TWiV 937: Pediatric hepatitis with Emma Thomson

This week in virology, the podcast about viruses, the kind that make you sick. From ESCV 20. This is TWIV, This Week in Virology, a special episode recorded on September 8th, 2022. I'm Vincent Racaniello, and you're listening to the podcast all about... viruses. ESCV is the European Society for Clinical Virology, and this is their first in-person meeting in a couple of years, and we're in Manchester.

England and for this episode my guest is professor in infectious diseases at the University of Glasgow Emma Thompson welcome back thanks very much Emma was on twice before 188 and 341, both recorded in Glasgow. Yep. Have podcast will travel. And you should check those out because... 188 was at the beginning of your career, right? That's right. And 341, much later, you talked about treatment of an Ebola patient in Glasgow, right? That's great, yeah.

And so today we're going to talk about pediatric hepatitis. But before that, remind us about your training, where you did what before you got here. I studied in Glasgow. I studied medicine at Glasgow University and also parasitology, actually. And then I went to London where I trained in infectious thesis.

And then I did a PhD between London and Oxford University with Paul Klenerman and with John Weber, Mary McClure, and Peter Kariannis on hepatitis C. So that's my background, but we've kind of moved a bit away from that now. And then I moved back to Glasgow around about 10 years ago to the MRC, University of Glasgow Centre for Virus Research. I remember you did a lot of hep C work.

I assume today you're doing a lot of SARS-CoV-2 and now this pediatric hepatitis. Any more hep C going on in your lab? We're still looking at hep C, particularly in sub-Saharan Africa. We've been describing some... more divergent genotypes in sub-Saharan Africa, which are still not fully characterized and still we don't know enough about how well they respond to treatment, although the studies that we've been doing are largely reassuring, which is great.

We just heard a talk on hep C vaccines, and I was surprised to hear that the really good antivirals don't make much of an impact on the global numbers. No. That's right. And also in countries where hep C is endemic rather than kind of with the epidemic founder strains, we... There are one or two which are much more difficult to treat. So genotype 4R, for example, which is really rare in Europe and the U.S., is quite common in East and Central Africa. So hence a vaccine is really needed, right?

I think so. Yeah. Vaccines still needed. Yeah. All right. Today we're going to talk about pediatric hepatitis of unknown etiology. And this is a preprint. that I found a couple of weeks ago from your laboratory. And let's talk about those results. And originally this outbreak took place in Scotland. So tell us how this...

this cohort of kids with hepatitis was picked up? It was picked up by a very astute pediatrician in NHS Greater Glasgow Clyde who noticed that she was seeing a larger number of cases. of hepatitis in children that were completely unexplained and so we're testing negative for the usual. Hepatitis A to E, autoimmune hepatitis and other causes. And she raised an alert with Public Health Scotland. And they subsequently spoke to the UK Health Security Agency and started to pick up.

that there were an increase in cases also in England and subsequently around the world, actually. And now there have been more than 1,000 cases picked up. How would a child... What would a child have? What symptoms would a child have, I guess, to go to the emergency room or to seek medical care, right? Yeah, so the sort of typical presentation is with a gastrointestinal illness first. And then a few weeks later, and there is a gap between the initial...

presentation with gastrointestinal symptoms, then presenting with jaundice and very elevated liver function tests. So I would guess the parent would see the eyes being yellow. Yeah, I think that's probably... how a lot of them have been picked up. Okay, and then you would go for medical care and then this particular... Yeah, and it comes up on the routine blood analysis. How many cases had she seen before she kind of...

I think it was a handful. I'm not sure of the exact number, but it was a handful, certainly well under the hundreds that we've seen now. So is this a system that's in place for... certain syndromes to when you get to so many, because I know, you know, in Wuhan, there's this outbreak of pneumonia. I was unexplained. So that raised the flag.

I think also Public Health Scotland are much more alert at the moment to outbreaks. I think our radar is working quite well just now. That's right. Well, as I was going to say, when you see something unusual like this... Do you say, oh, it's something new or we're just getting better at finding things, right? I think in this case, it's two things. I think we are better at finding things and also questioning when we see something that we can't explain.

But the second thing is obviously that there have been changes in the circulation of viruses following the... removal of measures that were in place to stop the spread of COVID-19. Right. So was Scotland the first country that reported this? Yeah. And then subsequently other countries have as well? Exactly. Many. Many. Yeah. Largely northern European countries or European countries in the US. There have been cases from all around the world, though, just in smaller numbers.

Do you know what the total numbers are? It's over 1,000, and at least a fifth of those are in the UK. And is the outbreak continuing or is it subsiding? It seems to be subsiding, actually, which is reassuring for now at least. And so, yeah, if you look at the curve, it's going down.

And I suppose that in a lot of countries, there's a lot of hepatitis that kind of is not reported or goes under the radar. So it could be an underestimate, right? I'm sure it's a huge underestimate, even in well-resourced countries. Yeah, for sure. So when you have a child with suspected hepatitis, as you said, you look for viruses, hepatitis A through E, right? You can run tests, which I presume you would do in blood, right? We do, yeah.

And you mentioned some other etiologies. So autoimmune hepatitis sometimes presents with jaundice, including in children, and toxins can cause hepatitis. Yeah, there's quite a wide range, but once those have all been excluded, then... Yeah, sure. I mean, there are other viruses as well, of course, that cause hepatitis. Epstein-Barr virus can do it, CMV and so on also.

Do any bacteria cause hepatitis? There are many, yeah. So Leptospira, for example. There are several other bacterial infections that we also would look for. And these were all ruled out? They were ruled out. Okay. And so when she had these kids with hepatitis, which were all about the same time, I guess, right? Temporally. Yeah, exactly. Ruled out. So then she raised the flag and alerted.

Other people. Exactly. And is that the point when you got involved in this? Yeah, that's when I got involved. So I'm part of the ISARIC consortium, which is a really nicely set up. consortium led by other colleagues who have put in place research protocols so that if something funny like this does happen that we can pounce quite quickly and we can take consent and take blood samples and other samples from.

whoever is affected with whatever disease. And so the protocol was turned to paediatric hepatitis. And we were asked both by the ISARIC Consortium and Public Health Scotland if we would have a go at... sequencing samples from all of these children. And the children by then had already recovered from their disease? Some of them had, some of them were still in hospital. So it was very much an investigation.

during the outbreak. And so you could, from the ones in hospital, you could easily request specimens. That's right. I mean, the other ones, we were able to go back and get stored specimens and also to take new specimens. But some of the samples that we have are from quite late on in the onset of illness, which is obviously something that's really important.

And WHO, for example, has reported the investigation from other European countries who have been less likely to report the finding of adenovirus in these cases. has been shown by the UK Health Security Agency to be very strongly associated with hepatitis in the UK in these children.

But you have to take the right samples, so it's better to take whole blood samples rather than plasma. We don't routinely take those and then store them in freezers. And also, obviously, we look at liver biopsies and so on. That can be helpful as well.

I remember a number of reports came out before your preprint with all sorts of different etiologies. One was adenovirus, which we... Puzzled over. Kathy Spindler on TWIV is an adenovirologist. She thought it's kind of rare to be involved in hepatitis, right? And I don't think it was the right serotype even. That's right. So we found type C and F, and the majority of cases have F41.

And that hasn't been associated with hepatitis before. Other adenoviruses in immunosuppressed children have been associated, but it did feel a bit strange, despite the very high odds ratio. UKHSA have found and described. Of course, there were the accusations of COVID vaccination and SARS-CoV-2 infection itself, but none of those really made any sense. And that's when I saw your preprint to say.

This makes perfect sense now. But one thing you do mention, maybe this is jumping the gun, though, but you say that before these outbreaks, there was a spike of adenovirus infections. Tell us about that. Yeah, so we looked at diagnoses. And I have to say that the way in which we did this has its limitations as well. But we looked at reported diagnoses of adenovirus infection in Scotland. So that's... when people present with symptoms and have the test run, et cetera.

there was a huge spike in under five-year-olds in Scotland, not so much in five to ten-year-olds, but in the under five age group. And we found that there was a large... rise in diagnoses reported to Public Health Scotland in that age group. And of course, that's the age group that is affected by this hepatitis. The median age is 3.9 years. These are young kids. And this will make sense in a moment, I think. But what would have caused that, do you think? So my guess about this is that...

There were lots of measures in place for COVID-19. Not just lockdown, which we had, but also extensive measures of hand washing and wearing masks and so on. And I think that will have... for sure prevented the transmission of many viruses, respiratory and gastrointestinal viruses. And so this peak in adenovirus infection probably comes with the release.

of those restrictions and the opening of nurseries. So you now have a large cohort of non-immune kids and the virus enters them and you have an outbreak, right? Exactly. And we're talking about adenovirus here, whereas... Without restrictions, they're just infected here and there. You don't see any outbreaks. Yeah, kind of sporadically, right? And it's probably not just one virus, but many that are increasing now. Now you're tasked to work on this.

I was thinking, wow, I could think of so many things to do. Did you have a big committee sit down and decide what to do? We did have – certainly. So there were – incident management team meetings which were led by Public Health Scotland and then also there were research meetings which involved the ISARIC consortium and internally at the CVR because we have a strength i guess in next generation sequencing we thought that we could offer that to see what we could find

any viral cause. And we were very specific to say we would find it quite difficult to look for bacterial genomes and so on, which requires a bit of a different approach. Although we had generated data that could be used to look for that as well. So we said we'll sequence everything, send us all the samples. So you said just send us whatever you have or did you request certain things? I said send everything. Send everything you have on these children. And what did you get?

So initially we got from nine children, and that's widened now to 30 children, but our first sweep and the preprint describes nine cases. We sampled serum or plasma samples from those children, sometimes multiple samples that were available. We also sampled four liver biopsies and we did stool samples and one rectal swab, I think we were given, and we had throat swabs as well. So those were the samples that we were initially given.

And what did you do with those? So then we ran those with two protocols. So one with standard metagenomic sequencing, where you sequence everything. in the room, if you like. And then the second was we used the Roche-Vercap probes to purify viruses. So these... basically pull out viral nucleic acid, and they're quite nonspecific. So the theory is that they can pull out anything from any viral family. So this is Vircap-Seq, right? Yeah, exactly.

Where you use antibodies to pull out viruses? It's actually oligonucleotides. So these are oligos which pull down. matching oligos, but they have a lot more give in them, for example, than primers. So as long as they match by at least 30% or so, you should pull down the virus. And we've used them quite a lot, actually. recently in Uganda as well to look for emerging viruses there. And we found a number of...

emerging viruses, which haven't previously been described, using this technique in mosquitoes, for example. So you extract the nucleic acid first, obviously, right? We extract nucleic acids, both RNA and DNA, and then we have a separate protocol. So RNA, we treat with DNAs to increase the chances of having enough RNA to pre-like full genomes. And then with DNA, we then... run with probes. You can deplete as well host DNA with these samples. Okay. Yeah. You also had controls, right? So we...

looked really hard for control. So initially we ran the cases and then we ran more cases and controls. But getting controls was really quite a challenge. We wanted to answer a number of questions with those. The first controls that we looked at came from the Diamond Consortium, which is a paediatric study run by Mike Levine at Imperial College. And they gave us samples from healthy children who are age-matched.

and taken from between 2020 and 2022. So not exactly the same time period, but very close, as close as we could get. So we ran those as healthy children. And then we thought, actually, we also want to know what's going on with children with adenovirus infection and to see whether or not they might have, after we've found our initial findings, we wanted to know.

if we were going to find the adeno-associated virus, which showed up in these cases, in children who had adenovirus as well. And then finally... We had a third group, which was of controls with severe hepatitis. These are kids. Some of them had been very critically unwell with very abnormal liver function. for other causes and the reason we looked at them was to see because AAV2 which I guess we'll come on to talk about in a minute can reactivate in severe illness and there was a sort of a

a view from many people saying, well, you know, AV2 reactivates in everybody with hepatitis. And so we looked at 33 children. who had severe hepatitis of other causes. Those were not age-matched, but they were still children. Okay. So your initial patients with hepatitis, these, had they been... Had they had COVID or SARS-CoV-2 infection? So some of them had. We find evidence that six of nine of them had antibodies positive. And yeah, so three of them hadn't obviously had COVID.

And none of them had been vaccinated. They were too young to have been vaccinated. Okay. And I think you also said none of them had immunocompromised conditions. Yeah, no, they were all healthy children, healthy children. And one other thing we noticed about the children, or in fact my colleagues, the paediatricians noticed about the children, was that they were ethnically not diverse, so they were all white Scottish children.

Which made us wonder a little bit about genetic associations as well. Is that typical for the outbreak in general or just the one in Scotland? I don't know, actually. My understanding is certainly in the UK that that's probably the case. But the rest of the world, I'm less familiar with the data.

Okay. So then you did the sequencing that you described and you used your cases first. So you got the result before you did your controls, right? Yeah, we did. And what did you find? Was it a Sunday morning at 5 a.m.? That sort of thing, yeah. We tried to do it very fast because the children were quite sick. A lot of them were hospitalized. And there have been quite a large number of transplants, including one in Scotland and several in the rest of the UK.

And obviously that's a life-changing event for these young kids who've otherwise been fitting well. So we wanted to do it really fast. And we looked at the data and found...

We did find adenovirus in some of the cases, so we found that in six out of nine of the children at various sites. We didn't have the most optimal blood sample type, so we didn't have whole blood from these children. We looked in plasma. But we still picked it up in six of nine of them. And we also looked, obviously, at throat swabs and stools. And we got a full genome from the stools of one of the kids, which was an F41 adenovirus, in keeping with other reports.

were adenovirus that had already been described by others. How was that transmitted, that particular adenovirus? It's usually a gastrointestinal infection. All right. And then we, but what was really striking was that we found this virus, adeno-associated virus 2, in all of the children in nine out of nine of the cases in plasma samples. Was that a total surprise to you? was a real surprise and my first thought was what have we contaminated our samples with and

We looked very carefully at all the reagents, what had been used. The extractions had actually initially been done in the clinical virus laboratory. We looked very carefully at what was used there. We repeated samples and then of course subsequently now we've done much more testing. And the controls as well, right? Yeah, it fitted. And the other thing that was important, actually, was that the sequences were not identical. So it looks like there are two lineages. Most of these were very...

They were different from each other. All of them had changes other than three, two of which came from the same household. So we know those were related. And one from the same time period, although we didn't know that was a related case. control cohort that you described did you find AAV2 in any of those we didn't find AAV2 in any of the controls so 58 controls all negative for AAV2 and then you had kids with

Known hepatitis, right? Other causes. Did you find it in any of them? We did not find it in any of them at all. And then I actually, I didn't put this in the preprint, but I looked through all 5,000 samples that I've sequenced in the last few years. from various cohorts, including hep C cohorts and also children in Uganda. And I found one positive. So tell us about AAV2. What is it?

How do you get it and so forth? So AV2 is a parvovirus, very distinct from the adenoviruses. It's a dependo parvovirus, so that's the genus. And it requires... a helper virus to replicate. So it was first described in cultures of adenovirus in around 1965 when they found it as a contaminant.

When they were trying to grow adenovirus, they found this AV2. So it's very commonly found in association with adenovirus infections. And there are various proteins from the adenovirus that can support the... The transition of the AAV2 virus, which can exist in latent form, integrated into chromosome 19 in a very specific site. It's quite interesting, actually. which is called AVS1, and that site...

There's a sort of preference for it to integrate in there. And it can also exist in an epizomal form as well. But when you get an adenovirus, that can allow the virus to become lytic and replicate. And there are several proteins that are produced by the adenovirus that will allow that to happen. And then you get a productive infection. Yeah. So how would you acquire AAV2?

So EV2 probably is acquired, I think, through respiratory route. It's been sort of, I guess, I don't know for sure that it's not also... acquired through a more gastrointestinal route as well. And most of us are infected, or seropositive at least, by... Yeah, most people are seropositive by the time that they're 18, so 90% of us will have had it. And most infections are asymptomatic, right? And most infections are asymptomatic, and of course...

The animal-associated virus has been used medically for gene therapy, which we might come on to, but it hasn't been associated with the disease before, and it's very common. So we were left with wondering... You know, is it just a sort of coincidental outbreak or something that we're picking up? Obviously, just an association like this doesn't equal causation. And we've gone on to do a lot more work now too.

try to explore whether or not the AV2 itself is causing disease. So presumably these kids would be infected in normal times and you wouldn't get any hepatitis, right? Yeah. So one of the things that we... was that the kids that are getting hepatitis, 89% of them have got a particular MHC class 2 HLA association, which is HLA DRB1. Oh, yes, I know that one. And, yeah, it's actually quite a well-known...

MHC class 2 allele. It's got an association, for example, with autoimmune hepatitis in adults. Okay. And it also has an association with things like Felty syndrome, which is the sort of extra articular manifestation of rheumatoid arthritis. So it has got associations already with the disease, not so much in children, though, and not with autoimmune hepatitis in children. But this allele is present in about 15% of Scottish blood donors. It is quite geographically...

differentially distributed. So it is common in Northern Europe, a bit less common in the US actually, and definitely a bit less common in other parts of the world. So this HLA association, you think, is somehow involved in the hepatitis? I think that's...

Really likely. What do you think is the mechanism? Well, I think it's pointing to an immune mechanism for the pathology. And so class 2 MHC associations usually indicate that CD4 cells are somehow... because MHC Class 2 is what presents to CD4 cells. And so my... and this needs a lot more work, is that there will be a peptide that may be presented, or more than one peptide, presented by this particular allele, and that that is inducing...

particular type of immune response. I guess there are other alternatives though. This could be rather than a direct immune response direct against the virus, it could actually be a... It could be molecular mimicry or something like that, and an autoimmune hepatitis triggered by AV2. Yeah, so there are definitely a number of possible explanations. But my feeling is that...

So now we've done further work, which is as yet unpublished. But we have actually found the virus in the liver of these children by in situ hybridization. So we can see... that the cells which are buried have a very typical appearance of being what's called ballooned. So the hepatocytes are sort of blown up and they have this feathered appearance. And right in the centre of those in the nucleus.

You can see AAV2 RNA, so we've stained for that. And you can also see it in the cytoplasm. So we've now seen that in all of the liver biopsies of these children that we've looked at. Yeah, and some of them have it in the cytoplasm as well, which would indicate replicating virus, I think. So as you said, AAV2 requires a helper. So do you find...

And that can be adenovirus or a herpes virus. Yeah, it could be a herpes virus. And we have found also in these children HHV6, so human herpes virus 6, specifically 6B. We found that in a couple of the liver biopsies, and we found it also in some of the cases in blood samples. However, we also found a very similar number in the controls that we looked at of HHV6.

I think that HV6 is reactivating as a result of severe illness in these children, either the controls who had critical illness of other cause or in the children who were very unwell with this. hepatitis. So we didn't find a significant difference between cases and controls with HHV6, but we did detect it in a number of cases. And certainly...

it's possible even that three viruses might have an interaction. We are staining for those viruses at the moment in the liver to see if they're in the same cells and so on. I don't have that data yet. We're doing it this week and next week. So, again, most kids get HHV-6 early in life, right? Yeah, they do. It's a rash disease? Is that the roseola subantham? Yeah, so it can cause a rash and it can cause hepatitis.

Definitely as well. On its own, right? Yeah, on its own. And then it becomes latent. And then it becomes latent and can reactivate during periods of illness. Some kind of stress, yeah. All right, so that might have infected them.

several years before, and then maybe the AAV2 came post-pandemic, right? And then in this particular HLA cohort, it triggered the disease somehow, right? What's not clear to me is if the children might have been co-infected. with the AV2 and the adenovirus at the same time, or even all three viruses at the same time. That's possible as well. You mean once...

After the pandemic, when they were starting to go out into the world. Yeah, and there's lots of viruses mixing in the environment. Yeah. You probably don't have any... older samples from any of these kids, right? No, sadly. Because that would be perfect. It would. You could say, when did you acquire your... But we are looking at IGM responses and also IGG to look at the trajectory of those. Since there's an AAV2, there must be an AAV1, right? There is. So why AAV2? Is there a geographic?

preference for circulation of this? Is it a serotype, I presume, right? Yeah. So there are at least 12 of these viruses that have been described and they can infect humans and other primates. AAV2 is one of the more prevalent ones, and I think it's fairly common around the world, actually. So we don't really know a lot about the epidemiology of AAV2, or at least we don't.

It's not the kind of thing that we monitor standardly by public health and so on, unlike other viruses that are monitored really frequently. We don't follow AV2, so the patterns... And the distribution are probably not very well documented. Are the other AAVs also hepatotropic? Yeah, so they do vary in terms of hepatotropism. So, for example, AV8 is even more hepatotropic.

These viruses have been used for those characteristics as gene therapy. And yeah, so the tropism is probably guided by the cap. So these VP1, 2, and 3 proteins likely determine which cells these viruses will enter. AV2 is hepatotropic, but there are some that are even more hepatotropic than AV2, AV8, for example. And it can also get into smooth muscle cells. Smooth muscle endothelial cells, particularly, can get into some of the neurological lineages as well. So we have two...

viruses that commonly infect kids. And then we have the HLA, which is out there. Yeah. And so why haven't we seen this before? So I think this is a triple hit phenomenon. Okay. And then it must be quite uncommon because...

So I think the circumstances have led to a mixing of viruses in the environment. There's a genetic underlying susceptibility. And we have now found that 25 of 28 children have got this HLA alley on. I would have been less confident with 8 out of 9, but 25 out of 28 does show that the allele does look like it's associated. And so, you know, that's roughly 1 and 9 or whatever of the population.

carry that. Is that Scottish or world? Yeah, it's commoner in Scotland. It's a little bit less common in other parts of the world. Your thought is that they got all three at once. And it wasn't a matter of them having one and then getting the other coming out of lockdown, say, because...

That would have happened before. You should be able to see that, right? Yeah, that's my guess. Is it something you can... We're trying to dig it apart. So we're looking at IGMs to see whether or not these look like acute infections. It would have been... perfect to have preceding blood samples from these kids. It is the age at which people do get infected with AAV2. So under five, this is when children are largely first exposed to AAV2.

And I think getting both at the same time might increase the risk of this. But of course, it could be that they've been... pre-infected with AV2 or even adenovirus, actually, and that the AV2 has reactivated rather than causing acute infection. Did you find adeno in any of them?

Because that can be a helper too, right? Yeah. In the nine initial patients. Yeah, we did. We found adenovirus in six of nine of those as well. So some of those kids had all three. Some of those kids had all three. All right. Yeah. Interesting. So I'm suspecting that this has happened before. We just never picked it up. I think so. We need to go back and look at samples from children with unexplained hepatitis. And I think...

If I'd been asked to go and maybe run a couple of samples of metagenomic sequencing of odd sporadic cases of severe hepatitis in children, I'd have said, I don't think we can do it because we won't know what to make of the data. And so I think this has led to a larger number of cases and a more solid finding. It makes you wonder before. metagenomic sequencing we wouldn't have been able to pick this up right because you very hard to culture these viruses right

I mean, I think it's how people felt when they first started using microscopes, right? It's like a subtle knife with Philip Pullman. You know, you're sort of opening a window into another world. This method. So outside of Scotland, has anyone else made similar observations? So my understanding is that other countries are now looking.

What I would say is that my colleague Judy Brewer at Great Ormond Street Hospital has also got a preprint out and we've been communicating very closely. And she has also found AV2. in the cases in England as well. So it's been found in the UK and then other countries are now looking. I looked on the CDC U.S. website. They said, we're still looking. Okay. I'm really keen to find out. I'm sure they will find something. So one more thing about AAV. As you said, it's been used.

being used for gene therapy. There's an eye, there's a blindness treatment already approved and many others. So does this have any implications for that? I mean, is there a problem with kids who might get it as gene therapy? So I think...

Medicine's always a sort of balance, right? And these gene therapies are extraordinary advances. And so gene therapy is just a phenomenal achievement. And however... A small number of children, and it is a really small number, have had fulminant hepatitis following AV-vectored gene therapy. And there have been at least four cases reported with one of the AV vector gene therapies. Now, also...

Deranged liver function tests in children who receive gene therapy is extremely common. And it's so common that people in the field know how to manage it and they use immunosuppression to treat. deranged liver function which is generally fairly mild and very treatable with immunosuppression and so it may be that we're seeing a kind of similar phenomenon and that

I do wonder if these kids who had the very rare side effect of fulminant hepatitis, what their HLA type is. I imagine people will look at that. So that could... That kind of hepatitis could occur in the absence of a helper, right? Yeah. So yes, it can, because the gene therapy is delivered with... with helper protein so they actually give it as part of the treatment so what what they do is the the cap proteins are made in trans in the laboratory and then

The virus particle is used to deliver the gene of interest. It's encased by these inverted terminal repeats. And the rep and cap genes, which are normally inside the wild type virus, are completely removed. And so all you're left with is the ITRs on either side. And that allows the... That's used to introduce genes which have to be of a certain size. So the labors, amorosis has been treated with this. And there are a number of hemophilia B, for example, as well. So anything which is a...

got a gene size of like around 4kb can be put in in this way. But it's a sort of transient exposure and it's thought that... The understanding in the gene therapy field is that the hepatitis that they sometimes see is associated with T cell mediated response against the... cells which have been, you know, in which the vector gene therapy is being delivered. And so, yeah, so it's possible that with this sort of very kind of...

controlled delivery, the likelihood of hepatitis or severe hepatitis is much lower because obviously with a wild type infection you can get uncontrolled replication for quite some time. But I think it may be a related phenomenon. In these cases of hepatitis after vector administration, they haven't looked at HLA types. Not as far as I'm aware. Maybe they're looking. I hope so. Now they should. I think the really important thing there will be to look at the ones.

who had fulminant hepatitis, because I think my understanding is that a little bit of hepatitis is extremely common in most people who get these gene therapy, AV-vectored. treatments. But there are no viral, I guess the captured proteins would be the ones that the T-cells are directed against, right? Which would be doing that, yeah. Okay. And this is the HLA association that we find, of course, would be in keeping with that as well.

Now, in terms of long term, once you have the vector, then it's my understanding that the DNA remains episomal for long periods of time. So there are no more capsid proteins left. So it shouldn't be a risk in long term. Exactly. Exactly. So they don't tend to integrate these gene therapies. They're manufactured so that they exist in episomal form for many years.

Yeah, I mean, it's extraordinary, isn't it, that you can replace these genes. Well, it's interesting that the non-integration was a chance finding because if you have a replication-competent AAV, that can be...

that has REP and CAP, it will integrate. But if you take all that out, it doesn't. Yet it remains episomal, as you know, for years because the blindness treatment lasts years. And the results are extraordinary. I think gene therapy, it's very controlled. They know what dose to give. In fact, they know hepatitis is usually associated with the higher dose gene therapies. And so the kind of doses that they're using now, these risks are very, very low.

I'm sure that you can modify the capsid to get rid of the hepatitis, right? I bet, yeah. If it's a different receptor than in other tissues. But the other vectors, AAV vectors are developed for like muscle. As you said, it does reproduce in muscle for muscle diseases and so forth. So it's an interesting consideration. So in terms of the pediatric hepatitis, is there anything that needs to be done that you can tell us?

Well, we need a large case control study, so much larger than we've done now. And we can do that with serology as well as with... So I think serology will be useful because not all children have been exposed to AV. It would be much more... tricky in adults because most of us have been exposed. So I think that's something that has to happen as soon as possible.

So that would involve more kids with pediatric hepatitis, unexplained pediatric hepatitis. Yeah, so all the samples that people have stored away from these kids, I think, should be tested, both by PCR and by serology. And then, of course, we need proper controls. And they need to be time and age controlled. Okay. And then you would look for what in these cases?

In the case control study, I think we want to definitely look at IgM and IgG responses to look and see if there's evidence of a more recent infection versus a kind of longer term infection. Although it is possible that reactivation might... cause a bit of a peak in igm as well but i guess it would help us and then uh yeah we obviously need to compare to good controls

I think there's a lot more to be done molecularly as well to try to understand what exactly is going on. And if we could maybe, for example, find a model for this. I think maybe a mouse model would be something. worth trying but it could be quite tricky we might want to you know use a humanized mouse model and think about expressing

MHC class 2 and so on, and it could get quite technically challenging. But then you could infect with two viruses or three and see if you duplicate the disease. Exactly. I think that's needed. Does this... So are you going to have a name for this syndrome? Have you thought of that? I haven't thought about it. Have you got a suggestion? I don't have one, but you need to make something that distinguishes it, right? Because it's an unusual syndrome. I guess you have to find out if it's...

AAV2, HHV, and perhaps adenovirus, O3. And I think that should guide your naming. right? AV2-associated hepatitis or something? I think AV2-associated hepatitis would work really well. Yeah, because you have hepatitis A, B. Et cetera. Yeah. Just add AV2 to the list. But I think we do have a little bit more to do. We've seen a strong association, but for causation, there's more work to do. Okay. But that leads to the other question, which is... You're going to find this in more kids.

How does that guide treatment then? Because when a kid comes in with hepatitis, this will be added to the list, right? AAV2, HHV6. So what do you do then? So I think there would be room for consideration of two approaches. I think the first thing to bear in mind is that these children, their illness is suggestive of an immune pathology. It's just the pattern of having had a gastrointestinal illness followed several weeks later by hepatitis.

would be in keeping with an immune pathology, but it could also be directly related to the virus. And so I think the two approaches that should be considered would be antiviral therapies.

and immunosuppressive therapies. And I think we probably can learn from the gene therapy field. And I am aware, and this is very anecdotal, that a number of the children have been treated with steroid therapy, which is often used in transplant units and so on to try to avert fulminant hepatitis and the need for liver transplants.

Trials are required for this, and so I don't think any recommendation on treatment right now is possible, but I think that immunosuppression would be one avenue and antivirals. So that would be important because... If you don't know what the agent is, you don't know what to do, right? But this would give you clear paths, especially if you've...

tested them in clinical trials. Yeah, absolutely. Are there any antivirals that could be used? There are one or two antivirals that could be used for DNA viruses, but I'm not aware that there's much data at all for AAV really to support their use at this time. The real question is whether by the time you see hepatitis, whether it's too late to treat, right? With an antiviral at least. We have found it in the liver.

and probably replicating in the liver. So there may be a rule for antiviral treatment. That's something you'd have to work out in a trial. Exactly. There's no other way of doing it. Yeah. I mean, as we clearly know from... SARS-CoV-2, when you can give an antiviral and then when you have to do immunosuppression. Absolutely. And yeah, I think we've learned so much about that in the last two years. Yeah.

What about surveillance? Yesterday in my TWIV with Richard Knight, we talked about the importance of surveillance and knowing where things are. What are the implications of this work for that? Well I think that we should be using sequencing to carry out surveillance more. We do have to work out what we're going to do with the data. So, you know, we look at wastewater, for example, in the UK and we find recently polio. What do you do with that information?

I think it's really valuable because as a physician, I do spend about 20% of my time working in the NHS. I think I would find it quite useful to know that there was poliovirus nearby in the sewers. If I saw somebody with paralysis, I'd think about it. You know, I hopefully would think about it anyway, but it would raise my awareness. And so understanding what's in the environment.

with wastewater looking at, for example, causes of severe respiratory infection in people in our intensive care or critical care units. I think we should be doing that routinely. There are viruses around that we don't. Of course, sometimes that doesn't matter. But I think we've underestimated viruses.

And we should be looking quite hard for them. So what you're saying is if someone has an illness that's unexplained, you should be doing metagenomics. Well, I think if someone has a mild illness that's unexplained, it doesn't matter. Okay. Unless perhaps they would like to go to some commercial company or something and send a swab or whatever. I'm not sure that it can be justified in the budget, you know, even of a high income economy. But people who are critically ill...

Yes, I think we should be considering it. It has to be at the right point in the illness. We know from our work that if you wait too long, it's quite difficult to pick viruses up. There are other methods that are interesting. I don't know if you've ever sort of like covered the kind of FIP-seek methodology, which this is a really nice method where you express.

For example, glycoproteins, you can do hundreds of viruses all at once by phage display. And then the protein is... expressed on the surface of the phage, and then you can pull it out with serum or extracted antibodies. Supposedly, you can get your entire life history of everything you've been exposed to. By looking at the antibodies they find. Yes. So we could be doing more high-throughput serology as well to diagnose patients. I think we'll see a revolution.

the use of sequencing-based diagnostics in the coming years. Well, you know, some countries still need to get their medical records in order. Yeah. Nonetheless, do sequencing. I mean, I agree with you, but... You know, in the US, we don't have a universal records, right? Right. Which you do have in the UK, right? We do. Yeah, to a certain extent, it could still be better. And, you know, actually one thing from COVID is that we've learned how to link records much, much better now so we can link.

records with genomic data, for example. And there have been huge initiatives, HDR UK and others, to try to get safe data linkage. I was looking at my... my badge here it says the united states but they're not united each state is individual and that's why we have fragmented health care and health care records but you know we have that in the uk as well so i can access all the scottish data but not the english data it shouldn't be that way

So you mentioned polio, which is an interesting example because you're obviously doing some wastewater screening here, but we have not in the U.S. ever. done polio wastewater screening. And, you know, we started to do SARS-CoV-2 for obvious reasons. And we had some of these people on TWIV.

I told them, you should look for polio. Nah, why? Why should we look for polio? And then, of course, when we had the case, they started to look. And they're all looking now, and they're going to find it. But that's an example of... Don't you want to know if there's polio in your neighborhood? Yes. I mean, if you find it in wastewater, it means it's in someone. Yes. Right? And so you might want to get vaccinated. So this whole idea of surveillance and monkeypox is the same thing. Yeah.

Do you know, what are you doing for monkeypox surveillance in the UK? Do you have any idea? Yeah, we're involved with that. CVR is involved with lots of centres across the UK. So we are sequencing monkeypox. It's trickier than other viruses. It's got a bigger genome. Also, it evolves less rapidly and in different ways.

Yeah, lots of us are now trying to learn a lot about pox virology. But we are looking and I think it will be helpful to understand more about monkeypox. I think sequencing is one route. It may not be as helpful as it has been for SARS-CoV-2, for example, although everyone said that at the beginning of the COVID outbreak as well. And I think that case has been pretty much made now.

But also we need to look for new treatments and screen. We're growing the virus, for example, at the CVR and also in other sites in the UK, Portham and so on, we'll be doing it. testing antivirals to see if we can improve treatment for people who get it. Yeah. The situation in the U.S. is that you can't get a diagnostic test for monkeypox unless you have a lesion. Ryan.

That doesn't seem right. Shouldn't we know where the virus is and just screen more people, right? Yeah, I mean, I think there's a good... Well, there have been some data recently showing that some people who are, you know... Some people may be relatively symptomatic. And there are also cases in... The virus seems to have found a niche in gay and bisexual men who have sex with men. And so there have been cases of isolated rectal disease as well, which obviously wouldn't be seen.

I think there is a very good case for doing a case control study in partners of men who have, or women who've had the disease and see if they're...

they're infected and asymptomatic. I think that would be where I would start. I mean, I heard that in the UK, in some STDs clinics, they're taking the same samples and looking for, you know... gonorrhea also look for money monkeypox just getting an idea but that's biased right yeah it's a biased population shouldn't we be looking more broadly like shouldn't we know if it's in kids in school yeah i think

It's tricky, isn't it? Because that requires a lot of resource. But I think we need to be looking very carefully at making sure that certainly at the very least that general practitioners, for example. who may see children, paediatricians, know how to recognise monkeypox versus chickenpox or other diseases that cause rash. Because it is pretty characteristic, that rash. There have been quite a lot of atypical cases.

there's a very characteristic appearance. And at the very least, people should be on heightened awareness. And obviously, if we start seeing this in children, that's a massive concern. And we could be doing... limited surveillance in kids, for example, with chickenpox-like rashes, without testing everyone, but just looking to make sure it hasn't gone into that population, which would be a big concern. My colleague Daniel Griffin.

told the story of a physician seeing a boy with a belly rash, which he then treated with freezing, and it was monkeypox, and it made it worse. Oh, right. Yeah. So there's an example where you should, if you're in doubt, you should just test and take a swab and send it in. I mean, we're allowed to do that. So there is a part in the guidance which says under physician discretion. So if you think the rash looks very typical or suggestive or you have a concern, it is possible to test.

Yeah, I mean, this is just an underscoring of how important surveillance is, especially if you're not sure where you are, right? We're at the point with SARS-CoV-2 and COVID where we don't need to do that anymore, right? If you're sick, we can... do tests, but for monkeypox and polio virus. And now you're AAV associated hepatitis. We should do it, right? Yeah, we should. One more question is, I wonder if you think we're going to start to see other unusual.

associations of viruses with clinical syndromes that have arisen as a consequence of the pandemic, right? Yeah, I mean, I think this idea of co-circulating viruses causing a serious problem is really important. I guess we don't know what we do when we introduce major measures. Not that I'm suggesting we shouldn't have done it because it saved a lot of lives, no question. But the consequence of that is unexplored.

The other thing is that if you get two viruses in a cell, who knows exactly what happens? I mean, lots of them interfere with them, interfere on pathways. the immune response to, you know, in many ways, and they've evolved to do that. If you have two together that do that in different ways, there could be all sorts of consequences. Now, there have been papers, Glenn Royal.

who's been on Twitter, has a wonderful paper showing when you have two viruses and you infect an animal, you get two different diseases. But when you co-infect, you get a third disease, which is different from the first two. And Daniel Griffin always says, You can have more than one infection. He says, okay, if you have a patient with a respiratory illness and they test positive for SARS-CoV-2, don't just say they have COVID. Maybe they also have influenza, right?

absolutely and I think our eyes are opening to that with these sort of enhanced methods diagnostic methods that we have it's tricky And, you know, we also pick up viruses which are not pathogens very commonly when we do metagenomic type sequencing. And it requires a real eye. It's a bit like as an infectious disease physician, you have to learn how to interpret blood cultures.

And sometimes you see bacteria there which have come from the skin surface and are of no consequence. And now you can see many more than what you get from culture. we do have to learn it's going to become a real sort of expertise and speciality how to interpret these data properly. Well, when you did the metagenomics on the kids with hepatitis, you obviously saw a lot of other viral reads. We did, yeah. But the ones in very, very high numbers were...

AV2, right? Yeah, AV2, very high numbers. All other viruses, very low, including adenovirus, actually, and also HHV6. But we also picked up one or two other viruses. We find norovirus, for example, in one of the children. And there are other viruses around, obviously, in hospitalized children. But this was the thing.

you know, let up because it was in all the cases and none of the controls. Well, there was a talk this morning about making panels, diagnostic panels for specific respiratory viruses, right? Instead of doing, you know... Massive multiplex, which would be confusing, or metagenomics. You could say, okay, it's winter. We're going to look for SARS-CoV-2, influenza, respiratory syncytial virus. Maybe that would help.

Yeah, I think syndromic screening is exactly the way to go. So somebody with meningitis might have one panel, some of the respiratory illness another. And for your disease now, you would add for hepatitis. Is there a hepatitis panel, syndromic panel? I mean, we have a list of tests that we run clinically for hepatitis, but a syndromic panel as a sort of diagnostic test.

No, I don't think so. I know what I might put in it. These biofires. Like biofire and so on, yeah. There are panels and there are also multiplex PCR panels and so on that people use for sure, yeah. of today's discussion is that now for hepatitis, you have to add AAV and adenovirus and herpesvirus. Yes, please. And I'd really like to know what... How many people are coming up with AB2 in the US, for example, and other countries?

Would this be just for pediatric hepatitis? Yeah, I think so, actually. I suspect this is just for pediatric hepatitis, certainly at this point, because most of us have had it by adulthood. I mean... There may be studies in the future of unexplained hepatitis in adults, but for now the data... really is for children. But if you have an adult with the right HLA type and all these viruses, why not? Why not? And I think that it would be good to look at recipients of gene therapy as well.

All right. And this is about to be published, right, in the next few weeks? I hope so. Not just the original nine, but more cases as well. More data, yeah. We looked at 30. All right. Very good. All right. That's a special TWIV at ESCV. You can find the show notes at microbe.tv slash TWIV. You can send us a question or comment to twiv at microbe.tv. And TWIV is part of a bigger network of science podcasts that we do.

to educate the public about science, and we need your support to do it. If you'd like to help us, you can go to microbe.tv slash contribute. My guest today from the University of Glasgow, Emma Thompson. Thank you so much for joining me. Appreciate it. I'd like to thank ESCV Organizing Committee for having TWIV here, in particular, Haley Harvilla, who suggested this particular session, which was a great idea. And I'd like to thank the American Society for...

Virology, the American Society for Microbiology, for their support of TWIV, Jolene for the timestamps, and Ronald Jenkins for the music. I'm Vincent Racaniello. You can find me at Virology. You've been listening to This Week in Virology. Thanks for joining us. We'll be back next week. Another TWIV is viral. Thank you. Gracias.