 All right, good morning, everyone. We're going to try to get started more or less right on time. I am Nate Mosier, Professor in Agricultural and Biological Engineering and Interim Department Head of Agricultural and Biological Engineering. It is my great pleasure to welcome our distinguished guest and speaker this morning, Joan Rose. Professor Rose is the Homer Nolan Chair in Water Research in the Departments of Fisheries and Wildlife, Crop and Soil Sciences at Michigan State University. She's had an incredibly interesting and varied career, and we'll get to hear some about her work here this morning. So with that, please help me in welcoming Professor Rose. Thanks very much. It's a pleasure to be here. I drove over and I missed the storm. So that was great. Had a nice leisurely drive without any ice on the road, so it's been as it has been in Michigan, that's been kind of a crazy winter here in the Midwest. But it felt kind of warm yesterday walking around here where it almost got 40 degrees. I enjoy coming to Purdue. I work with Andy Welton here on a project, and so I've come to the campus several times, and I always enjoy coming to this campus and seeing all the students and all the new things that are happening, so it's great to be back here. I thought I would talk about some of the work we're doing on viruses and give you an introduction to why I think viruses are important. So you may remember from your biology class that viruses are obligate parasitic biological entities. They replicate only inside a host cell, and they produce nanostructured virons. We call them virons, the particles. They have a very simple structure. So viruses are made up of a nucleic acid, either an RNA or a DNA. They don't have both. And they're surrounded by a protein capsid. And you can only see them under the electron microscope. And so I say that our environmental virologists were the first ones to study the nanoparticles before nano was good, so these bio-nanoparticles. Viruses are everywhere, and they're spread from people, plants, air, water, all kinds of things. They infect all living entities. In fact, the discovery of viruses really, we knew that they were in bacteria, the bacteriophage we call them. So these are viruses that infect bacteria. But we didn't actually recognize that viruses were in protozoa, amoeba, algae, until later on. We knew they were invertebrates and invertebrates in the animal kingdom. We knew there were viruses of plants early on, but recognizing that viruses then infect all these other entities. I think it's interesting. We have about 150 ungrowing known viruses that are spread through water, what we call public health enteric viruses. These are the viruses that are spread through the fecal oral route. They are excreted in the feces. The next individual gets them through oral ingestion. And we used to think, of course, that these viruses were very ho-specific, and some tend to be so. Only human viruses affect human viruses. But we all know about bird flu. Rabies was our classical virus that jumped from animals into humans. And now we're finding out there's these other types of viruses that are jumping into humans, which is quite odd, like this algal virus that was found to show up in human brain, slowing brain activity. So viruses are very transmissible agents. They used to be called. We knew about them, really, from their disease, like hepatitis A and jaundice. So we knew about water-borne jaundice, food-borne jaundice, this disease that got spread around. They recognized early on that they were contagious, that they could be spread, but they didn't know very much about it. Polyovirus was one of the first that really kind of, I guess, amended environmental virology studies because of the vaccine, the development of the vaccine for that particular virus. And because it was, fecal orelin was spread through water. So this sort of started the idea. Now, as much effort has been made to eradicate polyovirus over the last 60, 70 years, it still pops up in today's world, where we have outbreaks, where it's spread around. Now they're worried about other viruses, which are very similar to polio that are in that same family that are being spread around, hand, foot, and mouth disease, and other types of issues. This interovirus 71, currently identified as interovirus 68, especially in children, causing a similar paralytic disease. This polio-like illness found in five California children in 2014, and then it's been spread around. Now hepatitis A is also an old virus that we've known for a long time. We've known about. We have a vaccine. In many places, it's required that you get vaccinated. So if you work in the wastewater business, or you work in a water lab, maybe in schools now, they recommend hepatitis B and hepatitis A vaccines for kids. But it's not universal. The vaccine is not universal. But we didn't think we had much hepatitis A. And just recently, hepatitis A started showing up in the homeless. Its first appearance was in California, but it spread across the whole United States, into Canada, and into Canada, but into Kentucky. In Michigan, we've had a large number of deaths, and a high number of cases. So while they're saying, should we go in and try to vaccinate these programs, what are they doing about hygiene? This is fecal oral. In our own country, is this open defecation? Is there non-access to safe food, safe water, in our own country? How did it spread so quickly across the United States? From one state to the other. So here's something we thought we had under control in the US that we didn't. Now, being a virologist that looks at mostly human viruses, some of the work I'm going to show you has kind of even opened my eyes over the last few years to look at the viruses in that affect other animals. This circle virus, and I'll bring you back to why I'm interested in these viruses, how they came to my attention, being focused very much on human health. So this circle virus is a very interesting virus that affects dogs and pigs. They think this strain came over from China. They don't know how. And it was starting to cause mortality in dogs. The Rana virus came to find out that this is a virus that is causing some extinction in some areas of the frogs. And there's actually a website about saving the frogs because they're so important to our ecosystems. And this Rana virus apparently is being spread around. And I'll come back to why I brought these two viruses forward. The other interesting thing is, and that goes back to this algal virus that we found in somebody's brain, is that there are some recent studies that suggest that actually plant viruses can infect and replicate in mammals. And there was a recent review here that looked at why this is the case and why we've missed perhaps these plant viruses and thinking about the specificity. And of course, we're concerned with algae and toxic algae. And where there's algae and where there's bacteria and where there's blooms, there's always viruses. The viruses tend to follow their hosts. We don't know much about those dynamics. They seem to have a prey predator modeling effect that you might use in larger animals. But no one really understands what happens when the host starts to bloom than what happens to these viruses as they start to replicate inside the host. So this is quite interesting. This algal virus has been found both in humans and in animal models, mouse models. It's called the stupid virus in Europe because it impairs cognitive abilities. And yet, we don't know anything about how prevalent it is in the environment, how many people might be exposed, how many people might have been infected with this virus as it passes and attaches for some reason to the mammalian brain cells and enters in to cause death of these cells and hence problems with our abilities. Now, the new technologies, as you know, the genomic technologies that arrived with human genome studies and then the human microbiome studies, these are instruments and technologies that have allowed us to sequence, to look broadly where we couldn't look before. We actually had very limited tools to study viruses. And even with PCR, which came in in 1985, that seems recent to me. I'm sorry about this young audience and that 85 sounds like it was a long time ago. But when PCR came in, you could use PCR and we do, but we have to know the sequence. We have to know the gene sequence to develop a PCR methodology where we can use the molecular understanding of the DNA or RNA to design a diagnostic test. Now with these new instruments, we can start to look broadly at the unknowns. And in fact, they've used this to look at disease and disease in humans and animals. They've used a variety of these tools. And what that means is that you can see the expansion of knowledge on new viruses identified. New viruses identified. And it's a very sharp increase in growth of knowledge since the 1980s and 60s when these new techniques started coming in and we started using them. And I'm going to come back not to talk about the methodologies, but I'm happy to share that with anybody that's interested later, send you some papers, but about what we found with these new methodologies. So let me get back to the hundreds of waterborne viral pathogens that cause disease, because that's my interest. They cause a whole array of diseases that we already know of. Diarrhea, of course, is one. They cause respiratory illness. So there's waterborne viruses that cause respiratory disease, liver damage, kidney failure, heart disease, cancer, nervous system disorders. Just recently, they've identified some coxsacky B viruses that they think are associated with Alzheimer's. So these viruses that can move from the gut to other organs in the body, like hepatitis A, moves from the gut to the liver. These viruses move to other organs in the body, and that's where they cause the disease. And so this coxsacky B, which is a common enteric virus in sewage showing up in the brain and causing Alzheimer's. Now the other thing is, of course, that we know that these viruses are spread with feces. And where feces go, these viruses go. And they're quite stable, little nanoparticles, right? And we know that our waters are quite susceptible to fecal contamination. Why? Because our population lives near water. We live near a coastal system. We live near a river. We live near either a marine or freshwater system. And we know that this is a cause of pollution, and general pollution, a major pollution of our waterways. And in fact, we do not have proper sewage. We don't have proper sewers. We don't have proper sewage. Transmission, we don't have treatment around the world in many places in the world. And you can see in Cambodia on the left, in many of their waterways, they live above the water and their toilet empties right into the waterway. Or, as you can see in this other picture in Kenya, it's just an open sewer, right, that goes through the community. This may not be any surprise to people that these are the conditions that other people live in in the world. We know this, and with the sustainable development goals, there's an aspiration to change this, to change the way we look at sanitation around the world. Now, even in our own state, we've looked at wastewater. And we have a prohibition of discharging wastewater directly to the lakes. We don't have necessarily advanced treatment, because we got a lot of water. You know, I came from Florida. I worked in Arizona. I worked in California on reclaimed water, right, where we take wastewater. We treat it to a high level. We reuse it. We reuse it. And this is happening around the world. Michigan uses about 0.01% of their wastewater, reuses it. They discharge. But we do have a rule in the Great Lakes that says water that we take in the Great Lakes should stay in the Great Lakes. We should return flows. So these are our return flows. Here's our wastewater map over here on your right. And all those rivers go to the Great Lakes, by the way. And those are not advanced wastewater treatment plants. These are routine, activated sludge, traditional plants. We do use disinfection in Michigan year round, unlike some states. Some states have seasonal disinfection, because they're not swimming. It's the swimming that said, we've got to put disinfection on our wastewater. And so they start disinfecting Memorial Day to Labor Day. And if you're really hardy in Michigan, I think sometimes on Memorial Day, you can swim. But Michiganers, I found out, are pretty hardy. Now, not only that, but our septic tanks. And interesting enough, we don't really have good records on our septic tank. So we've had to model that by overlaying sewer maps against population. As to where there's no sewers and there's people, there's likely septic tanks. So we're trying to use population. And then verifying that in counties, because a lot of the data is in the political boundaries. They're not in watershed boundaries. What's interesting here is you'll see that we actually have high-density septic tanks around our urban centers. As they've rolled out from the urban, they've used on-site wastewater systems. So we have a lot of wastewater that goes into our environment. Now, we've been gathering data with the Global Water Pathogens Project around the world on concentrations of pathogens in sewage to try to focus on how engineers and managers might look beyond the indicator system when they're designing and adding to their wastewater systems to make sure that they're actually protecting public health. Because if we just use our indicator system, which is E. coli, it's a bacteria. It does not sufficient for viruses. A bacteria is so different from a virus. It does not help us understand that we're protecting public health from viruses. And one of the things we also found out is that viruses are excreted at extremely high levels as part of their strategy for transmission. So they can be excreted at 10 to the 11th per gram, 10 to the 12th per gram. And they end up in wastewater once it's diluted out between 10 to the 9th, 10 to the 7th, 10 to the 9th in wastewater. So very, very high concentrations. And viruses tend to be very potent. So just a few viruses. What potency means is that you don't have to swallow very many viruses to initiate infection. Even one rotavirus has a probability of a 40% chance of causing an infection. So if I gave each of you just one virus, which is excreted at 10 to the 12th per gram, half of you would get infected. Half of you would get infected. It's very potent. So we want to know with these new methods who's there. And we want to know, try to find these unknown viruses and not just look for the ones we know about the tools that we have. What are their concentrations? So concentrations are very important to risk assessment. They're important to our control. How much removal do we need? Do we need 90% or do we need 99.9999999% right? We need a lot of removal here to protect public health. And how do we control the flow of pathogens in feces from their excretion rate to waterways? So we have methods now where we can concentrate. I guess I could show you that. Concentrate viruses very effectively. These are ultra filters. We can get 60 liters, 100 liters. Some people do 1,000 liters of groundwater. It depends on how dirty the water is. It'll go through these filters. And we get a concentrate where we can look for viruses. From there, we can do a whole series of different types of tests. Traditionally, we use cell culture. We have to know what cell culture. And then we grow the viruses up. We can actually grow them in these cells. And we can look at their impact on cells. We can do EMs. We can do further assessments. So cell culture and isolation is still important. But more and more, we've turned to molecular biology tools. Now, the unique thing about viruses is that we need RNA detection as well as DNA detection. Because we've got DNA viruses, and we've got RNA viruses. And our methods for DNA viruses won't detect any of the RNA viruses. And so we've got to use reverse transcription. So we've got to do special things to get the whole array of viruses there. And of course, we have the metagenomic studies. That means we look for everything, all the DNA, all the RNA in a single sample. The problem with that is if you don't separate out the viruses from the other stuff, which is pretty big, a bacteria has a big genome compared to a virus, or protozoan. You miss the viruses. You can't find them. You can only find the most abundant viruses, which are the bacteriophage. And you don't see the other viruses, the rare virome, or the pathogenic viruses. So we must process our samples to remove as much of the other material, the big stuff, to look just for the viruses. And we must look for RNA viruses and DNA viruses. Now, I want to show you some data over the years of what we've done. We use traditionally cell culture to look at viruses. This is in raw sewage. The bottom is the month over a year. And then we've got the different kinds of viruses and bars that we were able to identify in the cell culture system. Now, originally, we would take antibodies, or we might do a PCR test. So that means we look at one virus at a time. This is a microarray system. It's set up to look at the whole genome of the family of viruses. So it's a big platform. And you could look and see what viruses were there by lighting them up, by their probes lighting up. And you could distinguish the RNA from the DNA viruses as a confirmation. And what we found for the first time in a survey is the increase in viruses showing up in sewage in January. Now, is this a seasonal effect? Others have said disease is seasonal. But often we don't have an understanding of the concentrations and how they may change, or the diversity of viruses that might change. So this was showing us that we had a whole array. And the RNA viruses were actually even more diverse. And this may be due to the way RNA can mutate in terms of creating new viruses compared to DNA. So there's all these viruses that we're showing up and showing up at certain times of the year that we weren't even looking for, you know, astroviruses and sapoviruses and things like this. Now, let's see if we can play this little video. This gives you just a visual of what probes showed up over time. So the time is at the top. And these are the probes lighting up. And so this was one of the first times that gave us an insight, a more dramatic insight, to this change in virus excretion from our population over these seasons and how much we were really missing. Now, through metagenomic techniques, then we started looking more broadly. The microarray had to be built with genomes that we knew about in the database, right? So we had to know about the whole genome of the virus and put it on a microarray, this new technology. But with metagenomic approaches, we could look for any viruses there and blast it against our known databases, kind of like a fingerprint testing, right? Well, what we found is not different from what other people have found in using metagenomics for viruses. 60%, 80% of the viruses, they're viruses. So they hit a certain part of the genome that says they're a virus. We don't know what they are. They're unknown. They cannot be identified in our database. So there's a whole big group of viruses in our waste while we don't know what they are. We have an occlu. The ones we didn't know, we found single-stranded RNA viruses, unclassified double-stranded DNA viruses. But what was interesting with these single-stranded DNA viruses? So not too many people were looking at the single-stranded DNA viruses. I'm going to come back to that because the circle viruses are single-stranded DNA viruses. And we found plant viruses in here. We eat plants. Now, are these viruses that are infecting humans and ending up in sewage? Are they passed through viruses? We actually don't know. So plant viruses are found in sewage. Are they coming through our food chain or are they replicating? And we found animal viruses. Now, we do have leaky sewers and we do have farms around. And maybe we're discharging these things to our sewers systems. Or are these animal viruses that are infecting humans that we don't know about? We don't know. And we started finding a whole array of different viruses. The polyomoviruses, which have been known to cause cancer, show up through urine. Circovirus, you'll see there. I don't know if I have a pointer here. But circoviruses is there. One of the bigger circles, the circoviridae showing up. And so this started having us look. In fact, that's why I started looking up the circoviridae family. I said, what is this group of viruses? What are they doing in sewage? So we found some of the typical human viruses. We found these animal viruses, plant viruses, and of course the bacteriophage. But the circoviridae is a virus that was mostly in animals and has been mostly associated with pigs. And I found out that actually our pork prices went up one year because there was a massive kill of pigs and piglets due to this virus clear across the US, enough to influence the price of pork in the market. As I said, it'd come in to Michigan and jumped into dogs. And now there's a new set of circoviridae viruses found in humans, a cyclovirus. It's a fecal oral virus. It causes a paralytic disease, moves into the central nervous systems from the gut. It's been found in Asia in terms of disease. It's highly related to pigs. Its genetic characterization is highly related to pigs. And I'm interested in it. Why? Because these single-stranded DNA viruses are our smallest viruses known. They're tiny, tiny, tiny. They're smaller than the introviruses, 27 nanometers. So they're in 15 nanometers, 16 nanometer size. They also have a single-stranded circular DNA. In fact, I don't know, Chip, whether anybody's tested them against UV. I'm wondering if they're more resistant to UV maybe because of their capsidermy or the way their DNA is configured. We have found it in some genomic studies where we followed through a wastewater reclamation system that had a microfiltration system. And so what happened is that the circoviridae, this is relative abundance. So this tells us how many viruses are there relative to each other. After a membrane process, the circoviridae was appreciably increased. That must mean in abundance studies that it went up. We didn't get the concentrations. But relative abundance, if you see that bar going up after this process, those number of sequences belonging to that virus were appreciable. And so other people have now found that these little tiny viruses are going through some of our membranes that we thought were very good barriers to viruses. We found, done some same things in Kenya, wastewater system, and they have some interesting viruses showing up. The Kobu virus, which is a serious virus. Hepatitis A shows up there. Cosaviruses. Cardioviruses, a virus that affects the heart. So these are some unique viruses. We don't know that. We don't see those very often in the United States. But we're seeing those, these types of viruses of concern in Africa. We also have, you know, a lot of studies going on to look at just general introviruses and rotoviruses. Rotoviruses are still showing up in our wastewater, despite us having vaccines for our young children. Still causes diarrhea. And there appears to be some seasonality. But these, this is the range in concentrations that we find through wastewater system over time. And this is across the US. This is at about five different wastewater plants in Virginia. Now, there's been a strong interest in trying to understand how we remove viruses in wastewater. These are fecal oral. They're going to end up in feces. They're going to end up in raw sewage. So how do we control them? How do we get rid of them? The colophage here on the far left is a virus that affects E. coli. So it's been used as a surrogate often. And it's easy to cultivate. And in fact, EPA has recommended that wastewater treatment plants use the colophage more frequently than just E. coli or what's in their standard. Most wastewater plants do not use colophage. In fact, if you go to a wastewater plant where they're monitoring, they don't even know what a colophage is. But it's an easy test. It's cheap. You can get the results in 24 hours, unlike some of the other viruses. So colophage is a good indicator for us, I think, to use in virology studies. And what we found is that between primary, where you see the top bar, is our raw sewage, and the bottom bar is what's in our secondary influent, you're going to see that in some cases, we have high variability in the influent, like for salmonella. So salmonella infections and excretion, we've got high variability coming into the plant. But we narrow that variability by our treatment process. So our treatment process is able to control salmonella to a greater extent in terms of the variability coming in is quite high. With the fudge, it's an indicator. We're always excreting it. It's like Giardia and some of our E. colis. What's coming in is quite consistent. We know what's coming in. But even what's going out, we don't always control with our treatment process. So our variability coming out might be higher. And so we see that with the colophage. Now I want to point out these last three bars here to the right. So this is total cultivatable viruses on cell culture. The next set of bars is cell culture with PCR techniques using molecular techniques, where we can identify which viruses might be in that cell culture. And the last bar is just QPCR, just viruses, particles, virus particles. So what you see right away is that total cultivable viruses come in high variability, but we're able to control them through treatment processes to a pretty low level and with low variability. Now when we start looking at which viruses are in these cell cultures, we start to see much greater variability. Obviously, the influence is lower because we're selecting certain viruses in our cell culture. But the variability is high. Variability of what's coming in. And that probably reflects what we saw in the microarray study. This whole different kinds of viruses, depending on the time of year, what's coming into the wastewater system. And they all have different resistance to our treatment process. But then when we got to QPCR, where we're just looking for the viruses, we've got a large numbers of viruses coming into our wastewater system. We don't remove them very effectively. Now we don't actually know if they're alive or not. Are these viruses we can't grow in cell culture? Are they alive? Are they infectious? We don't know. But we do know that if we look for the total virus particles, large numbers coming in, large numbers going out, and high variability, these are some of the distributions from that work. This is the QPCR, interovirus distributions. And we want to be at the end where we get great log removals. How do we operate our facility so that we're down at this end? How do we do the environmental monitoring with engineers at a full-scale system so that we can understand design, operation, variability and the influence, et cetera, et cetera? So this is our goal, is to go to full-scale systems, do enough monitoring that we start to understand this variability. This is our FODGE, not too bad as a surrogate in terms of the distribution and whether we can start to understand with an easier methodology with a surrogate on how we optimize treatment controls to minimize virus transmission through wastewater treatment. And the same thing, this is a conventional wastewater system, but we're looking at lagoons and other types of treatment. I'll show you our high-rate clarification system. This is a system that has been put up to take combined sewer overflows. So when it rains, the rainwater and the sewage comes into the plant, really high flows, into the wastewater plant. You can't put that whole high flow through the wastewater plant because it blows it out. Then your wastewater plant doesn't work anymore. So they have what they call a system where they capture the high flow. They then put part of the flow through this what they call high-rate clarification. So it processes high volumes. It's being looked at, I think, for storm waters and other things because it does process high flows. And it takes out the turbidity, takes out the big stuff. Then they disinfect it and they discharge it. The rest of the flow goes through the traditional wastewater plant. Now, the question was, you can meet your standard for fecal coliforms. When the EPA came into this particular plant and said, we want to know that you're actually removing viruses because this discharge was affecting a recreational site downstream. So this is what the E. coli looks like, our traditional indicator. The green lines at the bottom are the traditional method. Activated sledge, it works quite well. So we've got high E. coli coming in. After secondary, it comes down. And by the time we disinfect, we're below that red line where we're at the standard. With high rate clarification, it's a little higher. The E. coli's a little bit higher. But we can meet our standards if we optimize disinfection. We can meet our standards. Interalcoxy is a different story. Interalcoxy is another indicator that is suggested to be used. It's a different group of fecal indicators. And it's more resistant to disinfection. And in fact, many people have suggested we use interalcoxy when we're optimizing disinfection because it is more resistant. And you can see it follows a similar trend. But it's harder to disinfect. And so we've been taking this data to work with the plant operators to say, how do we optimize disinfection to bring that interalcoxy down below swimming guidelines? So there's swimming guidelines for interalcoxy. Now this is what the coli fudge looks like. They're nano, nanoparticles. High rate doesn't take them out. And in fact, our ability to disinfect them is quite different. And trying to optimize disinfection is difficult. This coli fudge method, by the way, is a culture method. So we can actually look at disinfection. It's not just doing particles. We think they're attaching to different sized particles in an upflow clarifier. We think there's a different kind of distribution of particles that these nanoparticles, these viruses, are attaching to. It's protecting them. Because these viruses should be quite susceptible to our chlorination process. But in this clarifier, they're not. And we don't know the mechanism of, what's the distribution of particles? Well, how are particles moving in this system? How are viruses attaching to them? And what does that mean for trying to optimize? Can we optimize disinfection? Do we need another type of disinfection? Do we need another process? Now I'm going to end by telling you the story on the work that we did in ballast waters. And this is really the work that I think has opened my eyes to the broader world of viruses, even though I've called myself an environmental virologist for a long time. I wasn't really looking at the whole biosphere, the whole array of viruses that might affect our planet. And this study really opened my eyes to this. We did some in the Great Lakes, but I'm going to focus on the marine water work we did. We took samples in Long Beach, and we took samples in Singapore. Now these are the locations. The green is the Los Angeles ships, and the red are the Singapore ships. And if you know anything about ballast water, it's water that's taken up to stabilize ships. Where the dots are is where the water was picked up. Now there's a law in the United States now about recording where you pick up the water, where you discharge the water, and also state agencies have access to the ships in the United States, because we passed a ballast water rule. So you can go on to a ship and get a sample with the state environmental agency. Now in Singapore is a different story. We do have a guideline from the Marine Commission, a global commission that talks about what we should do with ballast water. But it's up to the country whether they implement these guidelines. In Singapore, they're implementing them somewhat, but by the ship owner. So in Singapore, we had to get permission to go into the port and go on the ship by the ship's owner. Luckily, we had some connections in which the owners allowed us to come in and get many of their ships. And they were following guidelines where they recorded where they picked up the ballast water. So we did port water. We sampled the port, and we sampled the ballast water, and we recorded where the ballast water came from. So that's the story. It's not easy getting ballast water. I didn't know it was going to be so difficult, but the people on the ship really helped us out, set up pulleys so we could lower after we got the water, we could lower it down to the side. And some of the ballast water, you actually had to dip out with a bucket. They didn't have a sampling port. Some of the newer ships are going to have sampling ports because it's going to be mandated. In fact, in California, they're going to mandate a virus standard in ballast waters, which is one of the reasons we are working with California. They don't know what that standard is going to be, but they're going to have to develop ports. In fact, treatment on ships for ballast waters is estimated at a $70 billion business. So people are looking at it as a new market. Here's the Long Beach port, if you haven't been there. Quite interesting in them helping us collect port water. Now, virus richness. So again, the green is California. The red is Singapore. And virus richness is really about how many different kinds of viruses are there, right? How many different kinds of viruses are there? So what you see right off the bat is that Singapore is warm, it's tropical, it's near the equator, has a lot more virus. A lot more virus richness. And it's been known that places near the equator have more biodiversity. So California had less, and Singapore had more. And we started looking at whether we could relate these viruses to either engineering or other aspects of the ballast water. So time in ballast tanks, right? They picked up the water over here, and it took 10 days to get to the port. It took 20 days to get to the port. Over here they picked it up, it took two days to get to the port. We want to know if it was time in the environment. Distance from the shoreline. They're supposed to pick up ballast water. It's a guideline. And we saw that it wasn't always true, but they're supposed to pick up ballast water at 500 nautical miles from the shoreline. Well, they don't do that. And so we had a lot that were near the shore. Somewhat a relationship if you started looking at California, but not very strong. No relationship with the Singapore group. No relationship with other different parameters that we could find. Temperature. Somewhat of a relationship, but if you separated out California, the ships coming from in the Pacific mostly from Singapore, the temperature didn't make a difference. So the relationship was because we had warmer waters in the Singapore samples. And so all the reds were up there, and the greens were there. Salinity. Again, not much of a difference there in salinity. Believe it or not, ballast water gets picked up if it's near the shore or if it's rained. There's some fresh water. It's not all just ocean water salinity. And I was surprised to find that out. We started separating out. All these boxes in the bottom are the different groups of viruses. Fodge, vertebrate viruses, invertebrate viruses, plant viruses. And what we found was that fudge didn't give us very much resolution. Now there's been something said about everything is everywhere, just a different abundance. And that's what they've been saying in these marine studies about fudge. Everything is everywhere. Everything is everywhere. But when we started looking at very specific viruses that might actually represent that ecosystem, that particular environment, we started seeing relationships. And interestingly enough, it was with lat longs. It was actually with latitude for the most part. So the latitude may represent temperature at that particular location and may represent the environment at that particular location, what's unique about that. So this tells us that viruses, we are transporting viruses from their location, unique viruses when we start looking at plants or animal viruses. We can start to see these trends. It means that we are picking up viruses that might be unique to this one environment and moving them to another environment. You can't see it with fudge. You have to go to the more rare biomes. Now what we found was we identified these viruses. We found a human cyclovirus, a shrimp virus, and a human pycnobirna virus. Where it says H is harbor, where we found their color coded. So I think you saw that. The green is the cyclovirus. The pink is the shrimp. And the blue is the pico. So H is harbor water. That's where we found these viruses, these particular viruses in harbor waters. The B is ballast waters. And the D is where it's been identified as disease, causing disease. So you've got disease over here of the human cyclovirus. We found it in the port of Singapore. We found it in the port in LA in the harbor. But we didn't find it in ballast waters yet. It may be a sampling issue. It's very rare. It's a very rare biome. But we did find it in the harbors where the collective ships are all discharging. And the individual ballast waters, we couldn't find it. The human pico virus, though, on the other hand, is quite widespread. We've known that this virus is, as far as we know, it just causes diarrhea. But it's quite widespread in terms of disease. It's quite widespread in the harbors and in the ballast waters. The shrimp as well is the primary disease. You can see it's in Asia. Ballast waters there, the harbor there. We see a little bit in ballast coming across. But we didn't detect it in LA's harbor yet. So there's a lot of viruses there. They're unknown. They're moving around. And to get back, we've got this cycle virus. And to get back to that rhinovirus, we found our rhinovirus in some of these samples. And it turns out, I found a paper that the rhinovirus had jumped into fish from frogs. We don't know how common that is. But here, we've got these other viruses popping up that might be affecting the biosphere. So the rhinovirus, showing it in fish, these other types of fish, viral pathogens found, the shrimp viruses, and the human viruses found. So I'm going to end. I'm going to say that we've known about some of these viruses for a long time where they're still struggling to control them, either through public health programs like vaccination or through hygiene, defecation, open defecation, and wastewater treatment. But we've got hundreds of new viruses that we don't know about. They're emerging. Some of them we're just finding out about because we're linking our clinical studies to our environmental studies or through these metagenomic techniques. We believe that human, animal, and plant viruses are spread around. We don't know what the risk is. There are some models that talk about the invasion potential. How many times does an invader come into an environment and you get a hit where it takes off? We don't know. We don't have enough data to do any modeling on what the risk is of these viruses. There's potential for them to find the right host as they're transported. And we really think that we've missed some things about viruses jumping species, that we didn't know about before. And especially in the animal kingdom and maybe in the plant kingdom as well, these viruses are not well understood and studied. So we have the environmental virology group globally. We think that we start to have to look at these coupled systems, do more to look at exchange and what's happening. We have to characterize the rare biome. We can't just look at the most abundant viruses that are there. We're going to miss relationships. We have to use the new technology. And we're going to need to understand concentrations ultimately and what it means between particles and infectivity and how we actually optimize our treatment processes both in wastewater and in drinking water to control the spread of viral disease in the future. I'd like to acknowledge all our support and colleagues. It takes a lot of work to do these kinds of studies out in the field. And thanks for your attention. And I'll take any questions you might have. All right, so we do have a good amount of time for any questions that you might have for Dr. Rose. I ask anybody in the audience who would like to ask a question, please press the button on the microphone on the tables in front of you so that your voice can be heard. Did I talk too fast? We have more time. So how many of you have had a virology class? Anybody in here has had a virology class? Opportunity. Figure out how to press the button. Oh, I have to hold it. So ballast waters are also a potential source of invasive species that come in. So once those ballast waters with the viruses get dumped in the harbor, what's the chance of them now becoming indigenous to that region? Yeah, so that's what I was saying. We don't really have the models to do that prediction. We do have a couple of viruses that come into the Great Lakes through, they believe, through ballast waters, the VHS virus, which affects fish. And they've had several outbreaks that became indigenous. There wasn't there before. And then they had some outbreaks in the Great Lakes. It kills, it does a fish kill by affecting their blood system and necrosis. And so that one, they do believe, came in through ballast waters. So we've got that one example in a pretty confined area that they could maybe model. Now, is that virus typical of some of the other viruses in terms of its capability of jumping in and affecting the native fauna or flora? So that's what we don't know. But it is a start in terms of there's enough data there to probably get some models. So then what do you do to remediate it when it's in an environmental location like that? Because it's not like a sewage treatment plant where you actually go through a process. So right now with the new standards that are coming on and probably anybody that deals with California will have to meet the new standards. Any shipping agency from around the world that lands in California will have to deal with their new standards. Basically it means on board treatment, on board treatment. So they're trying to test a whole variety. UV looks very promising just because they have energy on board and the turbidities aren't as high as you might think in ballast waters. They thought maybe the ballast waters would be too high a turbidity and they'd need pretreatment before something like UV. But that doesn't look to be necessarily the case. Now the question is when they discharge, how would you treat it in the tank? Because when they discharge the flow rate is really high. I mean it's just spraying out there at a very high flow rate. They don't want to use chlorine because they probably have to denutralize the chlorine. And that's the cost. The cost is neutralization of the chlorination. Is ozone, ozone on board generation of ozone could be a possibility? And so they're looking at disinfectants right now. Whether they will go to a filtration and a disinfection approach, I'm not sure. There are some new types of filters that could perhaps fit on these ships. These ships are pretty big. So they can have. Now they were thinking of a pump and treat but it's not possible. Logistically you could not pump and treat all the ballast water that comes into this port. So it's gonna be on board. Joan, so human norovirus is a big deal. It's responsible for a large fraction of waterborne disease. Yet only recently were we able to culture it and my understanding is that's not a simple process. There's only a few groups that have been able to do it. You have hope that that's going to improve any time in the future? Because it is pretty important as a step. Yeah, the current culture methods are not easy. And in fact, the early attempts that were published were not replicable in other labs. And time will tell whether this new system can be picked up by a lab that has culture capabilities. But you do have to have a new system in terms of the way the cells are grown. So people are using QPCR. It's interesting though, even with QPCR you find norovirus to be quite diverse in different sewages. In Japan it's quite high. The norovirus is very high in terms of even just copies using PCR. And you can distinguish between norovirus one and norovirus two. So when the different genotypes started spreading around the world they could see these. I don't know if it's because they eat shellfish raw. You know they don't want a norovirus there and a lot of shellfish in Japan. Shellfish harvesting waters in their coastal systems. In the U.S. when we looked in Chicago and other people have shown this it may be something to do with the persistence of norovirus which we don't know very much about. In the raw sewage you find it once it got into the Chicago waterways the levels went way down, the non-detects rose. And so I think that we probably need to do more with QPCR in terms of understanding concentrations and removal. And there are some studies going on in Arizona that's trying to look at removal of norovirus. But also persistence because once it gets in the environment under certain conditions I'm not sure how persistent it is. So I think we're gonna have to fill in the gaps with molecular tools right now. Maybe some surrogates. We do have some surrogates. The marine norovirus which is cultivatable. And so I think we can use that surrogate. Maybe to look more at some of these issues. Joan I had a quick question. In your last slide you talked about the need for technologies. And you have it multiple points referred to metagenomics and the changes that allow you to investigate the phenomena you're looking for. Where do you see needs in the need space of what kind of technologies would really help make your work easier? Yeah, so I think even over the last five years there's some new concentration methods. And I'm just reading about a pipette system that once you concentrate it from the ultra filter you can take that volume and further concentrate it down. And it's fast, it's supposed to be efficient. It hasn't been pumped very much according to the manufacturers. So these technologies are out there that could make it easier. But ultimately with viruses, so there's so many steps even for the handheld sequencing, that is fast. We can use that. But the steps in between take us two days. And especially if we're gonna get RNA and DNA viruses. If we're just gonna get DNA viruses fine. But if we're gonna get the RNA viruses all these technologies are really geared towards having to do reverse transcription first. And so it makes that middle step. So the collection is progressing. It's gonna be fast. The analysis, the detection and analysis is progressing. But the intermediate steps are still very difficult in terms of purifying, concentrating, getting the reverse transcription all of those kinds of steps. So we're gonna have to figure out how we do that. That part better. We need probably a technical, maybe even automation. It's not a big space. So the companies, they don't see it as a strong market, right? Compared to the medical market. It's so small. Like how many people are gonna buy an automated system for water sampling? And so unless rules change where they say everybody's gonna have to do this. There might not be motivation. There are a few companies though helping out in that realm. And so automation and robotics in this area maybe we can adapt from what they've done in the medical world. And it might not be faster but then you can do more higher throughput and with more reliability. So I think that's going. But on the technology side, I have to say that for me safe is it's about science and our ability to control the hazard and public engagement or social engagement or however you want to frame that final bubble. And technology plays a role in all three of those bubbles. So information technology, technology for the science piece and technology for the control piece. So sometimes I just am talking about one part of that bubble in terms of technology but I think for us to ultimately say what is safe we have to think about technology in all three of those spaces together. Because ultimately we wanna say it's safe and that's a hard thing to say whether something's safe or not. All right, thank you. Before we wrap up I would like to welcome Dean Meng Cheng who is able to join us and may wish to say a word or two of welcome and thanks to the group. Thank you so much Dr. Rose for such a illuminating lecture and I usually do this at the beginning but I think it's better to actually do it at the end because I just learned over the past 55 minutes such a fantastic lesson about viruses and as you know that we started the College of Engineering Distinguished Lecture Series about a little over a year ago in order to bring the world leading experts from different engineering disciplines to Purdue and was so delighted to welcome June Rose to be our Distinguished Lecture today and indeed as we aspire to the pinnacle of excellence at scale at Purdue Engineering we realize how important it is to look at interactions with agriculture, with environment, with policies and we have our ABE school, fantastic school. We have our division of environmental ecological engineering and then there are also other faculty throughout engineering college from civil to mechanical working on related topics and again I wish I could welcome expertise such as Dr. Rose back to Purdue for as often as your busy schedule allows and as you know that Dr. Rose as a member of the National Academy of Engineering and recipient of both the Stockholm water prize and the recipient of both developing and developed countries honors. I know that you're working busily with Gates Foundation and UNESCO in Uganda and I think you are the recipient of this honorary citizen from Singapore. I don't know that means you have to pay their tax as well as US tax but it demonstrates the impact of our Distinguished Lecture today throughout the world in very different kind of environment. Some very developed cities, states and others are developing countries in a very different continent so the depth and the range of your impact is a true inspiration for all of our faculty and students here at Purdue Engineering. Again, thank you so much for joining us here, John. My pleasure. Thank you very much. Thank you so much. Thank you so much. You're welcome. Thank you. Thank you.