk will sort of be a mix of things that we've been involved in it's not intended to cover everything. Obviously it's focused on on the pandemic just because that's the perspective I'm coming from but there was so much more done during the pandemic with genomics that we weren't necessarily directly involved in and I just want to mention that you know for example wastewater surveillance was huge and there was some incredible modeling that was done with with genomic data by by other groups so I'll just be focusing more on the work that we've been directly involved in. And it is Friday afternoon I know that everyone has had a really really intense week so I'm going to try and keep it as light as you can when you're talking about high consequence pathogens. The structure has been before I guess I guess it would be would be kind of hard to make it interactive over the course of the talk but maybe I'll try and leave time at the end to have some discussion. I have a few slides on highly pathogenic avian influenza virus just as a heads up but if we need to drop those so we could have 10 minutes of conversation I will. Again starting from my own personal comfort zone. It's funny saying that about someone with SARS to but on the clinical side sonny book had the first case of of SARS coronavirus to which you might remember it seems like in eons ago we used to call it novel coronavirus or and and this individual on calling them patient one because when you call them patient zero it implies that they're responsible for the rest of the outbreak that's that's not the case whatsoever this individual may have transmitted to one other person but certainly wasn't the index case for for the region, let alone the country. And we had had a few cases coming in through sonny book that were what we call people are patients under investigation we use that term, not just for. Not just coronavirus to but also other high consequence pathogen so Ebola virus and most coronavirus and I have to say we were. I would say 100% prepared but we were certainly better prepared for this case because we had had so many POS for Ebola and for Mars, none of which tested positive but because we were had been preparing over the previous years for that possibility and you know when pathogen X literally walked through the door we at least were much better prepared than we had been when the first SARS came through the doors of sonny brook back in the early 2000s. We had with this individual case and I think you can see my can you see my pointer if I move it here or here. Maybe not. Okay, good. So, it's a split screen so I never know which one you can you can actually see it on so I'll just point to both but this individual that what we call the pretest probability was very high we knew that they had flown from from Wuhan dropped their bags off at home and pretty straight to sunny brook so pretest probability was high based on epidemiological exposure and then also based on clinical presentation including this chest X right here with bilateral infiltrates which was pretty classic from what we were classic classic for something we knew about for a month or two had been described previously I should say. This individual was was symptomatic I think we've since learned that you know the vast majority of individuals are either asymptomatic or posse symptomatic but this this individual definitely had some clear symptoms he had him he was coughing up bloody had fever and was admitted to hospital. We did some serial swabs on him and as you can see the CT value progressively increased which now we know is pretty, pretty classic but early on this was quite even an end of one was valuable information at least at least to us. And you can see that we were collecting samples from the big turbine it from from the nasopharynx. And he was admitted for just over a week and fortunately he he did not require ICU care and and was discharged home. At the time, we managed these samples like we would have an Ebola case so samples were not tested on premise they were sent to public health Ontario and then and then to the national microbiology lab for for confirmation. So then we didn't have any cases for several weeks and then we started seeing returning travelers from from Iran and and these were the samples that we use to isolate source coronavirus to so don't worry we'll get to the genomics but just a little little virology background if you'll indulge me. We do use tissue culture for this so this all has to be done in containment level three and the reason for that obviously this is a high consequence pathogen and and we were able to get in there because we had on our permit back to get the viruses because we've been doing some field work prior to the pandemic, and all credit to Aaron Jay Banerjee for for coming in and working with our group and Rob Kozak to take these patient samples and put them on to zero E6 cells and you can see the cytopathic effect here, you know, compared to, if I point over here do you see that. Okay, so so these are what normal cells would look like and then when their cytopathic effect that's what you see with the different specimens here and then we pass it again. And same thing we capitulated and of course we do PCR to confirm but there's nothing as satisfying as having a direct look at these viruses so it's old school but really kind of nice to use electron. And you can see here and you can see the sort of the corona or the spike that's on the surface of the cells as they bud. So these are actually from the cell cultures that that Aaron Jay produced. So not long after that and it's kind of funny I don't know Andrew this brings a smile to your face and I think Jay Lisa's on here to thinking back to how far we've come, you know, now the Arctic protocol is pretty ubiquitous it's been updated in multiple multiple versions but when you think back I mean this paper was was published in August of 2020 so you can imagine the work was done probably around March, March, February, March, April of 2020 we didn't really know what the best approach would be to hold genome sequencing of this virus so, you know, very fortunately we were able to collaborate with Julian his cockpit at Liverpool and tried a couple of different approaches, random heximers or, or bait capture. And just comparing the older, older, it was a month older sample from the returning traveler from Iran to to more recent returning travelers from Iran and seeing some pretty significant differences so in the main reason I'm putting this slide and also the next slide up is just to show how quickly people started using available technology and how a lot of that work became building blocks, if not for the actual technology that was used, and the protocols that were used longer than we continue to use, but the collaborations also that came together at that time. So this, this was a tool that was developed by Bo Wang and Hassan Man at the Vector Institute again in collaboration with Andrew MacArthur and ourselves and, and others to start like there were so few genomes available. Unfortunately, it was a very different situation with SARS to relative to the first SARS, where genomes were shared I know there's controversy around that we won't discuss the controversy around that but I don't think. I don't think there's any doubt that there was much more rapid data sharing than there had been with the previous SARS with the previous SARS I think the etiologic agent was under question for a much, much longer time for while people thought it was clementophila, which is a any typical bacteria that can cause respiratory infections and in this case, again, technology having having advanced significantly made things much more rapid but I think also there was a significant political will to share sooner rather again, not perfect and people could criticize how this was done but certainly an improvement relative to the previous Sarbeco event. So, I won't go into all the details about how else. Our lab has been using SARS Coronavirus to sequencing and level three but I do want to highlight something that Andrew Benetti has been doing with or Benette sorry has been doing with the Omores and and Jared Simpson. We rely very, very heavily on whole genome sequencing in containment level three because we very quickly learned that if you put this virus on to cells. It does its own thing. What comes out the other end could potentially not look like what came in on the patient samples what I mean specifically. There is that you may lose a poly basic cleavage site for example which is a key virulence factor for source coronavirus to, and then you can no longer really use that virus for any experiments either in vitro or in vivo. Unfortunately we caught on to this but unfortunately I don't think everybody did from the beginning and I think that there were probably a number of experiments done and papers published using viruses that really weren't wild type viruses they were somewhat lab because just one passage and that that can happen so well you know not showing any of that data but the data that I'm showing now is really an experiment looking at a different question so we know that variants of concern have emerged one after the other. And people have used an approach. Doing in vitro competition essays, and they do them in human nasal epithelial cells. So one round of a replication just to mitigate any gain of function and the other thing that we do to mitigate any gain of function is only use viruses that would have overlapped epidemiologically ideally. So we're not trying to do to do competition essays for example between that coronaviruses and SARS coronavirus to that that would not be the risk doing that would be too high. So, the idea here is to really combine different ratios of different VOCs in the same cell culture system and then determine which one wins out. So usually nine to one ratio. So we see that, for example, quite readily. Omicron be a one here. Just been here we go out competes D 614 G fairly quickly just as one potential example. So lots of D 614 G in the input, you know, one part Omicron be a one, and then by day three be a one has completely taken over and again this is just an in vitro cell system. It's quite translational translational in the sense that it's human nasal epithelial cells that are being used. So that's just another way that we've depended a lot on genomics within the bio containment lab and doing our in vitro and in vivo work and we continue to do so not going to show the in vivo work with with hamster models but very similar so we are trying to sequence now is much, much lower outputs from these experiments just to make sure that what went in was correct. And also to potentially ask some really interesting questions around what's coming out. So now I'm going to shift away from from some of the high containment work and talk a little bit more about some of the work that we did in the clinical setting. So this is actually led by Allison McGeer through the T. I. B. D. and it's a bit of a misnomer the Toronto invasive bacterial diseases network even before the pandemic they were doing a lot of work on influenza and other important respiratory respiratory pathogens so it was only natural to also take on SARS coronavirus to but it was it was great to have this cohort of patients that were identified quite early. So we're going to take on the virus to or or COVID and start collecting samples so even though that sounds like a very straightforward activity, it's actually pretty critical in terms of follow on research. And it's also not without a heavy lift, especially if you're collecting both acute and convalescent serum, and we're also collecting samples from respiratory tracts of patients and also the environment and a lot of those samples and the data from those samples got shared over the course of the pandemic. So I'll just share some of the results from from this particular study that John Katwa was involved in leaving the analysis. We collected a total of 848 samples from 78 in patients again this is all very done quite early prior to the VOC is emerging. And we were able to also collect samples from bedrails phones chairs tables and patient washrooms and we also collected bio aerosols. Just to point out only three out of 146 bio aerosols actually tested positive. The CT values of course not surprisingly were lower for NP swabs. So these are the patient swabs relative to what you would find in the air. And in addition, you can see that the closer to the patient's head the lower the CT value in many ways. Which really is an indication of how heavily the lab is sorry the patient's room is contaminated relative to where the patient is actually emitting virus. In terms of looking at what the determinants for shedding into the environment were. There were a number of different variables that were used. And I won't go through all of them here but I do want to pull out the ones that were found to be to be relevant and I don't think they would be of any a significant surprise to individuals. Those who are hypoxic patient who had an NP that was had a lower CT value patients who had a Charleston score that reflected a moderate to severe number of comorbidities so these are other illnesses that they had. And then onset dates so less than seven days were all associated with contamination environment. Of course we didn't really want to stop there we wanted to understand while you know how are the genomes actually related and you can see these really long branches because you know the data were pretty scanner kind of pulling out sequences from some pretty scrappy bits of RNA and not surprisingly both the completeness of the genome and the and the depth were very dependent on the CT value. At the time this was kind of enlightening now I think it's pretty common knowledge, but again it's all these small pieces of information which until you go looking. And it does take effort and time to do that we kind of take for granted. Now, but they become very important when you're trying to prioritize limited resources remember these were in the days where you know people were 3D printing swabs in their garages, because the supply was so limited so we have to be really careful about everything that we did. And again just indulging myself as a virologist to indicate which samples were actually positive by culture so when we say CPE in this case we mean cytopathic effect and not surprisingly. It was the samples with lower CT thresholds and samples that were collected earlier in the course of illness both for the patient here, and also from the environment so. This was also quite interesting even though we didn't isolate any infectious virus from the air. Thank goodness others have since using different methods. But we certainly were able to recover infectious virus from the environment which you know really underscored the importance of decontaminating surfaces. So I'm going to switch gears again and really move to move away from human health, but still staying with stars coronavirus to hence the title of the talk follow that virus because that's essentially what we did you know we started in the lab. The virus on hand didn't work there and then also followed it through some of the acute care settings that we were working in and then because of what was happening more broadly with stars coronavirus to ecology and epidemiology we started looking at animals. We know that pandemic pathogens are most likely going to circulate in animals before they cause widespread human disease the only pandemic agent that the WHO has acknowledged as a pandemic agent that isn't as zoonotic as cholera. So really everything else has circulated widely in animals prior to spill over into humans and influenza is a obvious example of that and also now stars coronavirus to and I just want to highlight the Canadian wildlife health cooperative here because the samples come largely from them and from the Ministry of Natural Resources as well. So really, really important partners in this work. And if anyone's interested in these data they are being collated under the CAS dashboard. That's the Canadian animal health surveillance system dashboard and we're gently encouraging them to figure out a way to pull in genomes into this so it is somewhat limited it doesn't include all the samples that have tested negative you're just seeing positives. And of course, the testing is batched and sporadic so you don't, you're not really necessarily getting a continuous flow of information here. But I think that this data set could actually be enriched significantly if the genomes from from these positive animals, where when they are available. Obviously not sequences sequencing isn't possible on all of them because of high CT values but I think that would really enrich this data set. Trying to decide which animals to actually screen for stars coronavirus to could be quite a challenge not for lack of options I mean fairly early in the pandemic. You may recall zoo animals were being reported as being positive companion animals. And some animals that were being experimentally infected so that gave us an indication so parents as an example, we're being used as a as a model and that gave an indication that musta lives in general could potentially be compatible reservoirs for stars coronavirus to people were doing some really important molecular simulation and modeling so again another place where genomics played a significant role I'll show a couple of slides. And then obviously in vitro screening and indigo models are key and then, and then doing surveillance both from a passive perspective but also from an active perspective. So really early on one of the first papers was this PNAS paper that came out in 2020 that just did some computational modeling computational molecular modeling looking at receptor binding with between stars we knew what the spike sequence was and we knew what the receptor binding domain looked like. But also combining that information with other genomic information from the host so looking at the East to receptors because these two is the host cell receptor for stars coronavirus to comparing all of those and they didn't directly look at binding affinity but but modeled binding affinity. And, you know, the ones I'm showing here the ones that were considered moderate moderate to high ones that were had high likelihood of binding I'm not even showing because those were non human primates and I don't think there was any surprise there. I'm doing this because there were a few surprises and I think that that's why people started looking for example at white tailed deer because of the similarity between East to in the end here in humans and and how well it could potentially bind source coronavirus to and then in the richness of time is more systems like over the last few years people accumulated these systems that took a more cellular perspective and this is a heap of work these are multiple multiple cell lines across both organ systems and species that were essentially tested with pseudoviruses so pseudoviruses are not full viruses they just express the spike protein and in this case for this set of experiments with SARS to the first SARS and Merz so this obviously isn't work that our lab has done but just showing it how just showing how important again genomics is because they could make these pseudoviruses based on the published genomes that were available. So, in fact that's a very important tool whenever a new variant emerges to be able to make these pseudoviruses because chances are you'll be able to make them much more quickly and taste test them much more quickly in these binding assays or neutralization assays then then in the time that it would take to actually isolate characterize and share the virus. So again, you know, don't genomics giving investigators a leg up in terms of understanding the implications of any new emerging variants. So going back to the deer. Having a sort of zooming in a little bit on the interaction here between the receptor binding domain of SARS Coronavirus to and, and the ace to a white tail deer you can see up here at the top. These are really key binding residues and they almost all like four out of five match and then across the entire domain. 18 out of 20 actually match. Yeah, 18 out of 20 actually match. So, you know, it wasn't a huge surprise knowing some of the this will call it pre analytical information really started to pay off because people did surveillance and deer as a result and the reason that's important is because of when you don't get sick right so people are not finding die offs of deer and looking for SARS to unless you're actively looking for so this is what we call active surveillance based on some of this preliminary biological and and modeling like a modeling data, we probably wouldn't have known that you're getting SARS Coronavirus to because again they don't they don't really appear to become unwell. So over time groups like Suresh's kushu putis group were finding more and more. Zero positive deer and also deer that were testing positive by by swab of the upper upper airway or retroferingial lymph nodes, and it's really important to get those types of samples. And the reason for that is because you need to be able to sequence the virus to really start to understand the genomic epidemiology and ecology of SARS Coronavirus to in deer and you know this has really been a significant finding in the sense that we now know that deer can carry some of the variants of concern forward over time in the absence of ongoing circulation in humans. So, initially, when people were starting to identify SARS Coronavirus to in deer, it appeared to be multiple spillovers from whatever was circulating in the community at that time. And you know, based on work from Pennsylvania and also New York that alpha and Delta lineages can still persist. Despite the fact that. So these are the cases in humans, but now you see VOC is persisting long after they, I won't say are extinguished we never know if they're truly extinguished but let's say that that broad circulation has stopped. And we still see it persisting in deer. And this is could potentially be pretty important for our vaccine strategies because think about how we're not sure that we even know what we're going to do exactly in the coming years but we certainly have changed over the last year in the sense that we've started adding Omicron to so we now have bivalent vaccines so different Omicron lineages to to the vaccine so if we're doing this based on surveillance in humans as we should. We always need to bear in mind the possibility of an older variant of concern or an older sublinear spilling over from what may now be a new animal reservoir. And I'll just go through this very quickly because people often ask whether deer get sick or not and certainly in the wild it hasn't been very well. It's not as well described to be unwell but even when you inoculate them directly. A slight temperature change but otherwise clinically look well but there is a certain degree of pathology in the upper airway but also in the lungs so some mucosal hemorrhaging cellular infiltrates for example so it is doing, it is pathogenic in in deer, but not necessarily severe or fatal. They do seem to shed for maybe actually I'll go up here they do seem to shed for at least five days but once by day 20 there's no more virus and there certainly seems to be more abundant shedding from the upper airway relative to the lower airway and certainly nothing really much at all. And we also know now based on these experiments that deer can actually transmit to each other. So certainly opportunities for the virus to transmit from one deer to another. And given that chain of transmission, the possibility of divergent viruses eventually emerging and that's exactly what we saw with our work so now to focus a little bit more on on Canada and this is really been a great project that that been McGuire here has been a very, very, very closely involved in and it's been a very multidisciplinary project I know there are a lot of acronyms there. And there are a lot of different partners involved in this but but it's worth highlighting how important these collaborations are so it all starts, you know, with the wildlife biologists and their teams in the field you know these data that magically appear someone's out there getting, you know, eaten up by mosquitoes and freezing and working in rain and sleep to try and collect these samples in the field whether they're from from bats or from from dear. We did leverage existing wildlife surveillance programs. So, in this case for deer it was a chronic wasting disease we leverage the existing program for that so that was incredibly helpful. And material at hunting stations were collected in November, both in Quebec and Ontario a total of four different hunting stations and sent to us. Here John Katwa was the lead here at Sunnybrook on this work and then and then from McGuire was the lead on all the bioinformatics and computational biology so we did the initial detection the sequencing. And then the sequence sharing and the virus isolation here and because, because of what we found, we sent original samples over to Brad Pickering and Oliver Lung, who are great collaborators at the National Center for Foreign Animal Disease, who replicated everything, which was, which was very reassuring and another key reason for sending them the material is we really have no, and I don't know if you remember this like the whole reporting cascade with humans, very similar. So you know, when researchers find things that are have public health implications. If there's no system in place for communicating those results and passing them on and sharing them with with public health agencies it becomes problematic and that got resolved on the human side but with the animal side again, you know we found this and so that was the other reason we learned our lesson and immediately sent materials straight to NCFAD so that they, once they confirm they could report to the World Organization of animal health. So, initially, it was pretty routine, pretty quiet, you know, over 20 species were sampled across two provinces there were 4000 samples that were screened not just from no deer actually kind of came later we did raccoon skunks quite a Noah's Ark. And again, John Kotwell was was leading all of this with with collaborators from the University of Guelph and the Ministry of Natural Resources. Lots of high prevalence areas were where we focused and also there was a lot of convenience sampling for rabies programs etc. But when we started looking at deer that's when we started getting some positive hits. So, just coincidentally we started with the Quebec samples and started finding some positives that were geographically restricted to the Astrid region so samples from both the Astrid region and and further west of Montreal were obtained but the ones west of Montreal were all negative So we started seeing positive samples in the from the Astrid region both in terms of serology but also in terms of PCR positive from from nasal pharyngeal sorry not nasal pharyngeal nasal swabs and also retro pharyngeal lymph nodes. The positive results were probably not that surprising when they were sequenced, and this is work that all of her lead. They were Delta VOC so that was not that surprising so this was November of 2021 so Delta had just emerged, and we're just about to start seeing Omicron so these deer theoretically would have been exposed to individuals who were infected in the region and so the fact that it was a Delta VOC was not all that surprising based on what was being described oops sorry in the US. However, what was a bit surprising was that the most, most closely related virus was not from Quebec and Quebec was doing a fair amount of sequencing or at that time. It was not lack of for lack of data but it most closely resemble resembled virus that had been sequenced from from humans just south of the border in Vermont. There were a couple of snips there that were sort of interesting so wasn't exactly well tied Delta but really nothing to to divergent. Where things got very, very interesting was when we started looking at viruses from deer from Ontario. We thought okay we'll probably see the same thing again here and it was a quite a different story. And I know I've kind of scribbled all over all over this but it's really to underscore how many different people have already highlighted a lot of the people who were involved. But once we started finding things we had to pull in other individuals by sending Cote Alison McGeer to do some neutralization assays at Andres Finzi and many others. And also, Jen Guthrie was really key in terms of pulling putting two and two together along with pin so I'll get into the details of what what was actually found in Ontario so. Again, two sites were sampled 300 deer. But really all the positives were from geographically restricted to Southwestern Ontario. Again nasal swabs and retroferential lymph nodes were submitted. And we know that they were all arenas P negative so not contaminated by human positives but we had about 17 animals that were that ended up testing positive overall so again, maybe not that different from Quebec so far, but it was when the sequence actually been maybe you present this kind of awkward presenting your analysis right here. No, it's all yours. You're the one that as much as Dr. Maburek is good at talking about the collaboration all the people involved. It was her that brought everyone together and actually led all of this. We're not going to let her get away with a giving credit to just everyone else. You can just take the hard questions afterwards. So anyway, this was where this was one of those, you know, slack messages orphans. I think something's going on here and at the same time I think Jen Guthrie was looking at this very interesting case. And we were actually I think very lucky that she picked that up before someone on Twitter did because there was a very, very divergent virus that looked completely different from anything that had emerged up until this time this is this is a little obviously Omicron has diverged significantly since this but you can see that it's not really related in fact it's descended from a B1 lineage. So that was the first, I think indication that something quite unusual was going on and then the fact that there was actually a human case that cluster very closely with this was the second indication that that something was going on. It's really interesting in that, you know, the mutations were peppered across the genome. So unlike Omicron they weren't really crowded into a certain, you know, into the spike for example. And fortunately, around this time and this really was very, very lucky. You know, the cancogen had built a lot of capacity across Canada for sequencing. And this is when Omicron was starting to emerge. So we were sequencing. I can't never say 100% but as close to 100% is technically feasible. I think that was sort of fading away and Omicron was starting to starting to be introduced and and really take over the human epidemiology so, you know, fortunately, in this abundance of sequencing, we feel fairly confident, saying that there was no transmission among humans, there was just this one individual. And again, this is where maybe I will go back to this slide I want to point out how having people like Heather McClinchie she's actually she's actually a vet who works in the office of the chief medical office of health. So she's really embedded having a vet embedded in in a public in a provincial public health office was incredibly helpful so she was able to look into this case and even though we can't discuss any details we can say that this individual geotemporally was associated with with these this cluster and certainly had exposure to deer. So we're fairly also confident saying that that there was dear to human transmission in addition to being able to say that there was no forward transmission among humans, we would not be able to say any of this. There's been that capacity built across Ontario and also at the national level for for sequencing and if, if that implementation hadn't happened and been actually operational by this stage. So really just to think we've got. Yeah, so I think we have a little bit of time just to talk about some of these mutations as I mentioned before, you know, they're sort of peppered across the genome. So this really interesting piece here is that, you know, again, not most closely related to human cases in Ontario, but rather most closely related to human cases in Michigan, which in turn, very closely related to an outbreak in mink. So this made things very, very interesting. Unfortunately, because of massive gaps in sampling, especially among, among wildlife, we really can't draw a direct line between a line of transmission between, you know, we don't know if it went from human. But then if it went from mink to human to deer, mink to deer, to human, you know, a number of different possibilities. In terms of what was actually happening, for example, in the, in the spike, there are only six changes and I think actually in the next slide, I have some of them highlighted. Many of these mutations were previously found in deer. But some of them weren't. And there's some really interesting things, including, including an insertion, for example, in ORF 1A that hopefully we can follow up on from a virology perspective, that that we'd like to work out. But yeah, if you have a closer look at the, at the spike, really not a lot, not a lot of differences we're, we're seeing here relative to, for example, how much that you would see with omicron. There does seem to be a degree of neutral selection. So, you know, based on on Finn's analysis, you know, the size of the genome and what you would anticipate just over time, having changed did change. So again, we're not really seeing a massive massive amount of immunological pressure here. And again, we know so so little about deer immunity, all we could really say is this is probably completely susceptible species except, of course, we also don't know what other coronaviruses circulate in deer. So whether there's any predisposing immunity from that perspective and how much that drives selection of, drove selection of this virus really, really can't say at this stage. So as I said, we don't know in terms of potential scenarios which direction things really went in. So first, when you find a new virus, you have to do a risk assessment, we really wanted to understand, you know, is this a variant of concern is this just a variant of interest. Is this nothing. So, you know, of course, you remember from the VOC is the first thing people do is look at virus neutralization by serum from people who've been infected or, or vaccinated and this is where Alison McGear, Marceline Coté and Andrews Finzi came in and again. The sequence made it possible even before, or as the virus was being isolated for Marceline to make pseudo viruses and express these and do neutralization assays to see whether or not it was escaping in immunity and fortunately no. So in Brown here is Omicron which doesn't neutralize as well as you can see and that's to be expected whether you've had two doses or three doses of vaccine or been previously infected. But, but the dear viruses seem to be on par with the ancestral virus so that was reassuring. And then Juan Corridor led the work in in cl3 isolating the virus with with other folks from the lab of course. And then we had two systems from theomores and high boozing we were able to look at viral replication both in human nasal epithelial cells these are the same type of cells that I mentioned before that Andrew had used and also lung organoids human lung organoids so again this is a virus now that's coming from dear, and we all want to understand whether there's any, you know, enhanced viral fitness or replication in human systems and fortunately it does not appear to be the case it's pretty much on par with the other viruses for a number of different cell systems and if anything really doesn't do a whole heap of damage in lung organoid so so far. It's still a variant of interest to us, but it's certainly not nothing no red flags to say that it's a variant of concern. I'm going to change gears again and this is john caught was work. And he's also here so you can answer all the hard questions that come from this. And actually be kind of interested to hear the groups, if anyone in the group has some suggestions as to how to move forward with some of this work. It would be very interesting to hear this so this is work that's done with Christina Davey she's a wildlife biologist at Carlton University and we started working with her when she was still the Ministry of Natural Resources. And, you know, we, we went out and sampled with her in eastern Ontario so these are, these are roots that are located closer to the Ottawa region and sampled for different types of that some obviously more abundant than others. So little brown bats and big brown bats representing the majority of the animals that were sampled. And thanks to john's work, we were able to, and others of course we were able to detect endemic bat alpha coronaviruses. So many many thanks to Winfield was also here during this, during this course who did a lot of the analysis around this now for this be used. We used a pancoronavirus PCR so that obviously limits what kind of sequence you're going to get out because it's focused on the polymerase gene so that's what you get out of polymerase sequence and part of the reason that we use that approach is because it's so well conserved so then you get out sequence that isn't really very different from, from what has already been been published but I do think that these are probably true. Truly, what has been detected before by people like Vikram Misra here in in Canada so he found what we call a milu. Alpha coronaviruses so that's for my own specific so. So he detected this virus, a few years ago actually now from bats, he's based in Saskatchewan, but these bats were from Manitoba. And also the other alpha coronaviruses that was detected has also been previously described so just to reassure everybody this something that is necessarily new or to be concerned about an alpha coronaviruses are very different from the beta coronaviruses that merbeco and sarbeco viruses reside in so again just to reassure people this is nothing to be alarmed about but just something obviously to pursue I think if you look in wildlife will find coronaviruses I mean such a divergent family of viruses and important for us to understand them better but also important for us to understand that, you know, not every coronavirus is necessarily going to cause an international outbreak or pandemic. So, looking at where they reside relative to other ones quite closely related to Rocky Mountain at least based on the small bit of sequence that we have available at the moment and I think you know this is really where he's been focusing his efforts in terms of building out the rest of the genome and he's certainly made some progress in that regard but of course we're all interested in that key part of the genome which is the spike. The gene that encodes spikes so that we could try to understand what the putative host cell receptor is so that in my mind would really be the next step in terms of understanding this for this virus. And, and I think that's going to be the focus of our work so if anyone's got any cool tools in their back pocket that they'd like to talk about I think we actually do have time now.