Uncovering Zika infection in corneal cells – an interview with Dustin Farr

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Since Zika broke into the news in 2016, new research has been uncovering more information about this virus – from its pathways of pathogenesis to methods of transmission.

In light of this, we spoke to Dustin Farr (Wayne State University, MI, USA) at the recent ASM Microbe meeting (7–11 June, GA, USA), where he was presenting a poster on Zika infection in corneal cells. Dustin told us about the research that led up to this poster, the potential for Zika transmission via corneal transplantation and the challenges facing the development of a vaccine for this virus.

Listen to the podcast, or read the interview in full below.

00:55 – Introducing Zika in corneal cells

01:58 – The role of ISG15

03:57 – Translation to the clinic

04:47 – Zika transmission

05:47 – Research towards a vaccine

06:36 – What does the future hold?

Martha: Hi and welcome to Infectious Diseases Hub, I’m Martha, the Editor, and today I’m joined by Dustin Farr, a Graduate student from Michigan.

Dustin: Okay so I’m from Michigan, Detroit area originally and I stayed in Michigan for my Undergrad, I went to Michigan State (MI, USA). I was a Clinical Laboratory Science major, so I was definitely more bacterial when I started out and I worked in a hospital for 2-3 years. Then I went back to school at the University of Michigan, I got my Masters in Public Health in Hospital and Molecular Epidemiology, where I did research in Gary Huffnagle’s lab who works on lung and gut microbiome, so that was my  first big research experience and that got me into wanting to pursue research farther.

Martha: Dustin is currently at Wayne State University, working on his thesis; however, he presented some research at the recent ASM Microbe meeting in Atlanta, which although not his main focus, was an interesting insight into Zika virus and its infection of corneal cell and we’re about to find out a bit more about this.

Dustin: So my project is looking at corneal cell infections. Most of the work that’s been done with Zika has been in retinal tissue, that’s the big cell type we’re all worried about, but a recent – well it was  about a year and a half, two years ago now – study in vivo in mice showed that not just retinal tissues being affected. They saw Zika virus RNA in tear secretions, conjunctivitis, lacrimal gland cell infections and corneal cells was the big one. So there are a number of other cell types and areas being affected in the eye, not just retinal tissue.

With those being really new findings we wanted to look at human corneal cells, see if those are being affected and so that’s what we based our study on. We did time points at control 24, 48 and 72 hours where we saw a really nice infection with a couple of  different strains of Zika virus, then we looked at the innate immune responses – recognition receptors on the cells, cytokines, interferons, things like that.

Martha: Dustin mentioned a protein termed ISG15, which among other functions has antiviral properties, and told us a little more about this, including the results of a study his lab did in mice which investigated the effect of ISG15 on Zika virus infection.

Dustin: So our lab has done a lot of work on ISG15. ISG15 does a number of things but it also has direct antiviral properties for a number of different viruses – HIV, Ebola, all kinds of stuff. So one study that we did, which I wasn’t a part of but was done before I was in the lab, was with ISG15 knock-out mice and with those knock-out mice there was a much higher rate of infection.

We kind of focused on ISG15 for this study [in human corneal cells]too, there was a huge increase at 48 hours of ISG15 so then I knocked out ISG15 with siRNA, I added recombinant ISG15 protein and with the recombinant protein you have a huge decrease in infection, whereas with the siRNA when you knockout the ISG15 you have a huge increase in infection. So it just shows that ISG15 has a big role in Zika virus infection as well.

I’m not quite done with the whole study yet! Going forward, I’m probably going to be looking at; what is the signaling pathway of ISG15? So TLR3 is a big viral receptor and I think it is maybe going through TLR3, I’m going to be doing those same experiments with TLR3 – knocking out TLR3, seeing what the ISG15 levels are, seeing what the infectivity levels look like. I’ll also look at RIG-I, RIG-I is a big viral receptor as well, so I’ll be looking at doing those same experiments.

But then we also wanted to compare with dengue virus, using all four strains of dengue virus I looked to see if any of them would actually infect corneal cells and dengue virus 3 was the only one that did. Then we compared the immune profiles for those, and there’s much earlier increases for cytokines, interferons, the recognition receptors, all those things – much higher earlier on as compared to Zika virus. That has kind of wrapped it up to this point.

Martha: With exciting results in in vivo models, I asked Dustin what implications his findings might have when translated to clinical settings in the future, including the risks of Zika infection could have in corneal transplantations and Dustin explained a little more about this.

Dustin: Yes, a big thing with this is the clinical significance because corneal transplantation is actually one of the highest numbers of transplant procedures performed, which is really surprising, estimates of around 200,000 last year. So with Zika virus, a lot of people don’t know that they have an infection, they’re asymptomatic; reports of around 80–90% of people don’t know they have it. When you don’t know you have it and it can be transmitted sexually you can pass it on. You can see a situation where someone has the virus, they don’t know it and then their cornea is transplanted and it’s passed on like that. So there is definitely big significance clinically.

Martha: Of course, there has been a lot of research around Zika since the Public Health emergency was declared in 2016, with new methods of transmission being discovered, including sexual transmission and this possible new route via corneal transplantations. Dustin told us a more about what he thinks is left to uncover in terms of Zika transmission.

Dustin: Yeah I think a big thing right now is figuring out reservoirs where the virus can persist, especially sexually transmitted ones. There are reports of Zika virus being detected in seminal fluid for up to 6 months, so that’s really a big thing.

For us in the cornea, how long is it going to persist in the cornea or in the retina because the eye is an immune-privileged site it could persist in corneal cells or retinal cells, so I think that’s really the big thing is seeing where its persisting, how long its persisting for and how that’s going to affect transmission.

Martha: Do you think that’s a big priority in terms of research on the virus?

Dustin: I think so, definitely. There have been studies in our lab looking at how long is the virus hanging out there, so it’s definitely something that’s very important.

Martha: Despite huge publicity, since 2016, Zika has moved out of the news slightly and Dustin shared his view on this and the importance of keeping Zika in the public eye to meet targets such as developing a vaccine.

Dustin: I think it definitely needs more focus, I think a lot of the times when there’s a big outbreak and you have things like microcephaly the media picks up on it and it’s kind of a hot topic – it was like we don’t know what’s going on, we don’t know why all these microcephaly cases are happening and it’s a shocking image so it’s kind of gone away because the dynamics in Brazil and Central America have calmed down a  little bit but the research definitely needs to be there for things like a vaccine. There have been some trials that I think have reached Phase II a couple of them, so there’s still not a good vaccine for Zika but a lot of work needs to be done

Martha: Thinking again about the vaccine, I asked Dustin what his vison for the future was, and what research he thought was needed in the next 5 to 10 years.

Dustin: Probably a vaccine coming out. The tough thing with vaccine development is the potential for cross-reactivity with other flaviviruses. That’s one thing where if you get one strain of dengue virus and then you get another one you get antibody-dependent enhancement where the second serotype of dengue could actually give you a much more severe infection. So there’s thought that with other flaviviruses you can actually have the same thing. So if you have an infection with Zika and then dengue you can get a much more severe infection. That’s one thing with developing the vaccine that you have to take that into consideration.

Then the other thing is that with live-attenuated vaccines is that if you give it to the pregnant mother it could actually be passed to the fetus and have detrimental effects that way. So, the vaccine field with developing in terms of Zika has got a lot of challenges and I think really within the next 5–10 years a lot of work needs to be done.

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