Cara Burns



CRAWFORD: Today is Wednesday, December 20, 2017. This is Hana Crawford for the Global Polio Eradication Initiative [GPEI] History Project. I'm with Dr. Cara [C.] Burns [PhD] in the Broadcast Studios of CDC [United States Centers for Disease Control and Prevention] in Atlanta, Georgia, and we are recording her oral history today. Thank you for being here.

BURNS: Sure.

CRAWFORD: Do we have your permission to conduct and record this interview this morning?

BURNS: Yes.

CRAWFORD: OK, great. Dr. Cara Burns is the [team lead, Molecular Epidemiology and Surveillance Laboratory] Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, CDC, [and was formerly the deputy branch chief of the Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, U.S. National Center for Immunization and Viral Diseases, CDC from January 2017 to May 2019]. To begin, would you introduce yourself by name, state where and when you were born and share a bit about your early life?

BURNS: Sure. I'm Cara Burns. My original name is Cara Lynn Carthel. I took my husband's name, so I was Cara [Carthel Burns]. I was interested in nature and biology when I was younger. I was born in 1962 in Texas, grew up in Texas. [In] middle school, I was kind of leaning towards some sort of outdoor career, biology something like that. [In] high school, I kind of was a little bit interested in medicine, still biology, I did trail maintenance for the Student Conservation Association a couple of summers, thought about being a park ranger--something like that and then kind of started being drawn more towards, not public health yet, but pre-medical kind of things. I got a full scholarship to Texas A&M University, President's Endowed Scholarship and National Merit Scholarship and another smaller one from Houston, the Jones Foundation. [I was] undecided originally and then started studying biochemistry in the Department of Biochemistry and Biophysics and did an undergraduate research senior project in the laboratory of Dr. James [R.] Wild [PhD], working on enzymology of bacterial enzymes. My mentor there was Dr. DeAndra Beck [PhD], who was a graduate student [at the time]. She had been through the same undergrad program I had, and so she helped me do the research for a senior project and then we presented that in a symposium at the end of the year.

CRAWFORD: Could we go back a little bit?

BURNS: Yeah.

CRAWFORD: What interested you in majoring in biology so early, and could you talk about your family a little bit? We had a pre-interview in preparation for our conversation today, and one of the things that you mentioned was your family and their values of education and respect for people.

BURNS: Yeah, so academics was really important in our household. My parents were public school educators and musicians. I started singing in the church choir, playing piano, then I switched to flute in middle school; high school, switched to oboe my sophomore year, because we really needed a strong oboe player, and flute and oboe swaps are pretty common, I mean.

Going back to my granddad, my grandparents were really hard workers, really strong work ethic and also, education-focused. Both of my granddads had to drop out of school around the eighth grade to help support their families. One was a farmer, homesteading in West Texas in the Panhandle, and he continued reading, mainly religious Bible kind of things. The other one dropped out and did jobs like being a streetcar driver, helping at the dairy, small dairy, at their house, and then eventually, taught himself enough math to get entrance to an exam for the Colorado School of Mines, and then he got a degree in mining engineering, worked his way up from the mines, up to being a manager of a zinc smelter in Texas. Both [were] really hard working.

My dad is extremely hard working. My parents are really creative, always doing various things, sewing, and my dad is a basket weaver, but he had this public school principal-teaching-band-directing career. My mom was a teacher and then an administrator, and they instilled in me some really core values about respect for all people and also respecting the interdependent web of all living things, and so camping and things outdoors. My dad grew up on a farm, gardening, all of those things were really important to us. I had one brother two years younger than me.

CRAWFORD: What was the Student Conservation [Association] like for you?

BURNS: It was kind of like the Youth Conservation Core from the '30s, '40s. It started in about 1956, so it's been around a long time, and it was a way for high school students and then later college interns, et cetera, to participate in outdoors projects--like trail maintenance was the main thing they did. Now, they do a lot of different things in urban parks, et cetera, but it was volunteer work; we didn't get paid. They gave me a little small travel stipend to get there, but I did Bryce Canyon [Utah, USA], and we got to hike in Zion Park [Utah, USA] afterwards. Then the next summer, we did the Appalachian Trail, so we were based out of the Dartmouth Outing Club in Hanover, New Hampshire. It brought people from all over the--you know, kids, roughly sixteen to nineteen, from all over the U.S. together to work in groups on these trails, just really hard outdoor work, physical work. That was really interesting, and it probably convinced me I didn't completely want to be a park ranger, but it was a nice experience, introduction. My daughter did that a couple of years ago, two summers in a row, so it's a really great program.

I should say, I mentioned that academics was really important. In the end, my dad, my brother, and I all have PhDs in very different fields. He went ahead and got his in educational administration; his early degrees were in music, primarily. My brother's is in math. He has a bachelor's in physics and math, also; and then mine is cellular, viral, and molecular biology.

CRAWFORD: At what point did your interest shift over into lab?

BURNS: Yeah, so I took Biology II, so I was already pretty much doing as much biology as I could in high school. We had more dissections in Biology II. I think we did a rabbit, and you know, a frog, et cetera. I thought a little bit about medicine in undergraduate; however, I was more interested in trying to prevent illness, rather than just put a band aid on the problems after they occur. That's partly why I was drawn a little bit towards biochemistry and lab research. The undergraduate research program gave me an opportunity to get a little flavor of it. I wasn't sure if I wanted to run my own lab at that point, and I did take the MCAT [Medical College Admission Test] and think about applying to medical school, but I did not apply. I ended up applying to graduate schools, really choosing mainly between Texas A&M, which had offered me to just stay there and have a graduate fellowship, or the University of Utah, which is where my boyfriend had ended up going in early 1984, so about a year before I graduated. In the end, I ended up following him, and then we got married, and I started graduate school at the Department of Cellular and Viral Molecular Biology at the University of Utah.

CRAWFORD: If you had stayed at Texas A&M, what would have been the focus of your work?

BURNS: I might have gone off towards like plant genetics, or something like that, but otherwise, generally, probably some sort of project related to biochemistry. [It's] hard to say what avenue, whether it would be public health related or not. They also had a very strong research lab there. They have a very strong agricultural program. That's why I mentioned I know that some of the plant genetics is really strong, and that was a pretty hot topic at the time. [It was] really kind of serendipity that after I got to University of Utah, did my rotations and chose the third lab, which was the poliovirus lab. I was very interested in the public health aspect of that, but at the time, the eradication program was really just getting going in PAHO [Pan American Health Organization]. I wasn't really aware of that. Polio was just a really good model system. The head of the lab was a really great scientist, was a very strong female scientist. Partly personality, partly the work was compelling.

CRAWFORD: Could you talk about your mentor?

BURNS: Just to say, I mentioned Dr. Wild in the undergraduate and then Dr. DeAndra Beck in undergraduate, and then Dr. [Elvera R.] Ellie Ehrenfeld [PhD] was the head of the poliovirus lab. She was a prominent virologist and biochemist. She taught in the medical school. That department had formed from her husband, and she, coming from Albert Einstein [College of Medicine, Yeshiva University] in New York, and it was a very communal department. We shared a lot of big equipment and stuff. She was just a really good role model. She taught. Her office--for a while, her desk--was literally in the middle of the lab. She was still very tied to the research going on.

We also had Dr. [Oliver C.] Ollie Richards [PhD], who had been an independent professor, but he liked bench research, so he really helped manage the lab and work with the new graduate students, et cetera. He liked to still work with his hands at the bench. Ellie, to this day, is still involved in polio; she sits on scientific advisory board for the new OPV2 [Oral Polio Vaccine, type 2] Consortium. She's been on the Polio Research Committee [PRC], which is run out of the World Health Organization [WHO], and also on an antiviral task force [Polio Antiviral Initiative, Task Force for Global Health].

After a long career in University of Utah in the biochemistry, teaching, professor, full professor, she went to be dean at UC [University of California] Irvine of the biological sciences--had to do a lot of budget cutting at the time. Then she went to NIH [U.S. National Institutes of Health]. She revamped the extramural grants program and then was able to settle with a small lab for the rest of her career and retire from there.

After that, I had [another] great mentor for my post doc, also a strong female scientist, Dr. Julie [M.] Overbaugh, [PhD], and she had just started her lab a couple years before. When I started looking for post docs, it was about a six-year graduate time: about four years in the lab, plus a year and a half or so with my rotations and classwork and stuff. I heard from another graduate student about Dr. Julie Overbaugh. She had just opened her lab a couple of years before--really dynamic, talented female scientist--and she had, I think, three graduate students already, a couple of technicians, so it was a medium-size lab. I looked at other labs there, and then some other possibilities for post doc, and my husband was also looking for jobs, so we were both able to get a job. I took the post doc in Julie Overbaugh's lab; he had a job with the State of Washington, which happened to be in Olympia [Washington], seventy miles south of Seattle. He did a one-way seventy-mile commute for a couple of years, and then he moved to an environmental planning company.

Julie was another great mentor. She actually won a mentoring award for the Pacific Northwest, national award last year, I guess given this year, for application last year for all the many graduate students, post docs, visiting scientists, undergraduates that she had mentored over the years. Her lab is going to have a thirtieth anniversary this coming summer, so it's hard to believe how the time has flown.

Yeah, I've really benefited from strong female role models. Ellie, when I was trying to pick post doc labs, she gave me advice to really look at places that were respectful: not terribly competitive among the members of the lab, so that it's a pleasant work environment and just feeling that life was too short to go into some of these cutthroat situations. Sometimes people put two post docs on the same project, and they're competing with each other within one bench apart. Then when I came to CDC, Dr. Olen [M.] Kew [PhD], in particular, was my supervisor; a really great mentor. Mark [A.] Pallansch [PhD], who is now our division director [Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, CDC], was our branch chief, he also served as a really strong mentor. Of course, there have been a lot of other people along the way.

CRAWFORD: I wanted to ask you about the science. When you were at the University of Utah, what did you know about polio?

BURNS: I came in knowing almost nothing, and in the lab we used mainly type 1 Mahoney [PV(1) Mahoney], which is a lab strain. It's also the Salk strain, so the inactivated polio vaccine [IPV]. It's the strain that's in the IPV, inactivated polio strain. To us, polio was type 1 Mahoney.

Then when I got to CDC, I was opened up to the three serotypes, and all these wild strains. I really didn't know much about polio. I was intrigued. Rhinoviruses, cold viruses, are related to polio, and I still get very annoyed every time I get yet another serotype of rhinovirus cold. [Laughs] I really didn't--it's not like I went to the University of Utah planning to work on poliovirus, or anything. I didn't know a lot about it.

At the time, molecular biology was still sort of new, and so I was introducing mutations into the polymerase protein and then studying polio replication. We did that, some through cloning and bacteria--expressing the polymerase protein, purifying it, studying it in the test tube. Also introducing mutations into the virus, into the full-length genome, and then you could reproduce the virus in that way, so it was pretty easy to make new polioviruses. For my graduate work, I worked with polymerase mutations. You could put these specific changes in the genome, generate RNA [ribonucleic acid] in a test tube, put the RNA into cells; the virus is positive-strand RNA, so it can make proteins and then make virus particles, and then spread throughout the cells infecting them. Polio kills cells, lyses cells. In a few hours, you can have killed cells; they spread to the next and make a nice virus stock. Then you can study that [virus] with these specific changes.

I studied mainly RNA replication. There were other people purifying various polio proteins, studying the shut off-of host protein synthesis. Polio's one of the most rapid lytic viruses around. It's really easy to set up your assays and run them quickly. At the time, it was easy to work with the virus--basically, BSL [biosafety level] 2 or sometimes even work on the bench. Now, we're really containing, starting to contain, poliovirus. But at the time, it was really a common model system, used in environmental assays, people just working with--water quality might use polio for some of their assays, just a really great model system.

CRAWFORD: Where did that lead you?

BURNS: I got a lot of skills in molecular biology, virology, genetics, and I still thought I might want to run an academic research lab, so in my post docs, I was looking at various labs where I would train to have a project I could take on my own and set up in my own lab. I would say I was sort of interested in public health, but I didn't really know about the options there, in terms of working in a government lab or some other sort of institution like that. As I was picking my post docs, I was still interested in virology, but also wanting a good working environment and looking at places that were interesting to live. Through this colleague, I explored a couple of labs at University of Washington in Seattle, which is a really great institution, also--has a great reputation, a lot of research grants--and then chose this lab, Dr. Overbaugh's lab.

My project was feline leukemia virus. I was trying to clone receptors. There were three main types of feline leukemia virus. I had a few projects; I was working on pathogenesis. Envelope processing was linked to pathogenesis, or the ability to cause disease in these feline leukemia viruses. It was a model system for feline immunodeficiency, different from feline AIDS [acquired immunodeficiency syndrome], but this particular set of viruses caused immunodeficiency. They are typically more like a cancer virus. Murine leukemia virus, feline leukemia virus are simple retroviruses--not as complicated as feline immunodeficiency virus or FIV [feline immunodeficiency virus]. But also in the lab, people were studying SIV, the Simian HIV, essentially, and also studying HIV-1.

I worked first on the receptor project. At the same time, I worked on this envelope processing pathogenesis project. Then, near the end, I worked on HIV-1 integrase, which is part of the replication enzymes, where we did a lot of sequencing of integrase genes from all around. There were international collaborations with Africa, particularly Kenya, in Julie Overbaugh's lab, so we had samples from Kenya.--[I had an] undergraduate working with me. We did a lot of sequencing of the integrase genes and looked at the molecular epi of those genes.

The HIV [work] broadened my skills, and I thought, potentially, that would be a direction I might go in. Feline leukemia virus receptors [work] was pretty risky. In the end, we did discover some, but it's a pretty high-risk project, and it didn't immediately yield the receptor. We were after the receptor of these unique viruses that cause the immunodeficiency. At the end, we figured out a few years later--they kept working on it after I left--figured out that there was a co-receptor, and so it was more complicated than just identifying the one molecule.

When I was looking for jobs--after a long post doc, multiple projects in the fifth year, or so--as I was starting to look for jobs, I interviewed for a number of academic positions at smaller, medium-size research institutes, still thinking about academic lab, but a little worried about all the grant proposals and funding the lab. It's like running a small business, and I wasn't sure I was ready for all those different managerial tasks. Then I saw the ad about the polio job at CDC in the fall. The government application process is pretty complicated. I put in my application; I didn't hear anything for a long time, and then I think it was in February that I heard directly, and then they wanted me to interview really quickly and move really quickly.

CRAWFORD: That was in 1998?

BURNS: That would have been in February of 1998, yeah, so I applied in 1997. I saw the ad in Science Magazine [journal of the American Association for the Advancement of Science], so they were doing an international search for somebody who could do a lot of the molecular work that I had done. Then there was kind of a vaccine project that wasn't advertised initially, because it was in the early stages. It was kind of confidential in the beginning. I was hired to build a central sequence database, which would be the foundation for more of the molecular epidemiology work, and then this vaccine project was like maybe thirty or forty percent of my time in the beginning and grew to about half of my time later.

CRAWFORD: I wonder, in your interviewing process, I wonder if you could talk--describe the interviewing process, but also what you understood the position to be?

BURNS: I came, I gave a seminar about my envelope processing, feline leukemia virus receptor work and met people. I met, certainly Olen and Mark. There were some people coming about the same time I did, and by the time I got there, there were about eight or nine polio staff, so they had increased polio staff a little bit at the time. I gave this seminar, interviewed; I talked to people like [Stephan] Steve Monroe [PhD, director of Office of Laboratory Science and Safety, OLSS], who is now in the top leadership at CDC. I understood that there would be some support for the Global Polio Lab Net [Global Polio Laboratory Network, GPLN], which was relatively young, through the molecular epi [epidemiology] work. Molecular epi is combining the genetics of the virus with the epi information to figure out how the virus is circulating: if virus is imported from another country, roughly how long the virus has been circulating or replicating; things like that--combining epi and lab, essentially. I had a sense that there would be some travel, but probably not a really good idea how much of my job would be helping to support the Global Polio Laboratory Network, or the GPLN. I knew I'd be involved in the sequencing part of the molecular epidemiology, probably didn't know how much support I'd be giving to the other sequencing labs as they developed over time. I started with just some of the international meetings, representing CDC with Olen and Mark at the time, mainly. Then of course, I just kept growing in the kind of things that I would consult on or help with: doing trainings, some on-site, some at CDC--and continuing to build our sequence database for the molecular epi work was one of the large parts. Getting people to relinquish their sequences and put them all in a central place was the biggest challenge of my first year. It was very interesting and challenging.

CRAWFORD: Do you have any stories about it?

BURNS: Well, of course, it's government information, but people use those sequences to develop techniques and things like that. Sometimes getting people to put all that in one central database is a little bit challenging. It took the flu group, probably ten years after we did it, for them to start merging their information into central database, where you could pull in the epi information, also, and you'd have it all right there together.

We work with the Global Immunization Division [GID, CDC] for the epi support, so for a while we were in the same center, but most of the time we've been in different centers. Unlike most of my colleagues in the Division of Viral Diseases, our epi and lab people are separated in different centers, and now they are in the [Center for Global Health].

But over the years, polio has made a really strong effort of combining lab and epi from the very beginning, and Olen Kew and Mark Pallansch are part of that. Olen showed that you could use a 150-nucleotide piece of VP1 and the beginning of the 2A gene and do molecular epidemiology for viruses. His post doc, Rebecca Rico-Hesse [PhD, MPH], had a paper in 1987 that was really seminal in molecular epidemiology for viruses. With this short 150 nucleotide region, they could show differences in the viruses circulating in different parts of the world and could really start doing molecular epidemiology. That was in '87, soon after that, the polio eradication program--Olen kept showing how you could use this information to help with eradication. After a decade or so, they really believed him and started integrating it into the whole program. By the time I came in 1998, you could already use this information to show, for instance, that there was some kind of cross contamination, instead of a real outbreak in China. They saved over $1 million dollars in not having to immunize, because they were able to tell that it was a problem that happened elsewhere--not in the actual populations, not in the field. That's kind of a trivial use of molecular epidemiology, but there were many examples starting to happen, where they could really track the virus.

Polio evolves fast enough, really as fast as any other virus, at a rate of about one percent per year, about one mutation per genome replication or division, so it's fast enough you can track it, but the proteins don't change much. That's why we're able to use the same vaccines now [that] we've been using from the '50s and '60s. Unlike HIV and flu, where for flu, you have to change the vaccine every season, and there's quite a bit of change happening at the protein level. The virus is trying to escape the immune system. Polio really doesn't do that much. There's a little bit of antigenic change, but generally, it's just the nucleotides or the RNA sequence is changing and the proteins are staying generally the same, probably partly because of the way the structure is. There's not as much room for this change of the proteins on the surface of the virus. That allows us to track it from this silent change that goes on, but yet the vaccines still work, because the proteins haven't changed.

Basically, that's a very powerful tool, and the eradication initiative took that up, and so for the last ten or twenty years, they're often demanding to see a family tree of the viruses--they're called dendrograms--they want that updated with every new wild poliovirus or every new vaccine-derived poliovirus. Sometimes we think [laughs] we've created a monster in getting this molecular epidemiology really embedded in the polio eradication program, but it has been a real strength. Since polio replicates in a lot of people that don't know they're infected--so ninety-nine-point-something--ninety-nine-point-nine, for some types, percent of the people who are infected don't even know they have poliovirus. That's one of the major challenges in eradicating polio, which is different from smallpox, where every infected person had lesions and knew that they were infected.

It is a really powerful thing to be able to track the virus. You can isolate it even from healthy people, but of course, AFP surveillance, the surveillance for acute flaccid paralysis, or AFP, that is the basis of the polio program, and environmental surveillance has been added on top of that. Generally, we're not sampling from healthy people, but if you want to, you can isolate virus from some of the healthy people in the area where there have been paralysis cases.

CRAWFORD: You mentioned before that in your first year you met some challenges in working with other labs and creating this database. Could you talk about that a little bit more, what the challenges were and how you met them?

BURNS: Yeah, so, initially it was really mainly our own internal sequences that were from viruses brought to CDC for sequencing. A lot of people from other labs would come to CDC, learn how to do the sequencing there, because it was still a very new technique, and then some of them took that technology back to their labs.

For instance, the South African lab director, Nicksy Gumede[-Moeletsi, PhD], came, I believe it was in the spring of 2000, and she had a lot of viruses from up to the 1999s from all over Africa because they were a regional reference lab for Africa and the lab is in Johannesburg [South Africa]. She came, learned VP1 sequencing and then went back and set that up in her lab, and I continued to help support them, visiting there in 2002 and then again a few years later, then later doing lab accreditations. Then we've always had sort of ongoing collaborations because that's one of the critical labs. It supports the entire African continent.

Because of the workload, we have supported Nigeria, in particular, and then sometimes some of the other countries, so Kenya and Somalia. Often when there was an outbreak, we did some of that sequencing. We're doing the Democratic Republic of Congo [DRC], VDPV [vaccine-derived polio virus] sequencing right now. We've continued to support the Johannesburg lab, but they've continued to develop their abilities and are really a solid sequencing lab, one of the main ones we rely upon. Certainly, the African continent is so important for polio eradication.

Other people came over the years: the lab directors from the Pakistan lab had come before I got there, and one of them ended up being the EMRO [Eastern Mediterranean Regional Office of the WHO] regional lab coordinator. He is to this day the EMRO regional lab coordinator, Humayun Asghar [MBBS, MSc]. He came and learned polio sequencing and techniques of CDC, and that lab has really developed over the last two decades. They've been doing their own sequencing for several years; they only send us samples if there's something really complicated. Since wild polio is only in Pakistan and Afghanistan this year in 2017, it's a highly critical lab. It's reporting to EMRO.

In terms of challenges, we're now finally getting a polio nucleotide sequence database. It's based on measles and rubella databases that are similar. That will truly be a global database, and that's in development at CDC. The MEANS [Measle Nucleotide Surveillance] database is hosted in the United Kingdom. That will bring together polio sequences from all over the world and will be a real legacy, having that database all available for researchers who are working on that sort of virus.

CRAWFORD: What did it take to coordinate this cooperative effort between labs? How did you convince people to participate?

BURNS: Just in general, Olen Kew and Mark Pallansch wrote a plan for the GPLN more than twenty-five years ago. They had been asked by WHO to come for three weeks, sit in Geneva, and write this plan of what they thought a global polio lab network would look like and roughly what it would cost, so they could see if it could be implemented. From the very beginning, they really had an attitude that we were going to empower the labs in their own countries to do this work themselves, that we would develop technology that could be transferred to them, and then we would build the public health lab capability in all of these countries.

In the beginning I think they envisioned many fewer labs. I actually didn't read that plan when I got to CDC. It was about ten to thirteen years old by then, I think, already--not quite that old, but the LabNet was already in place, and a lot of the cooperation was already started. PAHO had started polio eradication, Pan American Health Organization countries, and Ciro [C.A.] de Quadros [MD, MPH] was a really strong leader. They had had great results, and so by '91, they had wiped out polio. I came in 1998; the World Health Assembly had taken up the cause in 1988.

It was still early days, but we thought polio would be eradicated in the year 2000. I think the attitude that Olen and Mark had from the beginning, which we adopted--people like me and others who were recruited and came later--really showing a lot of respect for these scientists; for the challenges that they have in developing country labs; of trying to make techniques that were not radioactive, that would not cause hazards for their country--ending up just in some field somewhere because the radioactivity wasn't disposed properly; techniques that were simple enough that they could do and were robust and that we could train.

I think all that helped to make it a very collaborative environment. The labs trusted CDC and other global specialized labs to help them out, to help their capabilities, to help train the lab directors. Many of them earned their PhDs and went on to careers, some of whom ended up as regional lab coordinators in polio or in the global AIDS program or other public health international programs.

I really think we tried to carry on what Olen and Mark had started, just really fostering a collaborative environment. Email was really critical for sharing methods, information. We had these annual meetings--later the small working group [World Health Organization Ad Hoc Small Working Group] came about for the global specialized labs and the regional lab coordinators to vet new techniques, plan pilot experiments, involve the other labs to see if the techniques worked as well as they did somewhere like CDC or the NIBSC [National Institute for Biological Standards and Control] in the United Kingdom.

CRAWFORD: What was the occasion for introducing that role of the regional coordinators?

BURNS: I think they were envisioned in the original plan, but they've just been really critical. The regional LabNet coordinators serve as the spokes in the wheel that come back to the hub of the headquarters. Ousmane [M.] Diop [PhD] is the current global LabNet coordinator, based in Geneva, but he travels--he's like the second or third most-travelled employee at WHO. He's visiting these labs all the time. He tries to visit most of the regional labs, and I think there's twenty-five or so of those. Really, the regional lab coordinators, they understand the politics, the logistics in their region, and so they can try to implement these new techniques, plan workshops. They have their regional meetings.

One of my important roles is I'm one of two or three people that will go to the regional meetings, share the new techniques, give them updates, be a liaison available. Sometimes they'll ask for training at CDC or workshops there. It's all coordinated at the Geneva headquarters level, or that's the goal, but the regional lab coordinators really know how to implement things in their region. Many of them were lab directors before--polio lab directors or measles lab directors--so they know what it takes to get an international program implemented.

CRAWFORD: What does it take to ensure close work and cooperation between all of the labs?

BURNS: I think you need some physical interaction like these annual meetings and annual regional meetings. Then a subset of the people come to a global meeting. The small working group has been helpful to at least know what direction we're headed in, so with those seven global specialized labs and the regional lab coordinators and the global coordinator, we make plans. Last year we met three times, but normally, it's more like twice a year, so every six months or so in person, and then we'll have some phone conferences. I think Skype and phone conferences certainly have been great tools, but we do need to meet in person on occasion and really have deep discussions. I think just being sort of very respectful of their needs and their challenges and trying to figure out how to make it work in their situation has been helpful, rather than kind of dictating what exactly is going to happen from the top down.

Ousmane Diop was the head of the Dakar, Senegal polio lab. He was then working in SEARO [South-East Asian Regional Office of WHO] at a regional level. He comes from the African region; he has a very good understanding of the real-life challenges and great curiosity and respect for all these people working in the 146 global polio labs around the world. As I said, there are seven global specialized labs--I don't know exactly--twenty-something regional reference labs, and then the rest are national labs. That distinction has broken down some over time, because we've been able to transfer technology even to the national-lab level that previously we thought would have only been possible at the regional-lab level.

As I said, we always try to develop technologies that can be transferred, and one of the signs of success was when we were able to transfer the polymerase chain reaction, the PCR technology, real-time PCR, to both Nigerian labs, so Maiduguri and the Ibadan lab. Maiduguri literally is still worrying about bombing, and many of these labs have electricity challenges, so being able to do a high-tech technique like real-time PCR in these labs is a real big accomplishment. CDC scientists, my colleagues like A.J. Williams [team lead, STOP Program, GID, CGH, CDC], made multiple visits to that lab to make sure they had the foundations of good cell culture, good technique, setting up the lab right, inventorying their reagents, and all these basics that really are required to do a technique like real-time PCR. The lab methods developed over the years. [James H.] Jim Nakano [PhD] had developed a temperature-dependent assay to try to tell vaccine from wild virus, or vaccine and non-vaccine, a long time ago. Then, Olen Kew developed RNA fingerprinting, which was highly sophisticated, could not possibly have been transferred to most of these labs, but it was able to see subtle changes in, let's say, vaccine viruses or comparing wild viruses to each other.

Then, we came with ELISA [enzyme-linked immunosorbent assay], which is a standard protein-based technique that's used for many viruses. Then probe hybridization, which was the first real molecular technique that could be transferred to other labs. They made it nonradioactive, so that it was simple. You could use x-ray film, and you get the little black dots showing up, and the point was to see if it was like Sabin or not, generally. If it was not like Sabin, you could infer it was wild, and then you had some genotype-specific reagents.

From there, we went to the polymerase chain reaction, or PCR. Originally, it was gel-based. It required very thin acrylamide gels because the fragments were very small--it was technically fairly challenging--from there to real-time PCR, which required more of a sophisticated instrument that has to be maintained, so there were challenges with that. But the flu program also uses real-time PCR. Now many different detection assays use real-time PCR.

Some instruments were already available there; the polio program bought other instruments. That technology was really transferred around 2008-2009, starting in a small number of labs and has continued to this day. Ever Vega's [Everardo M. Vega, PhD] team, he's been the team lead [Molecular Diagnostic Detection Laboratory , Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, U.S. National Center for Immunization and Viral Diseases, CDC] for a couple of years, has been transferring the most recent version of that real-time PCR technology to now up to, I think, it's about 131 of the 146 labs. Most of the national labs are doing real-time PCR. That means there's less lag in figuring out if it's vaccine [vaccine-derived poliovirus] or wild [poliovirus].

First, we have an assay to see if it's an enterovirus. These are all run together: is it a poliovirus? Then there's a quadraplex that has the pan entero ["all enterovirus," a type of real-time PCR assay] and the three Sabins all together, so you can tell if it's Sabin 1, 2, or 3.

In the past we had molecular serotyping, which was developed by David R. Kilpatrick [PhD, former team lead, Polio Molecular Diagnostics Development Lab, Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, U.S. National Center for Immunization and Viral Diseases, CDC], who worked for many years in Olen Kew's group and retired a few years ago. He was able to accomplish molecular serotyping, so instead of using an ELISA protein-based assay, you were using a nucleic molecular assay, like the gel-based PCR, and then later the real-time PCR, to tell if it was serotype 1, 2, or 3.

Then from there, we didn't initially use wild-specific assays, but we could tell if it was Sabin vaccine strain or not. We could tell if it was an enterovirus or a poliovirus; we could tell which serotype it was. Then from there, we would either run a screening assay to tell if it was vaccine-derived poliovirus, or we would do sequencing, which told us even what kind of flavor of wild type 1 poliovirus it was, for instance. Then with the sequence we could break it down into genotype and cluster and then even sometimes lineage and chain of transmission. We could really get into figuring out where that virus had been circulating, and the critical part for the program was where those reservoirs were, where the virus would hide in the low season and then spring out from there and spread around in the higher season.

That's really what they're trying to focus on right now in Pakistan and Afghanistan, is hitting hard--immunizing very thoroughly--in those areas where the reservoirs are. They can see with environmental surveillance where those reservoirs are, even when there are no cases. Hitting those reservoirs hard then helps when the high season comes, for less virus to spread around. Of course, people move around a lot and spread the virus with them, so the molecular epi helps us tell where the virus is spreading. In places like Pakistan and Afghanistan, cross-border reservoirs exist, so it's really important that they coordinate and they understand where the virus is moving back and forth with the people, because some borders are just very porous and people are just moving across at will depending on what season it is and where their work is. The [Lake] Chad basin is like that, and if you have Nigerian virus, which has been a big reservoir, it can spread virus to the neighboring countries very easily.

CRAWFORD: I have a few follow-up questions.

BURNS: OK.

CRAWFORD: One of them is about your first-hand experience doing lab visits when you've done them. Could you just kind of illustrate some of what you've already been talking about, in terms of transferring technology, when these technologies have been absolutely pivotal when a reservoir has been identified and targeted?

BURNS: My first training, international training, was in February of 2000, so I had just been at CDC just a year and a half. I had not done lab visits. I had done an annual global polio LabNet meeting, so I went to Geneva in 1999 in the fall, so September-ish, [I] met a lot of the people involved in the region. I mean regional lab coordinators and some of the regional lab people.

Then, kind of out of the blue, Naomi [R.] Dybdahl-Sissoko [PhD, Molecular Epidemiology and Surveillance Laboratory, Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, U.S. National Center for Immunization and Viral Diseases, CDC] and I were asked to do a training in Mumbai, India at the Enterovirus Research Center, ERC, which was headed by Dr. Jagadish [M.] Deshpande [PhD, former regional lab coordinator in the WHO South-East Asian Region]. The person at CDC that was originally going to do that wasn't able to do it, and so we had two weeks' notice to plan and travel and get our visas and get everything arranged.

We had three big boxes. We had room-temperature items, which included even the printed-out lab notebooks, because it wasn't quite as easy to share digital files at the time. Then we had a four-degree box, which would have certain reagents that needed to stay at four degrees or refrigerator temperature, and then we had a frozen box where we needed to put dry ice. We had all these reagents, because we just weren't sure what all was available in the host lab where we would be doing this. We were using the template of a PCR workshop in PAHO from, I think, just a year or so before as most of the learning materials for PCR. The workshop was going to be for ten days, and it was for three techniques: ELISA, which was supported by the Netherlands lab and the Netherlands lab director came and did that part; probe hybridization, which CDC supported; and then the PCR that was gel based, which CDC supported.

CRAWFORD: All of which saved time.

BURNS: At the time, we were doing two techniques, one protein-based--so ELISA--and one of these other molecular-based techniques. But with the probe hybridization you didn't get quite as much information as you did with the PCR, but the PCR was more technically challenging. Like I said, you need these thin acrylamide gels; you had to be really careful about pouring them, et cetera.

Anyway, we crammed these three technique training sessions into ten days. We had one day off. We went to Elephanta Island and had some fun together on one of the weekend days. But, Dr. Nalini Withana was the SEARO regional lab director at the time, and she was very demanding. She expected a hundred percent scores on the proficiency tests, and she had this most ambitious workshop with three techniques. I don't think we did three techniques almost any other time. [Laughs] Naomi and I were trying to throw this all together and get our Indian visa, which today takes at least thirty days. CDC Travel helped us out, getting everything done.

It was a great workshop. The host lab had wonderful people helping us, making buffers and getting things ready for us. It was a little bit scattered in the beginning.

The people in the workshops are always great, often it's lab directors, but sometimes it's the actual staff who are running the assays on a daily basis. Both sets of people really need to know what's going on. The lab director needs to know how to interpret and report and troubleshoot with their lab staff. Typically, it's one or two people per lab. This was a pretty big workshop, several Indian labs, the Bangkok lab, the Indonesian lab, diverse set of labs from Southeast Asia.

One thing that we did that was quite a surprise and very funny was Dr. Nalini started off a singing session. We had this beautiful--really a banquet outside on the green lawn at ERC and then afterwards, it was not karaoke because there was no accompaniment, but we were expected to sing [laughs], and I had a singing background, but of course I had never needed to sing professionally. I remember she sang, "Daisy, Daisy, Give Me Your Answer, Do," a song I knew growing up. I started singing John Prine's, "That's the Way the World Goes 'Round," and I didn't remember all the words, and I thought, oh my goodness, that's a weird song choice, afterwards. But she said something to me like, "Well, Olen sang." In WPRO [Western Pacific Regional Office of the WHO], they do karaoke quite a bit for fun, but this was unaccompanied, and it was really quite humorous. I really did not expect that.

But really a collegial group of people. Many years later, I met some of the same people who had trained in that workshop from Indonesia. Three of the colleagues were still in the Indonesian lab when I visited; some of the same folks from Bangkok. One of the people from Chennai, she ended up being the SEARO regional lab coordinator after Nalini Withana left. Yeah, it was a great experience.

Ever Vega's team is running lots of workshops now. They are introducing the latest version of the real-time PCR, so the last two years, he's been travelling a lot to these trainings, to all the regions. Also, of course, people on his team also have been going with him. They've kind of finished that most intense training set for the latest technology, which is just an updated version of the real-time PCR. That technology has survived for a long time, and it's different from just a single clinical assay. It's really a battery of screening assays, as I said, to tell if it's entero or not, polio or not, which serotype--if it's Sabin 1, 2, or 3, the OPV 1, 2, 3 strains. Then there's a VDPV screening assay, which is not [a] definitive answer. You have to sequence the virus to make sure that it's a vaccine-derived poliovirus. But the screening means that we don't have to sequence all the strains; we can do about half of them instead of all of the ones that come up as potential VDPVs in the screening assay.

CRAWFORD: Before we talk about VDPVs, vaccine-derived polioviruses, is there any specific advice that you would give to people who in the future might be coordinating global training efforts, transferring technology?

BURNS: I mean I think there's quite a bit of information you can get from the literature online before you go to help you plan. We didn't do as much of that formal work, because people were already doing it for eight years or so before I got involved.

I just think that this culture of respect for what they're going through and what their situation is is really important. They can come and visit CDC and learn how to do things in our state-of-the-art lab, but when they go back and their distilled water is low quality, and their PCR machine needs to be calibrated, and they literally cannot get anyone to come into their city, or whatever, because of security concerns or just the way the technology is supported, those are things that are real bottlenecks for them. Kind of thinking ahead to, "What are the real-life logistics of this?" I think, is critical.

Then things like, you know, not using difficult technologies and radioactive waste and things like that, I think, have served the program really well, also.

CRAWFORD: Do you think it's best to find that information in documents and articles and manuals?

BURNS: Partly, but certainly talking to some of the people who do it on a regular basis is helpful. The Global AIDS Program, PEPFAR [President's Emergency Plan for AIDS Relief], they have lots of training resources. WHO, the Global Polio LabNet, has integrated different quality-assurance and quality-control measures from the beginning, but in the last five years, they've really upped that focus on biorisk management. WHO developed some videos that are available, a set of videos. Some of the videos are actually pointing out what not to do, and they are designed to be interactive, so that the laboratorians can look and say, "OK, I don't think that was a good practice," and then there's discussion about what the good practices are and what the mistakes were in the video. In addition, there have been some training sessions offered, and all of that's really critical for the containment that we're going through now, the Global Action Plan III [WHO Global Action Plan To Minimize Poliovirus Facility-Associated Risk After Type-Specific Eradication of Wild Polioviruses and Sequential Cessation of Oral Polio Vaccine Use], GAPIII, requires that type 2 poliovirus already be contained and then later type 1 and 3 will come, so all those quality measures are really critical for that.

I didn't mention that if you do a lab accreditation visit, there's a whole structure. There's a checklist and a scoring system. All labs are visited at least every three to five years. Most labs are visited every three years. If there's high performance and the paper review is high, then there's no need for an annual visit, but some of the critical labs are visited every year by a regional lab coordinator and someone from the CDC or the National Institute for Biological Standards and Controls in the UK, or the Netherlands lab. We're always trying to expand the number of auditors that are available.

The quality assurance measures have been really critical in being able to trust the results: the surveillance--getting the samples, getting them to the lab in a good condition and in a timely manner, that's all highly critical, and our epi colleagues and program colleagues really have to help with all of that. But once it gets to the lab, we know that the results are reliable. There's a system: if there are problems with the tests, [then there's a] referral to another lab. CDC is kind of the "lab of last resort," and if there are problems with a technique or a particular sample, it often does get referred to us.

Over the years we've developed easier ways to refer things, so we use these cards called FTA [Flinders Technology Associates brand] cards that are used for serum shipping and some other virus work. We really figured out how to put the virus on the card. The card inactivates the virus. We usually heat the virus beforehand to inactivate it partially before it goes on the card, and then the cards are dried, and then they can be shipped very easily without refrigeration to a referring lab to do either PCR or sequencing. Nowadays, it's mainly the sequencing that you would spot an FTA card and then put in the mail, so the [DRC] lab, [Democratic Republic of Congo] has been sending us these vaccine-derived poliovirus isolates that way, and it's really simplified referral of specimens.

CRAWFORD: We're almost to the vaccine-derived polioviruses. Is there anything you would want to share about what it's been like for you, personally, to work with LabNet?

BURNS: It's just been a really great professional satisfaction to meet people from other countries and then meet them again and again in other LabNet meetings, regional meetings, things like that--to watch them grow professionally, to see the public health lab capacity increased because of transfer of technology. I think it requires perseverance and optimism. You're not going to try to eradicate polio if you're not optimistic about the way things work.

CDC is just a really unique place to work. I don't think I said earlier, but when I saw the job, it matched my skills really well, coming from both the graduate work on poliovirus and then the work I had done with long PCR and molecular epi on retroviruses. It fit really well. But also, CDC is just a really unique place. You pull lab and epi together in a way that's hard in many places; really great scientists, well-funded--of course, polio has been really funded from the beginning. Although my husband didn't want to live east of the Mississippi, I dragged him along with me because CDC is really a unique place to work, and you can do public health work that's hard to do anywhere else.

CRAWFORD: One more question: when you mentioned earlier that you were interested in public health and in hard science as a way of preventing disease, rather than putting a Band-Aid on that, is that an understanding that developed for you later on in your career? Or was there a moment early when you were making your decisions about where to be?

BURNS: At the time that I was thinking that way, it was more like going to medical school versus going into research. I felt so many of the things that doctors have to do has to do with ameliorating symptoms, giving drugs, but vaccines could prevent having to give the drugs. I didn't know about polio eradication. I mean, I wasn't headed in that direction. I wasn't sure exactly what kind of basic research that I would get involved in, but I did like the idea of learning the root cause of what's going on and maybe being able to prevent something, instead of just giving cough syrup or whatever, after the fact. It was mainly research versus medicine, I think, at the time.

Later on, I began to learn more about what public health really meant, really, when I got to the CDC--somewhat in my post doc, because we had this collaboration with Kenyan labs, and HIV was a critical health problem. There were studies from mother and infant transmission. The Kenya work, a lot of it was sex worker studies, so a lot of women-focused work. I got a good flavor of public health there, but I was still mainly in the lab doing receptor cloning and things like that and later, HIV integrase sequencing, which was closer to the people in the field. But yeah, it probably took really getting to CDC to learn more about what public health international programs are really like.

CRAWFORD: Vaccine-derived polioviruses--

BURNS: OK.

CRAWFORD: You've done a lot of work on those.

BURNS: CDC was in at the very beginning for vaccine-derived polioviruses, and we wished they didn't exist, but identifying them and being able to track them has been really critical.

When I joined in 1998, the goal for eradication was the year 2000, and I wondered what I would do afterwards. Measles was one of the answers I was given, you know, "We'll move our polio to measles," and that has been happening. The measles network developed kind of on the model of the polio network, and they made tremendous strides. Unfortunately, a lot of what I've been doing after wild polio has been related to vaccine-derived poliovirus.

We, with WHO, we came up with the term "vaccine-derived poliovirus" with the first outbreak that was studied at the same time that was happening, which was Hispaniola, so Dominican Republic and Haiti were hit with type 1 vaccine-derived poliovirus. When they originally saw the clustering of AFP cases, they didn't know what it was, and there had been no wild poliovirus in that region for over a decade; 1991, was the last case in Peru. This was happening in late [2000], early [2001]so over ten years with no wild poliovirus. The chances of importation were slim, but it was a possibility. However, we could see from the ITD [intratypic differentiation], the molecular techniques, ELISA, and the PCR from the Trinidad lab that it was linked to Sabin, and we were worried that it might be somehow linked to the vaccine strain. We were starting to get some information about old type 2 strains from Egypt, which we originally thought were wild type 2, but it turns out they were vaccine-derived poliovirus that had circulated broadly in Egypt, probably for more than ten years earlier in the '90s. But we didn't have all that information yet; we just had a hint.

Everyone knew for a long time that the oral polio vaccine had some genetic instability. The live attenuated oral polio vaccine, OPV, strains 1, 2, and 3--one for each serotype--they were derived by passaging under conditions that were designed to weaken the virus, attenuate it, and make it less likely to cause paralysis. For instance, type 1, which is really the best OPV strain, was developed--a lot of work was done by Leanne Schaefer [and Dr. C.P. Li] at CDC, passaging this virus. Then they sent it to Albert [B.] Sabin [MD], and he did three successive plaque purifications, so just separating one virus from the other, and he called it "Sabin 1." It has the most attenuating mutations and is in some ways the most stable. CDC's history with vaccine research goes way back, decades.

Unfortunately, this first vaccine-derived poliovirus was studied right when it was happening in Hispaniola, it was type 1. We discovered it a few months after the first cases. The first cases weren't really picked up as AFP, and then from the virus we could tell it had been going on for over a year with the first cases. Anyway, when we got that first sequence, the Trinidad [lab] sent the virus to us. We got the first sequence of the VP(1) [Viral Protein 1]; we could see it was Sabin 1-derived, but it had mutated enough that it was going back towards a wild phenotype and was causing paralysis. It could recombine with other Species C enteroviruses in the area, and critical mutations that were attenuating had changed back more towards a paralytic phenotype.

We sequenced complete genomes of those; that was the first [done in] real-time. We used long PCR and Sanger automated [DNA sequencing], our standard sequencing technology, to get the full genome, approximately 7,400 nucleotides long. We did these twenty or so viruses and could really tell how they were related to each other.

Haiti was the bigger problem. The Dominican Republic viruses were more closely related to each other. Haiti had longer branches on the family tree, meaning there were more missed cases. Surveillance was not as good there. Probably what happened is it started in Haiti, and when it spread to Dominican Republic is when it was detected by surveillance and then more cases were picked up. The viruses were more highly closely related to each other, and then that's where it got, you know, flagged as AFP and sequenced.

CRAWFORD: That was wild--that was--

BURNS: All vaccine-derived type 1, yes.

CRAWFORD: Even though most of them are type 2?

BURNS: Right. Most of them over the next fifteen years have been type 2. There have been more individual--what we call "emergences," so cases where the OPV strain, we know it mutates in a single person. You can isolate stool from a person, sequence the virus, and some of these attenuating mutations have already started to shift. If those viruses then spread--we know it also spreads to the first contact, first layer of other people--to some other contacts, let's say-- and that can be a good thing, because then those people get immunized.

But if it starts spreading beyond that, it may recombine, additional mutations happen, and then it starts to pick up neurovirulence and become more like a wild poliovirus. The VDPVs that are circulating a while, when they've been tested in a transgenic mouse model or in primates, they cause disease that's similar to wild poliovirus. If you look from an epidemiologic sense, the VDPVs are similar to wild polio in their clinical characteristics--epi characteristics.

That outbreak was shut down pretty easily with immunization. However, some of the later vaccine-derived poliovirus type 2 outbreaks, for instance, in northern Nigeria have been harder to shut down. There have been more emergences going on at the same time, so more paths from Sabin to VDPV, and in Nigeria from 2005 to 2011, we identified about twenty-five different times where the OPV had started to evolve into VDPV. They were circulating at the same time in similar areas, different states in northern Nigeria. The main risk factor is the lower population immunity, so in those areas, the polio OPV campaigns were not immunizing enough of the kids. They were done too haphazardly; vaccine coverage was too low. This virus--these vaccines have been used safely for decades in the U.S., in the western world, in Asia; if you use them well, you have high vaccine coverage. You don't have many susceptible people, so the virus is not able to spread out to the next level of persons. Those people are protected, and the virus doesn't spread. But if you just immunize thirty percent, forty percent of the population, then you leave these vulnerable people, and the virus can start to spread, and then it mutates. Then it becomes a vaccine-derived poliovirus.

Our goal in calling things "vaccine-derived poliovirus" was to come up with a definition that would capture those viruses that were actually circulating and causing disease. We know that some mutations can happen in a single patient, and we don't really want to be sequencing all those viruses, so we were trying to come up with a way of saying, "OK, this virus has been replicating long enough that it could be a public health problem." If it's picked up in an AFP case, we have a screening method to flag those viruses, and then we sequence them. Many of them turn out to be ordinary vaccine virus and not extensively mutated VDPV, but that way we don't miss any VDPV. We capture some ordinary Sabin viruses, but then we are able to sequence all of the VDPVs and detect these critical ones that could be circulating.

As you said, type 2 has been the biggest problem. We hope that when type 2 is gone and we've stopped the VDPV circulation, that types 1 and 3 won't give us as much difficulty as type 2 has.

The WHO started planning to phase out oral polio vaccine because of the problems with vaccine-derived poliovirus and because wild poliovirus is going away, so it's harder to justify using a virus that could turn into vaccine-derived poliovirus, if you don't have many wild polio cases. Type 2, the last wild type 2, had been detected in India in 1999 and in 2015, it was certified as being completely eradicated. Type 2 was the first one to be contained, and as part of that effort, the WHO planned to switch from the trivalent OPV that had types 1, 2, and 3, to bivalent OPV that just has types 1 and 3. Removing that type 2 component removes the risk of vaccine-derived poliovirus 2 emerging, and since that's been the most common and frequent VDPV, that should really reduce vaccine-derived poliovirus emergence.

That switch happened in April of 2016. It went quite well, as the largest global immunization effort ever. They did discover some places that still had trivalent OPV upon inspection, later. Typically, you would pick that up, because you'd pick up a type 2 Sabin in AFP cases, and then you could go investigate. It's very hard to get the whole world to take trivalent OPV vials out of every clinic's freezer and destroy them by either incineration, autoclaving, and then replace it with new bivalent OPV stocks. You know, people tend to want to hold on to their precious vaccine, so it was a huge campaign to inform and educate.

Overall, it went pretty well. But there is a committee that examines every type 2 that's detected, and in cases with vaccine-derived poliovirus then they have to decide whether to respond with monovalent OPV2 [mOPV2], and then the director-general [of WHO] has to approve the use of it.

In places like Syria and DRC in the last year, they have had circulating vaccine-derived poliovirus type 2, which we sequence at CDC. Syrian viruses have been sequenced either in the Netherlands, in Egypt--which is a new sequencing lab; it's almost accredited--in one of the labs in Turkey, which is trying to become an accredited sequencing lab, or at CDC. That has been a collaboration of five different labs and three regions, I guess: EURO, EMRO, and CDC--PAHO.

Then DRC, those viruses have been coming to CDC, also, for sequencing. We're looking forward to the day that the type 2 circulation has stopped and hoping that type 1 and 3 won't be as difficult.

WHO did decide a few years ago that this vaccine-derived poliovirus problem was big enough [that] they would have to phase out OPV, and so they required a dose of IPV before the Switch in all countries, trying to get the population immunity--especially to type 2--up before the mOPV2 is removed from the trivalent formulation.

It wasn't a problem for many years when the vaccine was used well, but it has been a problem in some settings where risk factors--like the typical risk factors for polio transmission: high population densities, poor sanitation, crowding--seems like, for some reason, islands are common places for VDPV to emerge. We don't totally understand that. But the main thing is the population immunity, and if the population is vaccinated well, then the vaccine-derived poliovirus really cannot emerge and spread.

CRAWFORD: Is there a point in time at which the quality of vaccination efforts changed? You said, "when it was being done well." When would that have been?

BURNS: Obviously, it's hard to have really high-quality immunization throughout the entire world, and so different regions have their own challenges.

Now it's getting harder, because wild polio has been gone from many places, so the urgency is lower. Places like DRC, Syria, northern Nigeria--Pakistan had VDPV2 a couple of years ago. They typically have social infrastructure problems. Generally, these last countries have civil unrest of some kind--so the Boko Haram in Nigeria, some in Somalia, similar things. In DRC, there's some vaccine refusal. There's not a lot of roads. It's hard to get to all the people, and there's also some level of civil unrest.

In Syria, obviously, a huge war--the immunization system was very strong, and in the last few years of war, it's just disintegrated. Everybody knows what the conditions are like there. It's just really a hotbed, waiting for things like vaccine-derived poliovirus to pop up. Fortunately, it has been generally localized to the Deir ez-Zur area, a little bit in Raqqa, but very hard place to work, very hard to get samples, very challenging to know what the samples are once they come out. Multiple labs are involved, because samples can go in one direction and get isolated in one lab; other samples can go in another direction. You never know if the sample will make it there, so [there is] some redundancy built in, and then lots of different lab IDs [identifications] you have to compare to each other. It really has taken a lot of cooperation by email and phone to even figure out which cases go to which regions, for Syria, in particular.

CRAWFORD: Which is kind of a basic surveillance--or goes back to--Jon [K.] Andrus [MD] mentioned having a difficult time matching sample and case [in PAHO before the region was certified polio free].

BURNS: Yes, and frankly, they did some sample splitting, because they weren't sure the sample would get out. It often went in two directions to make sure that part of the stool would go somewhere and would be isolated into virus, and then would get the molecular testing and the sequencing. There are actually duplicates built in [laughs] that we had to figure out which ones were duplicates.

The other time when it was very challenging was the Republic of Congo, wild type 1 outbreak a few years ago. It originally was missed. I believe they thought it was kind of a chemical reaction, initially. By the time they figured out it was polio, it had spread quite a bit, and they didn't have the right samples, even. They didn't have stool; they had nasal swabs, some rectal swabs. Figuring out the IDs for that outbreak was also extremely challenging.

But generally, the surveillance system for polio is pretty good. I didn't mention much [that] the environmental surveillance has been expanding over the last five years, and that's been really important. Early on, [in] places like Egypt, environmental surveillance was implemented, partly through the efforts of the country themselves, and Humayun Asghar, who is the regional lab coordinator for EMRO. He's been a really big player in environmental surveillance throughout the whole world. He's taken what he learned in EMRO and has transferred it to Nigeria. He's been involved in selection of sites in many places. CRAWFORD: Could you repeat his name?

BURNS: Humayun Asghar. He was head of the Pakistan lab, and then he became the EMRO regional coordinator when Esther de Gourville [PhD] went to be the global coordinator. He still works very closely with us.

Anyway, there have been various environmental surveillance expansion plans over the last five years, in particular, and each region has tackled it. It's been quite aggressive in EMRO and AFRO lately, especially. Back in Egypt, basically, they were not having many AFP cases, and the environmental surveillance showed that there was still virus, especially along the Nile where people live.

CRAWFORD: At the beginning, could you describe what environmental surveillance looked like?

BURNS: The Finnish lab and Egypt and then some other labs--the Netherlands, a few other places--have been doing enterovirus or poliovirus environmental surveillance for a long time. The Finnish lab had done it for twenty years as of a few years ago. Egypt environmental surveillance was established pretty early on. Israel has a very developed system, but it's much more sophisticated than what you could implement in most places. For Egypt, it was basically using this two-phase concentration method that separates out the virus and concentrates it, so then you can take that concentrate and put it on cells and grow poliovirus.

The whole GPLN is based on growing poliovirus isolates, so it's right in line with that technology. Basically, you're taking raw sewage, and in many places the sewage is in ditches and open places. In places that have closed systems, they use a slightly different approach. But in open places, like a ditch where you can tell sewage and trash and stuff is all flowing into there, there's an art to picking a good site. You go and grab sample, so it's typically a liter divided into two five hundred [milliliter amounts]. What you process is usually half a liter at a time.

You take that back to the lab, keep it cold, process it as soon as you can or freeze it, and then you do this organic separation. It's actually in a glass funnel, and you let it sit overnight. Then you take the part that has the polio in it, the concentrate, put it on cells, grow it the same way you'd grow from stool to cell culture, essentially--slightly different algorithm, but very similar.

Then you do the same test on the isolate. It's a little more challenging, because the sewage has waste from ideally hundreds of thousands of people. You want a catchment area that's sufficient that you would detect polio if it were in the region. That's part of the art of selecting the site, is estimating how many people have waste draining into this gutter that you're collecting from.

CRAWFORD: How long does poliovirus survive in the environment?

BURNS: The half-life is in the order of days, so if you had a high titer, it could survive a week or so. It totally depends on the heat, the humidity, the UV [ultraviolet] exposure, whether there are chemicals in there. If you had an industrial site draining in, it would not be a good site, because it could kill the poliovirus in the sewage there--so highly variable. Mostly polio spreads from person to person, sanitation issues rather than from water, but it can survive in sewage or water quite a while.

Stool is kind of a stabilizing force for polio. If you study temperature inactivation and stool, you can see that the stool matrix seems to kind of stabilize the virus and let it survive longer. Sewage may have some aspect to that also. It's generally fine in terms of picking it up for surveillance. There may be some spread that happens that way in some places: kids are playing right near the open sewers and stuff.

There have been attempts to introduce more science into the art of picking the sites. The [Bill & Melinda] Gates Foundation has looked into various geographic tools that you could use to help estimate geographic features and catchment, for instance. But expansion has been pretty rapid, and a lot of it has been the people who had experience in the beginning, going to some of the early countries, trying to transfer their knowledge to other people.

Lab people were kind of at the forefront, because they developed the techniques and figured out what worked, and then we've been trying to get more of the epi people in each region to take responsibility and help with the site selection and all of that.

Then of course, sample collection, transport, and all of that is very critical, just like for stool. You've got half a liter of raw sewage, so typically, you're either taking that in a car to the lab. In a few cases, they have been frozen and shipped on dry ice. We have a project with Haiti where we're doing that. Angola sewage has been shipped to Johannesburg, but it can be complicated shipping across country borders, and it's a large liquid sample, so it needs to be kept frozen and it needs to be contained. Obviously, that's a little challenging.

There are some other methods out there. One of the filtration methods developed by the Japanese has been transferred to many China labs, and it may be adopted soon as an endorsed WHO method. There's one method, the two-phase method, that has been endorsed. In the past there's been enough quality control and implementation to know that's a good solid method, and labs like the Finnish lab have helped in doing that method development and transferring, et cetera. There have been efforts lately by the University of Washington to try a bag-mediated filter system that tries to attempt to solve some of the problems with the two-phase method. It does have its own challenges, but it has been piloted in the last couple of years and still kind of being improved and trying to figure out how it might help in this expansion of environmental surveillance, which has been quite dramatic.

Countries like Nigeria, Egypt, and Pakistan and Afghanistan--well, Pakistan in particular--have had over thirty sites that are collected either monthly or twice a month for several years. Newer countries in Africa, they start out much more modestly, so six sites or something.

It's really critical to do ongoing sampling, because you don't know the value of a negative result, unless you know that site could produce poliovirus if it were there. That has been a challenge lately. You really need a baseline in order to decide if it means anything if your environmental site is negative for three years. Well, if it's not a good site--it doesn't have a lot of people; the catchment is small; you don't have a lot of people's waste going into that site; if there are industrial toxins that are killing the virus; or whatever--you don't know if negative means anything. For instance, in one of the sites in India, they had a new site, a couple of wild polio detections, and then it went silent for three years. They were lucky there that they caught it right at the end of the circulation, established that it was a good site, and then it went silent, and they believe that actually meant something. I think that's part of the challenge in this rapid expansion of environmental surveillance, trying to make sure the sites are good enough that you know what a negative result means.

CRAWFORD: Is there anything else about environmental surveillance that you would like to include?

BURNS: I guess just that in certain places, it has been a really helpful tool to tell that polio is still circulating. If either AFP surveillance in small pockets wasn't as good as we'd hoped, or in the case of Egypt, where they were actually trying to say wild polio was gone, and the environmental surveillance kept picking it up and made it difficult for the politicians to say that wild polio was gone.

It's expensive. It's labor intensive. It's easy to tell the lab to do a lot of environmental surveillance, but it actually impacts their workload and their ability to do all their AFP surveillance, so it's definitely supplemental. AFP surveillance is the gold standard, but it has been critical in a few places. We did analysis in Pakistan and published a paper to try to show in a more quantitative way when environmental surveillance could contribute where AFP had not detected, either detecting earlier or detecting more often. [These were] their efforts to kind of put more of the quantitative information into the scientific literature.

CRAWFORD: The third topic that we thought we would cover today is your vaccine project.

BURNS: [Yes.]

CRAWFORD: Where does that begin?

BURNS: Basically, it begins with the genetic instability of the OPV strains, which I mentioned, for vaccine-derived poliovirus. It makes sense that if a virus has been attenuated and scientists tried to make it weaker that biological selection would play, and the virus would keep trying to recover higher fitness--and fitness meaning the ability to divide and grow and spread.

CRAWFORD: When did you begin working on this?

BURNS: Olen had the idea before I was hired, and so as soon as I was hired and I was going to do molecular epi and make the central sequence database and things related to sequencing, I started immediately also on this vaccine project. I made a copy of the Sabin 2 strain, so I had a plasmid that had the full-length virus, and we started making specific mutations and then studying it in the way that I described before, like the polio with polymerase mutations. I did that in my graduate work. The long PCR I had done in the post doc and the work I had done with the poliovirus, just molecular biology. Generating mutants was really helpful in the beginning for this project, and Jackie Quay [JD] was a technician who started with me on the project. Really, from the very first month that I came in late August of 1998, we started laying the groundwork for this project.

The idea at the time was that we would change the codons without changing the amino acid sequence of the virus. That's where the nucleotide sequence--the gene--changes, but the protein stays the same. That way, the function of the viral proteins are all the same, but the RNA or DNA, the genes, are different. Our original idea was that we would use codons that the virus rarely used. We would pick codons that the virus, for whatever reason, didn't like to use and stick them in there and thereby reduce the fitness of the virus--kind of like feeding spinach into a system that really liked dessert or something like that.

We originally thought that that would affect the amount of protein made and would, in that way, affect the fitness of the virus. What we found, however, was that the protein production wasn't affected very much, so the actual translation of RNA into protein that makes the viral proteins, that wasn't affected very much, but the structure of the RNA and the RNA sequence was affected. Still to this day, we are trying to figure out the exact mechanism, but definitely, there are reasons why some of those codons were rare, why the virus didn't naturally select those codons, and as we got more into it and made more and more kinds of codon changes--so, the codon changes is where you change the nucleotide sequence but you leave the amino acid sequence the same. If you go back to basic biology, there's the third position. Codons have three nucleotides, and at the third position there's what they call "wobble," which means that more than one codon can code for a single amino acid, and that's why this works. You can fiddle with which codon you choose for any given amino acid. When you do that, you change quite a bit about the nucleotides that sit beside each other--we call those "dinucleotides"--and then the codons themselves change. You can do it within a codon or across the codon.

What we started to see was that the CpG dinucleotides that were in a lot of these rare codons, and also some UpA dinucleotides, seemed to be as much responsible for the decrease in fitness as the codon changes themselves. In all of this, the protein stays the same. It's just the gene or the nucleotides, the RNA, that's changing. In some ways, it's a fairly subtle change, but it did have very profound effects on the virus fitness. We could push it to the level of extinction and get so many changes in there that virus could barely grow.

We passaged the virus a lot. We saw what sorts of changes biological selection did in response to all these mutations that we put in there, and that gave us hints that these CpGs and UpAs were pretty critical.

For a vaccine strain, you don't want a virus that's almost dead. You want a virus that's pretty robust, that can induce an immune system response, so it works as a vaccine, but the goal would be to have one that is more genetically stable, that doesn't emerge into vaccine-derived poliovirus or doesn't cause paralysis in a given person.

Over time, we learned more and more about the kinds of changes that we could make, and as I said, it didn't seem like it was much effect on the protein itself, but more on the way the cells sensed this new virus. There have been other people proposing for flu and some other viruses that something like CpG could be a signal as a foreign RNA. Viruses tend to minimize CpG, and that may be driving the rare codon affect, so you get less of these codons that have CpG in them or UpA. You have less of those codons, and the virus is trying to kind of come in incognito and keep lower levels of these CpG and UpAs that are signals for foreign material.

Exactly what happens in the cell once it senses the foreign material, we're not completely sure. There's a post doc working on that, and other labs have been working on it in other viruses. But we coined the term "codon deoptimization" and applied for a patent for it, and then the exact application we've been tweaking, in terms of how to change the codons exactly: how many, where, et cetera. We got the technology, we got the patent for all viruses for the U.S., Europe, Canada, India, and there've been some divisional applications after that.

The idea behind patenting this is just to secure it for public health, so that pharmaceutical companies are not going to make a lot of money on this technology, which is fairly universal. You can apply it to viruses other than just poliovirus, certainly enteroviruses, but as I said, we got the patent for all viruses. It certainly applies for RNA viruses, and other people have applied it now to HIV and some other virus families. It seems to work fairly universally. For FMDV [foot and mouth disease virus], another group showed you could get really pretty broad dose ranges with these kinds of changes, and in an animal system it seemed to work quite well.

What we've done for polio is initially we just had a little bit of effort going into it, kind of a small operation as part of everything we were doing, and then we got a Gates Grand Challenges award, $100,000 dollars for a grand idea. CRAWFORD: In 2009?

BURNS: Roughly, yeah. Then the WHO Polio Research Committee gave us some funds to work on this idea for alternative seed strains for IPV. The idea there would be the original inactivated polio vaccine, IPV strains, or wild polio 1, 2, 3, Salk MEF-1--sorry, the Salk strains are Mahoney MEF-1 and Saukett. In a post-eradication world, having wild polio in a factory in a pharmaceutical facility is a high risk for containment.

At the same time, while we were working on this idea, WHO was inching closer to Sabin IPV, so you use Sabin strains--

CRAWFORD: In IPV?

BURNS: To make inactivated IPV, yes, and that initially had some problems. They weren't sure it was going to work very well, but over time, some of the problems have been sorted out, and they weren't quite as bad as they thought. Now there are commercial Sabin IPV manufacturers, and it's licensed in Japan and China and several places. WHO started really pushing towards that, once they figured out it was going to be a viable path.

All along, we were thinking our technology would work with the original wild strains, because you could decrease their fitness and keep the wild proteins the same, but you can also do that with Sabin, so over time we also applied it to Sabin. You could do Sabin IPV and still make the strain even a little safer, because you could decrease fitness from Sabin down to a little bit lower level. Ideally, you would just have it be more genetically stable but still able to induce an immune response.

OPV and IPV are different in the way you would apply it, but we have applied it to both strategies. The Polio Research Committee was for IPV alternative seed strains. We started with wild IPV strains, moved to Sabin strains, but either way it could be used for inactivated vaccine, and you would just have less of a problem. The Sabin strain if it was released, it would be a safer Sabin strain if it was released accidentally from the IPV facility or if someone got infected. It wouldn't be wild polio; it would be a Sabin strain, a safer Sabin strain, even.

Then that morphed into--the Gates Foundation kind of learned what was going on, and they're really into technological advances. They had funded the Gates Grand Challenges for our technology, and so they started getting an idea of having a new OPV2 consortium of scientists that would put all the best techniques together and try to make a rationally-designed polio vaccine, which would serve as a backup plan. We hope that it actually won't be used, but the idea is that if this vaccine-derived poliovirus, particularly the type 2 that's circulating in Syria and the Democratic Republic of Congo--and circulated last year in Nigeria and Pakistan--if that continues to be difficult to shut down with the existing monovalent OPV2, which can always emerge into a new VDPV--that's the OPV paradox--then, this new OPV2 strain, or new OPV1 and 3 would be available in a vaccine stockpile that could be used instead of the standard mOPV2 to fight vaccine-derived poliovirus.

One reason that might be critical is [that] IPV does not stop the circulation very well; OPV can stop [it], because you get gut immunity. When the virus is ingested, your gut can respond, and IPV has mostly immunity in the blood system. In Israel in 2013, there was wild poliovirus circulating. It was detected in environmental sites, but it caused no paralysis. That was the first really clear demonstration that polio could circulate in an IPV-immunized population, and the spread of the virus would not really be stopped by the IPV. There were concerns that you might need a better OPV to stop circulating VDPV2 instead of just having IPV, which is the only available type 2 containing vaccine right now, because OPV2 was withdrawn from use.

[The] director-general has to approve use of monovalent OPV2, so it's used very sparingly now after the switch. If you could come in with a licensed, safer, more genetically stable mOPV2, which we call "new OPV2", from your vaccine stockpile, you could potentially do very directed outbreak response. So, [it's] not for routine use. We believe that IPV is the way of the future. Live attenuated vaccine strains still carry risks, but for specific indications, it might be really critical.

Gates [William H. Gates, III]--Bill Gates, himself, was interested in making sure no stones were unturned in having technological solutions to polio eradication. With all the effort that's happened, if we still have circulating VDPV2 a few years from now, if you had a licensed, better OPV, a new OPV2, you might be able to shut it down and not make new vaccine-derived poliovirus happen. That's the goal.

The consortium consisted of five members. We developed new OPV2 strains, two candidates, and new OPV 1 and 3 development is ongoing. Jennifer [L. Konopka-] Anstadt [PhD, team lead, Vaccine Development Laboratory, , Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, U.S. National Center for Immunization and Viral Diseases, CDC] joined our team two and a half years ago, and she's also worked on the daily operations of that team, continuing with new OPV 1 and 3. But there was a contained clinical trial at University of Antwerp this year--in the middle of the year--and they're making plans for a more extended, real phase 1 clinical trial for 2018. That's been the first test of new polio vaccine since the '60s. That's very exciting. Of course, it was challenging to do a contained study. They made a small facility in a parking lot at the University of Antwerp near the medical center with a bunch of containers. There's all the living space. Fifteen people received each of the two candidates, sequentially. There's a video that shows "Poliopolis," the village that they built and that these vaccine recipients had to stay in for roughly a month.

CRAWFORD: Just to keep it contained?

BURNS: Yes, yeah, while they got the initial information about excretion, et cetera.

Another group in our branch has been doing the testing, because it requires really quick turnaround. These people had to stay in the facility and monitor their excretion of virus from their stool in almost real-time. They had twenty-four-hour shipping and then a couple-day turnaround to do virus excretion assays to see how much virus was in the stool, or feces, and then also to see how the immune response was from the blood. All of that is still be analyzed, but [William C.] Will Weldon's [PhD, formerly of the Population Immunity Lab, Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, U.S. National Center for Immunization and Viral Diseases, CDC] team has robotics to help with the serology and the excretion studies, semi-automated assays, and so they're one of the only places in the world that could handle the volume and the turnaround time. The development and the testing have been kept very separate, since we're both at the same institution and blinded as much as possible, et cetera. We waited to hear the results with everybody else and were quite excited to hear when he unveiled it in a small meeting of the clinical collaborators, et cetera.

CRAWFORD: I think you've covered most of what we set out to talk about today, but I wanted to see if there are any--we always miss a lot of material--

BURNS: OK, sure.

CRAWFORD: But two of the things that you mentioned during the break that we missed today are computational and data management technology transfer?

BURNS: [Yes.]

CRAWFORD: And then next-generation in Ghana.

BURNS: As part of the molecular epi and sequencing technology--building family trees of these viruses or dendrograms, doing phylogenetic analysis, knowing how to report these vaccine-derived polioviruses, estimate the timing, et cetera--all those things require software tools, and we invested about a decade ago in using MATLAB® [matrix laboratory] software to try to make some compiled stand-alone programs we could transfer to some of the key labs, so Pakistan lab and South African lab, in particular. Things these programs did was build the trees and then label them automatically, so pulling the epi information about the onset date or the specimen date of collection, the location, the district, the year, et cetera, and automatically populate that on this family tree. Many years ago, Olen Kew was in his office manually typing out all these identifiers that have PIDs [primary immunodeficiencies] or whatever, and we were like, "OK, we have to solve this problem." We don't endorse MathWorks [company] software, per se, but this was a tool we were able to use, and because they have a compiler that the CDC has--it's very expensive, but it allows you to take the software you develop that you would run on MathWorks software here at CDC and make stand-alone programs that could be run in Islamabad or Johannesburg without them having to buy the license for this very expensive software.

We have modules that analyze the sequence. They'll annotate files and help keep them organized. They'll make the family trees and label them. They'll do mapping and then some other more sophisticated analysis. That, along with the data management kind of training--I would say, a mindset--that's some of what we've tried to transfer to some of our partner labs, and that's been a big part of it. Once you get your central sequences, you need to be able to put that epi information in a form that's digestible, and the program has been using that.

The maps were really important. When there was a lot of wild polio, we would--there's another tool that would help us do clustering to figure out which viruses are most closely related to each other and then give them names and update the names over time, because the virus keeps evolving. Then we could put those with symbols on a map and see much more than just serotype and genotype; we could see a cluster, and then from there you could infer even finer level of resolution, like lineages and individual chains of transmission that you could kind of trace on a family tree to see how the virus has been spreading and moving around. That's one other big area of transfer my team has been involved in.]

For Ghana, I just want to mention that that's one place where we transferred some of the technology. We had kind of a challenging time transferring sequencing technology, one of the key lab people had come and learned in roughly 2000, the VP-1 sequencing, and he had been doing that off and on. He went and got his PhD somewhere else; he came back.

A few years ago, someone from my team went to do an onsite training, and they were going to be another critical sequencing lab in the African area, in addition to the Johannesburg lab. She got there, and they had decided to do preventative maintenance on their sequencing instrument, and so the ABI [Electronics] tech actually showed up like the week before, or whatever, and worked on the real-time PCR machines, and that was fine. He worked on the sequencer. He disassembled it. She was there; she needed to do the training; it was disassembled, so she was very disheartened, and they did all the other steps. Then, like the day before the final day, he put it back together again, but it didn't work. [Laughs] She was so upset. This challenge with the level of technical staff and the maintenance and all that, and they had been trying for a long time to get someone to come and service the machine, and it just happened to be right before the visit. That was very challenging. Now, since then, they've passed their sequencing PT proficiency test. They are an accredited sequencing lab; there are plans for them to start taking over some of the workload, so that part has been successful. There's been a lot of email, remote coaching, and then one of the lab people has been able to come to a sequencing workshop in another location.

Then what I wanted to say, that's more recent, is the next-generation sequencing technologies have been developing. Our ability to use them for viruses at CDC in our division has improved over the last few years, and so there's not a lot of transfer. That technology within the GPLN, we're still relying on the Sanger automated sequencing technology from the last twenty years for a number of reasons. But we're starting to help, so Rachel [L.] Marine [PhD] in our division core, that does the next-generation sequencing, has visited labs like the Ghana lab and has started to--they have a sequencing instrument, and she's started helping them to transfer technology.

We're trying to figure out--the small working group is trying to figure out--how the next-generation sequencing technology will really feed into polio. We sort of hoped that it would just wait for measles or what came next, but we do have some things we can answer with complete genomes, and it is much easier to do the complete genomes with next-generation sequencing. It's not quite as fast; the turnaround is a little slower, so we're working on that aspect. We're also able to sequence deeper, so we can get into some of the samples that were virus mixtures and get a little more information.

However, next-generation sequencing doesn't fully solve the problem of virus mixtures, so there's still some technological development going on there. Generally, the next-generation sequencing was a huge boon to bacterial labs or labs that had huge genomes--their organisms have huge genomes--they just couldn't do it the old way. But for viruses that are only 7,500 nucleotides long, the old technology actually is still pretty good. Certainly, just for the VP-1 region, which is about a thousand nucleotides long, nine hundred nucleotides is the actual VP-1 protein. That technology is still pretty good, so we're trying to see how next-generation sequencing technology will be implemented in the polio program and trying to help some of our colleagues that have acquired new instrumentation to figure out how they could implement it and use it.

When I visited Ghana the first time for my first regional lab meeting, they changed the dates, so I was a day late. My luggage was lost, because I was in JFK [John F. Kennedy Airport, New York City] only an hour, and that's just not long enough. I got there, and I didn't have clothes, so we went to the local market. We bought like an African--just a flowing gown, just a single, you know, very traditional looking gown. I was supposed to chair the meeting, so I came late, the second day. I got up there, and it was just me and this big table, and Humayun Asghar joked that I looked like a Supreme Court justice, [laughs] because I was up there in this flowing robe, and everybody else was down.

Then the guy from Nigeria, Festus Adu [DVM, MS, PhD, former laboratory director of the National Polio Laboratory at the University of Ibadan, Nigeria], the lab head, he had this purple hat from his traditional costume, so I put that on for a while. That was my first introduction to the AFRO [African Region of the WHO] regional lab meeting, and my colleagues from Ghana were there. It was in Accra, Ghana and then all the African labs were there. That was just a funny bit of history. Then when I went back, because my connection was so tight in JFK, my luggage was late again, getting back, so I clearly learned a few things about coaching the travel people, not to put me on tight connections.

We're continuing to work with the Ghana lab, and the South African lab, in particular, also, that I said I supported, starting in early 2000s, to make sure that the African continent is covered for the molecular epi. We hope that wild polio is completely gone from Africa, and VDPV may continue, but Nigeria does have pockets of inaccessible people, and there was wild polio there last year. The jury is still out a little bit about whether wild polio is fully gone from Nigeria or not.

CRAWFORD: And the clock restarts, at least for certification?

BURNS: Yes, yeah, exactly.

CRAWFORD: Well, thank you so much--

BURNS: Sure.

CRAWFORD: --for meeting with me this morning and doing this interview. Would you be willing to do a second one as follow-up?

BURNS: Sure. Yeah. CRAWFORD: OK. Next up will be to have this transcribed.

BURNS: OK.

CRAWFORD: Before we close, I wanted to ask if you have any final thoughts?

BURNS: Well, thank you so much for the opportunity. I'm very excited that you guys are doing the oral history project and also talking to some of my colleagues. I wanted to also acknowledge Lina De [PhD, formerly of the Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, U.S. National Center for Immunization and Viral Diseases, CDC], who worked on the probe hybridization for many years; Dustin Yang [PhD, formerly of the Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, U.S. National Center for Immunization and Viral Diseases, CDC], who worked on the Sabin assays and many other aspects, the Egypt VDPV2 number of projects; and really acknowledge the teams involved.

Right now, there's about sixty people in the Polio and Picornavirus Lab Branch. Most of us work on polio. There are about five people who work on other picornaviruses. It's still a huge endeavor. If you divide the number of wild polio cases by the number of staff working on polio at CDC [laughs], it sounds a little crazy, but we're still trying to develop direct detection technology, where we don't have to use virus isolation. That's been kind of the Holy Grail for a while--and working with containment, getting ready for our own GAPIII audit sometime in the next year; working on inactivation methods, so that we can transfer that to other labs even outside the polio LabNet, because stools in rotavirus labs could have polio in them, depending on when and where they were collected, and so they need to know what to do to contain their potentially-infectious materials. Other sorts of things like that are still keeping us really busy, but yeah, just to really acknowledge all the team members over the years.

CRAWFORD: We're asking for names to be submitted of other people you'd like to see included, if possible.

BURNS: OK, sure.

CRAWFORD: So, maybe your top five names.

BURNS: OK. Esther De Gourville, who was the EMRO and then the global lab coordinator before Ousmane Diop; Humayun Asghar, who has been the EMRO lab coordinator, and he was the Pakistan lab director before that; Nicksy Gumede[-Moeletsi], who was the polio lab director in Johannesburg and is now the main AFRO regional lab coordinator. Maybe, Harrie [G.] van der Avoort, who was the Netherlands lab director and did a lot of African support of the ELISA technique, and they also did a lot of environmental surveillance.

CRAWFORD: That's four.

BURNS: OK, and Gene [V.] Gavrilin. He came and did polio sequencing in 1999, I guess. He was in the Russian lab at the time, and he's been the EURO regional lab coordinator for several years. Also, [he] is an expert in biosafety, biosecurity issues. CRAWFORD: Great, and that's again, not comprehensive, but just the first five that came to mind in this very moment, so thank you for that.

BURNS: OK.

CRAWFORD: Then if we do have the opportunity to do a second interview, what have we missed today that you would include?

BURNS: Let's see, I mean I think we covered things pretty well. Maybe a little bit more about the technique development over time, or the most recent techniques. But I think we hit on that pretty well. Oh yeah, I probably should have said something about distribution of the kit.

We developed the technology for these PCRs and sequencing methods, but we also--Ever Vega's team also distributes those kits of primers, essentially to all these 130 labs and then the sequencing--it's like thirty labs. I didn't talk much about that kind of support, just really every day replenishing the kits, distributing the kits, shipping the kits. I talked some about training, but they also do a lot of the recent training events.

CRAWFORD: Yeah, it would be great to have that. Anything else that isn't written down somewhere. The oral histories are part of the record--

BURNS: Right.

CRAWFORD: They're not--we can't replace document trails, but is there anything that occurs to you that isn't written down somewhere?

BURNS: Maybe a little bit more about the small working group, about those interactions and how that came about. I don't know if Mark Pallansch would have mentioned that in his. He has he done that one already? Yeah, he's done one already, OK.

CRAWFORD: Then we'll do a follow-up one.

BURNS: OK, yeah. I think that's all I can think of right now.

CRAWFORD: Well, thank you.

BURNS: Sure, thanks.