Interviewers: Lydia Morrison, Marketing Communications Writer & Podcast Host, New England Biolabs, Inc.
Interviewees: Charlotte Houldcroft, Assistant Professor, University of Cambridge
Thanks for joining us for this episode of the Lessons from Lab and Life podcast, brought to you by New England Biolabs. I'm your host, Lydia Morrison, and I hope this episode offers you some new perspective. Today I'm joined by Charlotte Houldcroft, self-proclaimed lover of viruses and lecturer in the Department of Genetics at the University of Cambridge, who joins us to talk sequencing, from double-stranded DNA viruses to ancient DNA. Charlotte, thank you so much for joining us today and taking the time out of your schedule to be here and talk with us.
Thanks for having me, Lydia.
I know you study double-stranded DNA viruses. Could you give us a quick primer on these viruses, like what diseases they're responsible for, and could you also point out the key differences between DNA viruses and their recently more popular cousin, the RNA viruses?
I'd be delighted to. The first clue is in the name. I'm really interested in double-stranded DNA viruses, and they're made of deoxyribose nucleic acid instead of ribose nucleic acid, and they are double stranded. And this is really important, because it means that they are higher fidelity when they copy themselves the RNA viruses, so they mutate more slowly.
There will be lots of DNA viruses that listeners will have heard of, and perhaps, unfortunately, experienced at some point in their lives. The viruses that I work on the most are the herpes viruses, and that's not just the herpes that you might have heard of, but it's also viruses like Epstein-Barr virus that causes infectious mono or glandular fever as it's known in the UK. Another famous herpes virus is Kaposi sarcoma herpes virus, which is one of the AIDS-defining illnesses. But we're also looking at viruses like Varicella zoster that causes chickenpox and sequelae in older age, and then there are a number of herpes viruses that cause really mild symptoms in healthy people. So that's the herpes virus family. They're some of my favorite. But monkeypox, which is currently causing lots of problems both in Africa and around the world, is also a DNA virus and causes really extreme skin manifestations, it can be very painful.
But I love the DNA viruses because they're so diverse. The herpes viruses and the monkeypox family, the pox viruses, they are so different from one another. They are hundreds of millions apart in their evolutionary history and it's that diversity that fascinates me.
Yeah, that's really interesting. I've never heard somebody say before that herpes is their favorite virus, but you definitely seem to be someone who really loves viruses. Can you tell me more about how next-generation sequencing plays a role in your lab?
Sequencing really has transformed virology for everyone, but I have loved virus sequencing since I did my PhD. In 2009, I started my PhD as the swine flu pandemic was rolling through the UK, and back then sequencing, it was all Illumina, it was much shorter read, and it would take weeks and weeks to get virus sequence back even from a pandemic pathogen like swine flu. But now during the SARS-CoV-2 pandemic, for example, we started on a Monday morning, a clinician brought samples down from upstairs from the clinical laboratories, 24 samples that had SARS-CoV-2 RNA in them, and by the next morning we had 24 SARS-CoV-2 whole genomes. So, sequencing has absolutely transformed the way that we can study what's going on in epidemics.
Now, for me, what I'm really interested in is how DNA viruses evolve, and they tend to evolve more slowly than RNA viruses. The only exception to that, really, is in people who are immune compromised. If, for example, you've had a kidney transplant or a bone marrow transplant, either you are being given drugs to suppress your immune system or your immune system doesn't work properly because it's growing back in from the bone marrow transplant. And in that period where you don't have an intact immune response, viruses that normally mutate slowly under strong selection pressure mutate much more quickly, and that might be to evade the emerging immune response or it might be to evade antiviral drugs that are being given.
And so, I've been using sequencing to understand this kind of dual pace of evolution, that you've got these viruses that are normally slowly evolving, perhaps passing from mother to child or from partner to partner, spreading slowly around the globe that then have these bursts of rapid evolution in immune compromised people that can be incredibly dangerous for people in hospital. You can learn about these evolutionary processes, and I think in quite a complimentary way, by sequencing both virus from healthy people and virus from people in hospital.
That's really interesting. What are the pros and cons of different methods like metagenomic sequencing and multiplex PCR or capture-based approaches? Do you feel like those are best used standalone or interdependently?
It really, really depends on the question that you are asking and the type of sample that you have. For example, there's another double-stranded DNA virus that I work on called adenovirus, and I have sequenced this virus using metagenomics, using overlapping PCR, and using bait-based capture. And it really depends what you want to know about that sample and that virus as to which message you choose.
So in one example, we were looking at stool samples, so this is a very microbially-rich mixed sample, and there was a strong suspicion that these stool samples were from individuals with a bacterial and viral co-infection. And so, using metagenomic sequencing from those samples was the best choice in that situation, because we were able to recover whole E. coli genomes at whole depth and whole adenovirus genomes at great depths from a single sequencing reaction. So that was very powerful.
But then, in contrast to this, I might have been working with samples from eye infections or samples from blood where an individual has a viremia, they have virus in blood, and there you might find that the host DNA component is very high, and the viral DNA component is very small, so there might be not very much virus there, or it might be low mass depending on the sample. And that's the situation where going in with bait-based enrichment, and there's now lots of different technologies and brands for that, it's very powerful. It's very, very sensitive, but it tends to be perhaps a little bit slower. It can be, depending on the method, a bit more expensive, but you get beautiful high quality genomes back.
And then finally, I've also used the overlapping PCR approach when I needed to get adenovirus genomes from a lot of samples very quickly and very cost effectively in a single nanopore run. And so the PCR approach, at the moment, we're still developing it. It's not as flexible if you don't know the genotype well of the sample that's going in. It doesn't always deal well with novel recombinants, which is where metagenomics and baits do better. But, if you know that you have, for example, SARS-CoV-2 viruses that are broadly quite similar to one another across the population, you're put inputting lots of the same thing, the virus loads tend to be quite high, and you need your answers in a hurry, that's when the PCR approach is absolutely fantastic.
As you mentioned, there's no reason that you can't go in and say, "Well, we've started with some PCR-based sequencing and we think there's more questions that we want to ask and now we're going to drill down to the samples that are most interesting and use metagenomical bait-based sequencing. So I certainly see in the future of my laboratory research that I'll be using all of these methods in the future.
Yeah, that's really interesting. It sounds like these methods can be very specific to the needs of the project or the experiment, and they can all be really quite complimentary, as well.
I think that it's very interesting about viral sequencing. There's no equivalent of 16S for virology. We haven't, as a field, home in on what method to rule them all that will be the way that sequencing goes on in the future. I don't know whether we will ever get there or whether we'll continue to use all of these methods and new ones that, perhaps, are being developed at the moment. But it's great to have a toolbox.
You're also among a small group of researchers making discoveries from ancient DNA. Can you tell us a bit about that research and about the challenges of working with DNA from the Bronze Age?
I'm really lucky to work with some excellent ancient DNA specialists, archeologists, and historians who occasionally send me really exciting emails that say, "Charlotte, have you ever heard of such and such a virus? We think we've got a monk with herpes." That was an email that I received a few years ago, and as we said earlier in the podcast, someone mentions herpes and I get very excited.
But you have to be incredibly patient to work with ancient DNA, and particularly if you are interested in ancient viruses. It has made me find depths of patients that I didn't know I have, because you might need to screen 1,000 or more skeletons to find a virus that you are interested in. And because the field is relatively so young, we still don't know what is out there, is it possible to find an ancient samples, and which viruses just won't preserve.
But I was lucky enough to be collaborating with projects where they were looking for ancient plague caused by Yersinia pestis in samples from Cambridge in the United Kingdom, and one of those individuals happened to have herpes simplex one DNA in his teeth, and this is a virus that causes cold sores. And then hundreds of other skeletons from all over Eurasia had to be screened to find three more individuals going back over 3000 years who also had herpes simplex DNA in their teeth. It was actually our individual from here in Cambridge, just a few hundred meters from my office, who had the best genome of any of the samples that we sequenced. And we still don't really understand what it was about this young man that meant he had so much of this pathogen in his bloodstream at the point of death. We tested lots of hypotheses, and perhaps he was just unlucky.
It's very, very exciting, but then you have this highly fragmented DNA, you require really specialist laboratory and sequencing facilities to get the most out of it, and then you hope and pray that you get a high-quality genome at the end where then you can go off and do some virology and ask what has changed about this virus? Does it look like modern viruses? Does it look like something different?
We, actually, found some really exciting things in the ancient HSV dataset. Your listeners may or may not be familiar with this virus, but lots of people around the world are infected with herpes simplex virus. It causes mouth ulcers, but after primary infection, you tend to control the viremia. It's not there in your blood anymore, and it's just hanging out in a nerve that connects from your mouth back to your spine. And when you are feeling a little bit run down or sleep deprived, the virus wakes back up and causes a new blister. The hypothesis has always been that the patterns of diversity in living people that we see today comes from the migration of humans out of Africa, perhaps 60,000 to 100,000 years ago.
But what we were able to show with these ancient genomes that we recovered and sequenced to high depth and combined with modern data is that, actually, the modern patterns are much more recent than the out of Africa migration. This still means that herpes simplex virus is a very old pathogen, it just means that diversity we see today is more recent than that. Actually, we think it probably dates certainly in Eurasia to Bronze Age migrations, as you mentioned, where you have new technology, perhaps new behaviors, new ways of living coming in to Europe and Asia. And we think that these people in their behavior helped new strains of HSV-1 to spread and overwrite all the diversity that was there before. And so that's been a fantastic study to be involved in, where ancient DNA has really changed our perception of what we think herpes virus dissemination and evolution looks like. So I've loved being involved, but you have to be very patient.
Yeah, absolutely. I can really hear your passion for it, and I can really understand, actually, a bit more how interesting and fascinating herpes is as a virus, partially because it's so old and you can really follow those evolutionary footsteps of it. That's very interesting.
How do you plan for discoveries from ancient samples? Is there a lot of planning and prep work that goes in before you receive this sample? And do you have a good idea, when you're going in, what you're going to find? Or are there unforeseen happenstance happening constantly around those samples and those types of experiments?
I think that the ancient DNA field, particularly where it touches onto ancient pathogens, has probably changed its approach over the last few years. There was a big push, perhaps initially, towards looking at plague pits. You're looking for evidence of epidemic type events, of course, mass burials, and then you have a strong suspicion that you are going to find lots of pathogens, perhaps plague, perhaps something else in that context. But with increasing use of metagenomics of really high-depth sequencing to get good quality human genomes and then use of metagenomic classifying software to look at what are all the other reads that are not the human genome, I think now researchers are more understanding that you might find both endemic and epidemic pathogen DNA in ancient remains from all kinds of settings. So that might be a plague pit, it might be a mass burial, or it might just be isolated skeletons buried with great love and care and planning, and you might find pathogens as diverse as plague, smallpox, salmonella, all these endemic, particularly DNA, viruses within these contexts.
One of the projects that I've had a small contribution to, it's in pre-print form and it was sequencing some human milk teeth, so baby teeth that had been found at a site in Siberia, and these teeth were 30,000 years old. They've been sequenced at very high depths to get high coverage human genomes, and then the researchers had gone and run the metagenomic classification on all the reads that were left that weren't human. And they discovered that these baby teeth from 10 and 11-year-old children, they had exactly the same cocktail of endemic human viruses that a 10 or 11-year-old child living in the UK or the USA today would have at the same age.
They had adenovirus, they had herpes simplex one, they had Epstein-Barr virus, and they had cytomegalovirus. So that's three herpes viruses and an adenovirus, so another DNA virus. That was not an expected finding. That was a propitious finding in samples that have been sequenced to something else. But it gives us a real insight into what does a normal infection history look like in really ancient individuals. But I don't think you could have planned for that in that study. It was a fortuitous finding.
And so, I think now there's much more of a trend towards saying, "We have these remains. We're interested in their human DNA content, but we should also be aware that they might have pathogens that might not leave any trace on the bones that might be epidemic pathogens, but might be associated with a burial that looks planned. Let's go in and see what we can find." There's been amazing work, for example, on hepatitis B virus, which has been found dating back at least 10,000 years in human remains and has really been found all over the world. And I don't think you could have gone out and designed a study to look for that explicitly, you have to say, "We are screening these human remains for other reasons, and then we're going to do an in-depth look at what else they have and then take advantage of the data that these studies are giving us."
Yeah, so interesting. Really phenomenal. And to think about the timelines of human evolution and viral evolution happening at the same time. It's so interesting. Do you ever find viruses in these ancient DNA samples that we don't see today?
That's really challenging. It's the unknown unknowns. I know that there's lots of work going on in other research groups to go through all of the dark matter in ancient DNA data sets trying to work out, are there pathogens present that we don't recognize today or that have gone extinct? And that's very hard to do, because we still don't have a complete picture of what viral diversity in living populations and animals looks like. So we suspect that there will be evidence of pathogens that are extinct in the samples, but it's actually quite hard to screen through the data in the right way to find them.
But we certainly know that we can detect extinct lineages of known pathogens. There was a very exciting study recently from another group, partly here in Cambridge, where they were looking for variola viruses, this is smallpox viruses, and they actually found that a number of Viking era sets of human remains had smallpox or smallpox-like viruses within them. But this is a lineage that was extinct by the 17th and 18th century, and we don't know whether the disease that this extinct lineage caused would've looked like the smallpox that gets into more historical medical records hundreds of years later.
So, ancient DNA definitely has the power to detect lineages that have gone extinct to things that we know about, and I think we will have to push the extent of our computational software and our ability to predict, for example, protein families from ancient and DNA sequence to find those viruses that we no longer know about because they've gone extinct all day or they don't cause a disease that we recognize today.
Thank you so much for taking time out of your schedule to be here today. I really appreciate it. And thank you for sharing your passion for viral research, specifically DNA viral research, and viral evolution with us. It's been very interesting to get a peek into some of the information that the field has gathered over the last several decades about where viruses are, where they've been, and how they've changed.
Thanks. I feel really lucky to be a virologist. The field of viruses and virus sequencing has changed a lot since I started my PhD over a decade ago, and I think it just gets more exciting. I wasn't planning to be a virologist in a global pandemic, but it was nice to be helpful. But I'm hoping that we can see a little bit more stability in which viruses are circulating, things that we know about and have a good way to tackle over the next few years. That would be something that I would hope for going forward.
I couldn't agree more. Thanks again, Charlotte.
Thanks for having me.
Thanks for joining us today. For the next few episodes, we'll be focusing on sequencing. I'll be joined by New England Biolabs Development Group Leader Brad Langhorst, and we'll talk about how bioinformatics can help inform sequencing and its analysis.
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