Jeffery Taubenberger - Full Transcript

Jeffery Taubenberger Senior Investigator in the Laboratory of Infectious Diseases at the US National Institute for Allergy and Infectious Diseases

Interview location: The National Institutes of Health campus in Bethesda, Maryland, United States.
Interview date
: 27th November 2007

 

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I am senior investigator in the laboratory of infectious diseases at the National Institute for Allergy and Infectious Diseases in the NIH, Bethesda, Maryland.

SA:  Jeffery, how did you first get interested in doing science – and what sort of family did you grow up in?

JT:  Well, I grew up in a German immigrant family, actually.  My father emigrated from Germany as an adult in the early 1950s, after World War II, having survived the war as a teenager.  (My father is German, but my mother is American.)  My father was a computer scientist, a systems analyst, and having had some electronics background in Germany, he was in the United States less than six months when he was drafted into the army – "Welcome to America!" – for the Korean War. But luckily for all of us, he wasn’t sent to Korea; he was picked out because of his background to be involved in an experimental project the army had about building a computer.  He helped build and install the first large main frame computers that the US Army used in Europe, in Germany, so he was stationed in Germany for a number of years.  I was born in Germany, but in a US Army base.  

My father was an army officer and then ended up spending another 25 years or so, after retiring from the army, as a civilian working for the Department of Defense doing the same thing.  So he spent his entire life in public service working for the Department of Defense doing computer work, which involved two years in Vietnam as well, and lots of moving around with the military.  We were a military family.

SA:  And did you get fascinated by his line of science?  Did science thrill you as a kid?

JT:  I’m the youngest of three children.  I have two older brothers who were not interested in science, and still aren’t.  But I was very interested in science from a very early age.  I have to say that from maybe the age of four or five I already knew that I wanted to be a scientist.  I guess I should say that, having made that decision, my interest in the kind of science I wanted to do sort of varied.  It started out (this is in the early 60s) with a view that I would like to do nuclear physics, or something like that.  That seemed very exciting…

SA:  But as a really young kid, what fascinated you about science, what did you know about science?

I just liked figuring out how things workedJT:  Well I don’t think I knew very much about it at all, actually.  And what fascinated me was that I just liked figuring out how things worked.  That was really it -- I just wanted to understand how things worked around me.  I was drawn to that.  I ended up, as a kid, reading lots and lots of biographies of scientists and inventors.  I remember as a child being fascinated by biographies of Thomas Edison, and Henry Ford and Pasteur, and Koch and others like that.  

As I got older I became quite interested in chemistry and I went through a phase that probably many boys go through, with interest in rockets and fireworks and things that go boom. But I actually acted on them with friends -- we made rockets and rocket fuel, and all sort of things that I wouldn’t let my children do! [We laugh] We did have a lot of fun with that.

A precocious scientist

The systems that control biology are just fascinating to meAnd then I became quite interested in biological things.  Animate things seem so much more complicated than inanimate things, and the systems that control biology are just fascinating to me.  So I started working in a lab of the National Cancer Institute as a volunteer when I was in ninth or tenth grade in high school -- that would have been aged 13 or so.  

SA:  Doing what in the labs at that age?

JT:  I was doing molecular biology actually!  At a time when molecular biology, in a sense, was being invented.  So, for example, in tenth grade, my sophomore year in high school, I did a project in which I extracted DNA from a variety of sources, with the question: is the total amount of DNA per weight of tissue related to how complex an organism is?  I grew bacteria, and I took DNA stands for deoxyribonucleic acid.  This is the material inside the nucleus of the cells of living organisms that carries genetic information (see also RNA). from a plant (green beans), and from an animal (I think we went and bought liver at the butcher shop), and then I calculated the weight of the tissue and how much DNA was recovered.  The hypothesis is not correct, actually: there’s no relationship between total amount of DNA per dry weight of tissue and the complexity of the organism.  But anyway, that was my tenth grade, high school science fair project.

These viruses can become part of your DNAAnd what was going on in that lab was quite interesting.  I met a man named Dr William Drohan, who mentored me.  He was a molecular virologist who was studying endogenous retroviruses – that is the same class of virus as the HIV virus, but these viruses can actually integrate themselves, that is incorporate themselves, into the Genome The complete package of genetic material for a living thing, organised in chromosomes. A copy of the genome is found in every cell.  of cells and become resident, become part of your DNA.

SA:  For ever and ever and ever?

JT:  For ever and ever and ever, yes.  So all humans and all animals have copies of these ancient viruses as part of our own DNA…

SA:  And are they doing anything?

JT:  Well, that’s a difficult question.  Most of them are inactive. They have mutations such that they cannot actually make most of their genes.  But they do move around; and they do have the ability to disrupt other gene function.  There was a big theory in the ‘70s that many cancers are actually caused by viral infections.  The hypothesis was that many cancers might be caused by endogenous viruses – viruses that you aren’t exposed to like a typical virus, but that you actually have encoded in your DNA stands for deoxyribonucleic acid.  This is the material inside the nucleus of the cells of living organisms that carries genetic information (see also RNA). .

SA:  And have we had these for hundreds of years?  I mean, they just are part of us now, are they?

JT:  Right.  In terms of humans, probably for tens or hundreds of thousands of years, or even longer.  They have been incorporating themselves into animal Genome The complete package of genetic material for a living thing, organised in chromosomes. A copy of the genome is found in every cell. s forever, basically.

I was about 15 or 16, when I started universitySo that was a very interesting time.  William Drohan mentored me, and I was working in his laboratory throughout high school.  When it came time to go into university -- and I skipped some grades so that I was rather young, I think I was about 15 or 16, when I started university – I decided that I wanted to stay here in the area, because it was such a fantastic opportunity being in his laboratory.  This was the 1970s, when a lot of the basic techniques of molecular biology were just sort of being invented.  So I went to George Mason University, which is in Fairfax, Virginia, very close to my house, and I worked in that laboratory half-time during the fall and spring semesters and then full time in the summers, for the four years.

SA:  And what were you doing at university – were you doing medicine?

JT:  I was studying biology as my major.  I obtained a Bachelor of Science degree in biology in 1982.  And Bill Drohan strongly counseled me throughout my entire time with him that I really should go to medical school and do a combined…what they call an MD/PhD degree.  I don’t know if they have the exact equivalent in the UK, but here you can, after getting your bachelor’s degree, go to medical school for a four-year course of study to earn a medical degree. Or you can go to graduate school and get a basic science degree in some discipline or other, a PhD.  But they have some programmes where you can do both, so it’s a combined thing, and it saves time because some of the courses overlap.  

Medicine as a discipline was enormously interesting and rewardingSo I entered a combined MD/PhD programme at the medical college of Virginia, in Richmond, with the idea basically that this would augment my research career.  I really didn’t envision myself as a 'physician' at that point in time.  But what I discovered, shortly after arriving in medical school, was that medicine was really fascinating! [Laughs]  Just sort of intellectually, it drew me in.  I never saw myself, never had as my role model, the sort of private practice physician -- I mean, I don’t think that ever entered my mind.  But medicine as a discipline was enormously interesting, intellectually interesting, and rewarding. 

Science and medicine: complementary worlds

The rewards of medicine and of basic science are usually quite different. Instant gratification is very rarely, if ever, seen in scienceThe rewards of medicine and the rewards of basic science are usually quite different.  In a sense they’re complementary.  In medicine you can get satisfaction in a much shorter time period, in that you do something and there’s an outcome and you receive satisfaction.  To take a very simple example: somebody comes with an appendicitis and has an appendectomy, and is saved when otherwise they would have died, and that’s enormously satisfying – hence the egos that surgeons develop!  [Laughs]  That kind of instant gratification is very rarely, if ever, seen in science. You know, if you have that sense, it’s probably because it’s wrong! [Laughs].  

Science moves at a much slower pace. The thing that's challenging about medicine is that it’s an uncontrolled experiment Science moves at a much slower pace generally.  That doesn’t mean that science is slow  -- it’s just the mechanics of the way science unfolds, because of the difficulty of doing things, the length of time it takes to do experiments, and to analyse the data, and so on.  You have a much longer-term sense of gratification, whereas in medicine it's shorter-term – things happen, somebody gets better or they don’t. The thing that's challenging about medicine, of course, is that, unlike a good experiment in the laboratory, it’s an uncontrolled experiment, there are too many variables, and you don’t really know what’s going on.  I think it’s always a mistake to be overconfident.

SA:  So working in the two things, which most fascinates you?  Or do you find you pick from both?

JT:  Well I guess I pick from both.  So I usually…If people ask me what I am, or what I do, I usually say I’m a physician, actually.  And I do definitely feel like a physician, even though I’m now in a position, having moved to the NIH last year, where for the first time since finishing my medical training I don't have some direct clinical role.  So this is actually a change for me.  And in one sense I still miss that, but I’m also, at this point in my life, quite happy with this decision right now.  I feel comfortable with it.  But I’ve been very lucky to have been able to do both patient care-oriented work as well as basic science.  I think that’s one of the tremendous advantages of being a pathologist, actually.

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So, going through medical school I did a PhD in the anatomy department.  And I ended up doing my research, my dissertation work, under the direction of Professor Jack Haar at the Medical College of Virginia whose interests were developmental immunology.  He was trained as an anatomist, as a histologist, but his interest was looking at how stem cells from the bone marrow become mature lymphocytes – T-cells mainly.  What has to happen is that stem cells from the bone marrow have to migrate to the thymus gland, which sits in front of the heart and produces the T-lymphocytes that you have.  This process occurs predominantly during fetal development, so that by the time you’re born most of the T-cells that you will ever have have been made.  And by the time you’re a teenager your thymus gland is actually atrophying away so that you can no longer produce new T-cells: they have to be either the old ones, or clonal descendants of the T-cells that have already been made.

This just seemed like a very interesting project to me.  I was shopping around literally to find a laboratory to work in, and I interviewed a large number of faculty members about projects.  I was looking for a project that would pique my interest and this just seemed quite exciting.  Plus Jack Haar was just a wonderful man, just a very nice and great personality, and I think that’s very important.  

It was really quite beautiful to look at the structure of different tissues and organsI identified him within the first year of my medical school.  I began working in his lab right away, so started at least to get some experience.  His group worked in the summer breaks and so on.  (That's the advantage of being young and energetic -- I think it would be hard for me to replicate that now!)  So I ended up doing my training, doing immunology work but in an anatomy department from a person who taught Histologythe study of cells and tissues, usually carried out with the aid of a microscope..  
I had a lot of exposure to histology -- looking at tissues through the microscope -- and ended up, as part of my training of course, being a teaching assistant in histology labs to medical students and others, such as dental students, and so on.  And I rather liked that.  It was really quite beautiful, actually, to look at the structure of different tissues and organs, stained with dyes and things.  It’s really quite lovely.

Public perceptions of pathologists

Most pathology is “pathology of the living” pathologists have done themselves a disservice by being so behind the sceneThat was, I think, a good grounding for pathology.  But I have to say that during my time at medical school I did not really have a clear idea of what pathologists actually did.  I think that most medical students think of pathology as something that either happened long ago, as in: “Virchow described this tumour…” and so now we’re done, we don’t need to do that anymore; or that it's autopsy pathology.  I sort of missed this whole idea that most pathology is what people have come to call “pathology of the living” – that is, the idea of the biopsy, and that it’s not the surgeon or the internist who says that this lump is benign or malignant, but it’s the pathologist.  It's this sort of magic thing that happens behind the scenes -- and pathologists have, in a sense, done themselves a disservice by being so behind the scene.  In the medical world, of course, they understand and appreciate the role, but I think the public often doesn’t.  

I think the public perception (which was the perception I had) is that pathologists are either doing autopsies or are forensic pathologists -- that is, doing crime scene investigations and so on.  (In the last 10 years in the United States, the number of detective television shows that involve pathologists looking at crime scenes has proliferated.)  So, I didn’t think of pathology as a career.  Because of my research work looking at T-cell development – and I also had an extraordinarily positive experience during my paediatrics rotations in medical school, with excellent attendings (consultants) -- I thought that paediatrics might make sense for me. I could combine interest in the lymphocyte development with childhood leukaemias and things like that.  So I thought I would do paediatrics, and I investigated it quite seriously.

Paediatrics or pathology? That’s the question

I finished my combined MD/PhD degree and would then have gone to do my post graduate residency training in paediatrics.  But I decided to delay my residency and stay in the laboratory just one more year to finish up some projects that we were writing up.  And during that year, actually, I had some doubts about whether I wanted to do paediatrics.

SA:  Why?

I wanted to have a very lab-centric career. a ridiculous dreamJT:  Mainly because I realized that I wanted to have a very lab-centric career.  While I liked (and still do like) medicine, I really wanted a basic science career – informed by medicine, but more of basic science-oriented.  And I began to wonder how practical that would be.  I think many people do a combined degree believing they can do 50:50 -- that is, that they can work in the laboratory in the morning and see patients in the afternoon.  Of course that’s a ridiculous dream. The demands of both careers are just too large, and their worlds, in a sense, are very different, so that it’s just impossible.  And of course, if you're to be paged in an emergency to see a patient, that always has to take precedence over what you're doing in the laboratory.  

So that’s when I thought, "Well, I’ve had all this experience looking at tissues under the microscope.  I really like that; maybe pathology would make sense to me”.  But I had not done any rotations in pathology.  (It was, unfortunately, not a required rotation in our medical school.)  And during my elective period I'd spent all my time in the laboratory.   So I sought out a number of pathologists in the department in the school to ask them about careers and programmes, and then I looked through lists of places that have pathology residency programmes.  But I looked for places that had strong research components as well.  

It turns out there are a handful of locations in the United States where people who want to do pathology residencies but who have a very strong basic science commitment often go – there are the Harvard programmes, Stanford, Washington University in St Louis, University of Pennsylvania, Johns Hopkins, and other places like that.  But as I was going through the list I noticed that the National Institutes of Health actually had a pathology residency programme.  I was amazed because I had no idea the NIH actually had residency programmes at all.  And it turns out pathology is the only residency offered on campus, and they only have positions for three people a year, so it looked to be very competitive.  But I applied and was accepted.  It was kind of thrilling because I had worked in the Cancer Institute previously, and here I was coming back to NIH to be a pathology resident, and it just seemed like a wonderful fit.  

SA:  And was this just pure teaching or was it a clinical setting where you actually saw patients as well?

JT:  It was a clinical service.  The National Institutes of Health has a hospital, what they call the Clinical Center, and it's an unusual hospital in the sense of its patient population.  The only patients seen at the NIH, or who are hospitalised as in-patients, are patients who are involved in clinical research studies.  So all of the institutes here – the Cancer Institute, and the NIAID [National Institute for Allergy and Infectious Diseases] where I am now, the NHLBI [National Heart, Lung and Blood Institute], and other institutes – they all have a certain number of beds and clinic spaces allocated to them, and they have clinical studies on-going.  So for example, they had melanoma protocols, or renal cell CarcinomaA type of cancer that starts in epithelia, the tissues that line or cover most body organs.  At least 80% of cancers are carcinomas (see also sarcoma, leukaemia, lymphoma). protocols, or congenital heart disease protocols -- you know, different studies.  Patients would be recruited into these studies and if it was a study that needed tissue confirmation -- for example a tumour protocol – of course the original material or biopsy done in the hospital would have to be interpreted just as a standard pathologist would do it.  So there are frozen sections, surgical biopsies, there are autopsies… It was a standard pathology residency, but just with an unusual patient population.  

There was a much higher concentration of “interesting cases”, we saw every kind of sarcoma imaginable, and all sorts of unusual thingsIn a sense there was a much higher concentration of “interesting cases”, so that for example, whereas in a traditional hospital as a pathologist you would see a lot of hernia sacks from hernia repairs, or placentas, or tonsils or things that are not really very rewarding, we saw every kind of sarcoma imaginable, and all sorts of unusual things.  So it was quite an interesting experience.

SA:  Can you remember some of the most interesting cases that came past your microscope at that stage?

JT:  Well I mentioned hernia sacks -- I only saw one hernia sack in a five-year residency, and it actually had a metastatic rhabdomyosarcoma, an unusual SarcomaA type of cancer that forms in the connective or supportive tissues of the body such as muscle, bone and fatty tissue. Sarcomas account for less than 1% of cancers., in it.  I’ve had gall bladders taken out that had melanomas in them and all sorts of amazing things.  I saw a really amazing case where a person had a kind of liposarcoma, which is a malignant tumour of fat. It’s a rather low grade tumour but it was infiltrating around the abdomen such that it could never be totally removed, and what would happen is that this person would come in and have as much tumour taken out as possible and then it would recur, and then they'd come in and have surgery again.  This went on and on, but then an amazing thing happened.  This person developed a kind of LeukaemiaCancer of the white blood cells, which are a vital component of the immune system (see also lymphoma)., and the leukaemia seemed to home in on the tumour so that you had the sarcoma, the fat tumour, and then infiltrating inside of it were the leukaemic cells.  It was a remarkable thing. So yes, I saw things that would be very hard to find anywhere else. It was a very interesting and unusual experience.  And it allowed me not only to be trained and board-certified as a pathologist, but to continue my research interests.

With colleagues outside the high containment lab at NIH, Bethesda, 2008I ended up doing research -- initially the same research as I had done my PhD on, T-cell development.  I was working with a colleague of my old professor, Dr Ada Kruisbeek, who was from the Netherlands, who spent a lot of time at the Cancer Institute and who also had an interest in T-cell development.  In my final years of residency I began to work in her laboratory, and continued my research on early T-cell development.  Then she decided to accept a position at the Netherlands Cancer Institute in Amsterdam and was interested in having me follow her there.  But I was just about to get married and it didn’t seem like the right thing to do at that point.  So I left the lab and went back to pathology, finished my residency, and ended up accepting a position as an attending (consultant) surgical pathologist in the department.  I had a lot of free time -- about half of my time I was not on service, and so I started doing independent collaborative research with a colleague, Kevin Holmes, who is still here at NIH, on lymphocyte DifferentiationThe process whereby an unspecialized early embryonic cell acquires the features of a specialized cell such as a heart, liver, or muscle cell. .

A person who was a mentor to me during my residency is a pathologist by the name of Timothy O’Leary.  He is an MD/PhD who also did his residency at the NIH and then went on to become an attending pathologist (ie a consultant). When I was a resident he had left the NIH to take a position at the FDA (US Food and Drug Administration), but he still came back to do autopsy attending pathology.  He was an excellent teacher and a great man. He knew a huge amount about autopsy pathology, but also was a person who had very basic science interests – had a career in which he had clinical pathology contact but was running a basic science lab.  I was very impressed by him.

He showed me that my dream was possible – you could still keep a hand in pathology, a hand in clinical medicine, but also do basic scienceHe showed me that my dream was possible – that you could still keep a hand in pathology, a hand in clinical medicine, but also do basic science.  I was inspired by the fact that, although he was doing a lot of basic science, he was still an excellent and caring physician.  He still has excellent diagnostic skills, and if I had a medical issue that I wanted to run by someone I’d feel comfortable running it by him.

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AFIP: “one of the oldest and most venerable pathology institutes in the world”

Timothy O'Leary subsequently accepted a position at the Armed Forces Institute of Pathology, the AFIP, which I don’t know if you know anything about…?

SA:  No I don’t.  Do tell me about the AFIP.

JT:  The AFIP is one of the oldest and most venerable pathology institutes in the world, actually.  It was founded in 1862 by executive order of Abraham Lincoln during the US Civil War, with an order to the Surgeon General of the army to study diseases of the battlefield.  Pathology as a medical discipline was sort of being invented at that time, the mid 19th century, with advances in the understanding of basic patho-physiology -- Virchow’s work in Germany, of course, and others.  So the AFIP was founded as an army institute, but it transitioned somewhere around the early part of the 20th century, while still being part of the US Army, into an institute that became a repository of pathology expertise that also tapped into the civilian world.  

This tissue repository has been growing from the 1860s to the present, so that they have millions and millions of casesThe Institute developed a programme very early on -- I have to say it was extremely foresightful -- in the early 1900s, and the idea was that they would serve as a consultative base for difficult cases, interesting cases in pathology. People would send them cases which would then be kept in their repository as a permanent and ever-growing collection.  And this tissue repository -- of fixed tissues, blocks, slides and so on -- has been growing from the 1860s to the present, so that they have millions and millions of cases.  

SA:  And beautifully written up and recorded, I imagine?

JT:  Yes.  They have computer-searchable archives for key words and diagnostic information, and they've gone backward in time at least to about 1915 or so.  So you can do key word searches and find things and know exactly where they are.  I’ve seen old pathology collections in a number of places -- the collection at the morbid anatomy department at the Royal London Hospital, for example, where they have also a large number of autopsy tissues going back to about the early 20th century.  But it’s in this horrid room in the basement with steam pipes and mould everywhere and old books that are falling apart and are difficult to read.  So the way the AFIP’s tissues are stored -- in warehouses with these enormous, unbelievable racks, and they have these automated machines that retrieve a tray of slides, for example… You punch in the number of the slide you want and these machines move, and out of tens of millions of slides, this one tray comes shooting out -- it’s quite impressive to see.

Many tumours have characteristic genetic changesSo the AFIP ended up being one of the largest places, and Tim O’Leary took a position as chairman at the department of cellular pathology in, I think, 1991.  He wanted to create a molecular diagnostic pathology component, with the idea that molecular biology tools would be useful as an adjunct in anatomical surgical pathology.  Many tumours have characteristic genetic changes, mutations or translocations or things that [he believed] could be diagnosed using molecular means, and that you could use molecular genetic means to diagnose infectious diseases, for example.  And of course there are genetic tests.  He was looking to recruit people who had dual interests in anatomic pathology and molecular biology to develop clinical diagnostic assays that could be used as an adjunct for classic histologic diagnosis, and he recruited me to come and head this new group.
That happened in 1993, and it was just a fabulous opportunity, as molecular pathology --as a sub-speciality of pathology -- didn’t really exist at that point.  (Now in the United States it’s a formal sub-speciality with its own board exam).  

It ended up being a fabulous nicheIt was sort of a natural progression for me, and it ended up being a fabulous niche.  What happened was that I arrived in 1993 and continued to try to keep my own interest in lymphocyte DifferentiationThe process whereby an unspecialized early embryonic cell acquires the features of a specialized cell such as a heart, liver, or muscle cell. alive, but was asked to set up a clinical molecular diagnostics laboratory.  We started with a handful of different assays – tests -- that we wanted to run.  For example, certain kinds of LymphomaCancer originating in lymphoid tissue, a key component of the body’s immune system.  Cancers of lymphocytes (lymphomas) and other white cells in the blood (leukaemia) together account for about 6.5% of all cancers.s have very characteristic chromosomal translocations where bits of one chromosome break off and re-attach to another one, so you can design a molecular assay that identifies that 'break point'.  And these changes are absolutely diagnostic.  

These molecular tests might actually be definitiveSometimes when you look at a lymphoma or a LeukaemiaCancer of the white blood cells, which are a vital component of the immune system (see also lymphoma)., for example, under the microscope it may be so classic in terms of its Histologythe study of cells and tissues, usually carried out with the aid of a microscope. that you can say, "Well it’s this sort of lymphoma.”  But very often it’s difficult, so you look for additional tools.  Immunohistochemistry A technique that uses antibodies labelled with fluorescent or pigmented dyes to identify, or indicate the presence of, specific proteins in tissues when looked at under the microscope., which looks for the expression of certain proteins, is an adjunct tool, and there are certain other tests you can do too.  It became more and more apparent, as basic science was advancing, that these kind of molecular tests could be used diagnostically -- and in fact, in a sense, would be the definitive diagnostic tools.  Histology might not be definitive, but these molecular tests might actually be definitive.

Our task was to develop assays that could be used on typically-processed surgical pathology or autopsy pathology material.  (This was mainly surgical pathology, because we were doing diagnostic work.)  You want to extract genetic material -- that is DNA stands for deoxyribonucleic acid.  This is the material inside the nucleus of the cells of living organisms that carries genetic information (see also RNA). and RNAstands for ribonucleic acid.  RNA, like DNA, is found in every cell of every living thing on earth.  The relationship between the two, in summary, is that DNA makes RNA, and RNA makes proteins.  In other words, DNA is the director of the process of protein synthesis and RNA carries out the instructions. -- from a tissue that has been fixed in formaldehyde and embedded in paraffin, but  while this process preserves the structure of the tissue, it actually does terrible things to the DNA and RNA inside: chews it up and causes cross-links and all sorts of bad things. So what we had to do was develop techniques that would allow us to reliably get nucleic acids (DNA and RNA) from such fixed tissue and then figure out ways we could ask the kind of diagnostic questions we wanted to ask, such as "Is this translocation there or not?”, given the constraints of the poor quality DNA and RNA we were going to retrieve.  So it was kind of an interesting technical question.  Most laboratories that were doing these kinds of tests would only do them from fresh tissues.  They said, well, these assays just couldn’t be done from fixed tissue.  

It was an interesting technical challengeAt that time, in the early 1990s, most people thought you actually couldn’t even recover nucleic acid from fixed tissues.  So again, it was an interesting technical challenge, a practical challenge, and we thought it was very exciting.  My colleagues and I (obviously I wasn’t doing this alone, there was a group of us) were doing this project and we worked out techniques that we could reliably extract both DNA and RNA from fixed tissue and we developed assays to do a lot of these diagnostic tests.  We became the only facility, at least initially that we were aware of, that could do these kind of tests, so people would send us cases from all over.  It was quite an interesting time.

Of breast cancer and dying dolphins

SA:  So how did your interest in flu arise?

JT:  Right.  So we started thinking about doing research projects that would take advantage of this technology, and one idea that a colleague of mine, Dr Jack Lichy, and I came up with was to do a series of projects on breast cancer genetics, which was quite exciting, (I’ll get to flu in just a minute!)  If you look under the microscope at a typical breast cancer biopsy, what you find is that it’s often not very uniform -- that is, there's some in situ cancer here, and some invasive cancer there, and the cancer over here looks a little different from the other one, and there’s normal tissue in between, and so on.  

Most molecular biologists who are not pathologists don’t see those complexities, it could never be worked out unless you looked at it at the level of a pathologistWhat we wanted to know was, could we find some molecular markers that would inform us as to how the tumour was progressing?  How did it go from being contained within a duct to actually invading through a duct?  What we reasoned was that most molecular biologists who are not pathologists don’t see those complexities.  They get a piece of tissue from a surgeon or a pathologist that they are told is breast cancer; they put it in a blender and make DNA stands for deoxyribonucleic acid.  This is the material inside the nucleus of the cells of living organisms that carries genetic information (see also RNA). out of it; and they do their thing; but all of that complexity is lost.  So what we thought we would do is actually micro-dissect – under a dissecting microscope often, with a needle – individual foci of cells. (There are actually instruments to do that now, but we were doing it before those were invented, so just with a needle, and often taking just a few cells from here and putting them in a tube, and a few cells from there and putting them in a second tube, and doing DNA analysis.) And what we found was that you could, through genetic analysis, construct family trees of certain things -- this bit of the invasive cancer was actually derived from this bit of introductory cancer over here, but not this one.  So it was absolutely clear that what was going on was just enormously complicated -- and that it could never be worked out unless you looked at it at the level of a pathologist, at the level of what was going on in the tissues.  So that was a very interesting project.

Another project that took advantage of this technology came out of the blue.  The AFIP has a department of veterinary pathology, so there are veterinarians who have done fellowship training in pathology who look at animal tumours and other things.  There was a veterinary pathologist there, Tom Lipscomb, who had a real interest in marine mammal pathology, and he came to me and said that there was this huge die-off of bottle nose dolphins along the US Atlantic coast in the late 1980s, and that they had initially speculated this was so-called 'red tide' – this is a sort of algal bloom that produces neurotoxins – but, he said, “This is not red tide.”  He said, “If you look at the pathology they have what look like viral infections.  They get encephalitis, they get pneumonias.”  He said, “This looks like distemper in a dog,” which is like a measles virus.  So he said, “Could this be some kind of a novel measles-like, distemper-like virus in these dolphins?”

We thought it was a ridiculous idea that one could [hope to] extract RNA from such horribly damaged tissueOf course the only dolphins they could find were ones that died and were washed up on shore.  Often the tissues were extremely degraded, so even looking at them under the microscope was difficult.  And these were RNAstands for ribonucleic acid.  RNA, like DNA, is found in every cell of every living thing on earth.  The relationship between the two, in summary, is that DNA makes RNA, and RNA makes proteins.  In other words, DNA is the director of the process of protein synthesis and RNA carries out the instructions. viruses -- RNA is considered much more fragile than DNA stands for deoxyribonucleic acid.  This is the material inside the nucleus of the cells of living organisms that carries genetic information (see also RNA). , and we thought it was a ridiculous idea that one could [hope to] extract RNA from such horribly damaged tissue.  But we found that you actually can, if you look for teeny tiny fragments.  We were able to isolate teeny tiny fragments of RNA from that virus, and we were able to sequence parts of the Genome The complete package of genetic material for a living thing, organised in chromosomes. A copy of the genome is found in every cell. , and it turned out actually that there are two highly related viruses that were causing these outbreaks, and that were novel viruses – they had not been seen up to that point.  Professor Albert Osterhaus in the Netherlands had also isolated these viruses and was beginning to work on them at the same time.  So there was the appearance of these new distemper-like viruses in seals and dolphins and porpoises that caused outbreaks in different parts of the world over the 1980s and ‘90s… That was a very exciting and interesting project.

John Dalton’s eyes

What could you do using molecular biology tools?So we were doing, in my lab, all kinds of different projects independently that all linked around the idea of starting as a pathologist and asking: what could you do using molecular biology tools?  And at some point I read an article that was published in Science magazine in which some very clever people had done an analysis of DNA extracted from John Dalton’s eyes to prove that he had classic x-linked colour blindness, classic red/green colour blindness.  So John Dalton -- the very famous chemist, the person who came up with the modern atomic theory of matter -- had red/green colour blindness.  

As a scientist he had had all sorts of interesting hypotheses to explain his problem, and when he died in the mid 1840s he'd instructed his physician to do an autopsy and to examine his eyes and test his hypothesis.  His hypothesis was that the vitreous humour, the liquid in the eye, was blue, and that it affected the transmission of the different coloured light.  And what his physician did, I thought, was just extremely clever. I don’t know that I’d have been clever enough to think of this, but he took one eye and opened it and saw that the vitreous humour was clear.  But with the other eye he put a nick in the back of it, the retina, and then looked through it.  He saw that there was no distortion of colour or vision through it, and so he concluded that whatever the problem was, it wasn’t a physical problem with the eye, that the problem was post-retinal.  I thought that was kind of an amazing thing to do!  

I’m sitting on a collection of millions and millions of cases going back over 100 years – there must be something that I can do that’s exciting like thatBut anyway, because it was John Dalton, famous man, the eyes were preserved.  The John Dalton Society of Great Britain owns them; there’s a mason jar with his eyes in it.  These researchers got permission to take a tiny piece of an eye, and since the mutations associated with colour blindness are now known, they analysed his DNA and showed that he actually had Daltonism [laughs], which is the old term for colour blindness.

Anyway, I presented this at our group – we had a Journal Club where each of us, one every week, would take a turn finding an article in the literature, presenting it and discussing it.  So I presented this and it just made me think, “I’m sitting on a collection of millions and millions of cases going back over 100 years – there must be something that I can do that’s exciting like that.”

SA:  It really thrilled you, did it?

The 1918 flu would be something that would be enormously usefulJT:  It really thrilled me, yeah!  I thought, you know, “Maybe we can find someone famous in our collection and do something kind of cute like that”!  It was just kind of a hobby project idea.  I had a brainstorming session with Tim O’Leary, and the idea that we came up with was to go after the 1918 flu -- which immediately resonated with me as a great idea.  Rather than just a trick like the John Dalton case, which was clever and cute but didn’t really advance medicine, the 1918 flu would be something that would be enormously useful, potentially of practical importance. 

SECTION 4

America’s Forgotten Pandemic

40-50 million people around the world died in the space of one yearThe 1918 flu epidemic was something I briefly heard about in med school, but I didn’t really know much about influenza viruses.  It was a huge outbreak that at the end of World War I killed millions of people.  Actually numbers keep going up as people look deeper into the records -- the current thinking is that about 40-50 million people around the world died in the space of one year in 1918 of influenza, which is an absolutely shocking number.  You’d have to go back to the Black Plague to find a disease outbreak that would kill so many people.

For one reason or another the 1918 flu epidemic was really not part of the cultural memory,this started literally as a hobby project, as an idea that I thought was really interesting, but the chances of success seemed extremely remote I had a molecular biologist working with me in my laboratory, Ann Reid, who expressed an immediate interest in this project and wanted to work on it.  So together we started reading up what we could.  We read a book by Alfred Crosby, who’s quite a famous historian, called America’s Forgotten Pandemic, which described the outbreak predominantly in the United States, and the effects.  But he was intrigued by the idea that here was something that happened 80 years before (his initial book was written in the 70s, so it was less than 60 years before) that basically no one had ever heard of, whereas every elementary school student has heard of the Black Plague.  So his basic question as a historian was, how could you have an outbreak in which tens of millions of people die, an unbelievable thing, and yet it doesn’t become part of the cultural memory?  He doesn’t actually have a definitive answer to that question; he speculates it’s because it was too fast -- that it all happened sort of within one winter season and it was the end of World War I which was a horrible thing anyway, and that all this horrible stuff happened at the same time and people just went on with their lives afterwards and didn’t think about it.  I’m not sure if that’s true or not, but for one reason or another the 1918 flu epidemic was really not part of the cultural memory.

So this started literally as a hobby project, as an idea that I thought was really interesting, but the chances of success seemed extremely remote to me.

SA:  Describe a bit about what that flu virus did do to people – I mean what was it like?  Because as you say, it’s not part of the folk memory, and people are always saying, “I’ve had a touch of the flu”, but that was something quite different, wasn’t it?



JT:  Right.  This virus seemingly came out of nowhere, and it caused disease that was absolutely recognized at the time as influenza.  Now influenza was not known to be caused by a virus at that point – though the idea that viruses existed was beginning to be accepted by the scientific and medical literature.  Virus, of course just means “poison” in Latin.  They knew a lot about bacteriology by 1918 and they were able to culture and identify and speciate a large number of bacteria.  They knew how big they were, and they developed filters that should block the passage of all the bacteria that they knew about.  

But, starting with experiments in the 1890s, they found that you could filter out everything that should be infectious, and yet the liquid coming through at the end was still infectious.  So they had the idea that what was infectious was a chemical, a poison, a “virus”; it wasn’t actually an organism.  Of course they had no electron microscope at that time and you couldn’t see a virus through a light microscope, so they had no way of knowing really what “a virus” was.  There was just this infectious “thing” that slipped through the filters.  Whatever these viruses were -- infectious organisms, agents, teeny tiny bacteria or whatever -- they were so small that they couldn’t be seen, couldn’t be cultured, couldn’t be filtered.  

So they didn’t know that influenza was a virus; they thought it was a bacterial disease
.  Clinically they knew what influenza was, an 'influenza season'.  When influenza hits a community in the winter months it has a very distinct course through the population.  It’s extremely rapid; it hits a community and within just a couple of weeks lots of people get sick at the same time.  And it goes away equally rapidly.  Even when you get ‘ordinary’ flu, it’s quite a serious illness.  People misuse the term all the time.  People say they have the flu when really they have colds or other upper-respiratory infections caused by dozens and dozens of viruses or other agents.  But when you have influenza you get extremely high fever, massive amounts of muscle aches and pains, and it’s not the kind of thing you take some Tylenol for and get up and go to work.  You are flat out in bed for 10 days; you feel miserable; it’s a really awful disease.  But most people recover.

It had this enormous impact on the healthiest, most robust segment of the populationWhat happened in 1918 was that lots and lots of people got the flu, as ordinary flu, but the amazing thing was that enormous numbers of people that got the flu ended up having really devastating viral pneumonia and secondary bacterial pneumonias and died.  And what was absolutely mystifying to everyone was that the people who had the highest chance of dying were young healthy adults, people in the 15 to 35 year age range.  And this is still the huge mystery of the 1918 flu – why it had this enormous impact on the healthiest, most robust segment of the population.  Of course, this had a devastating impact on the military, because that’s exactly the age range of people who were fighting in World War I. The peak of the pandemic was in the autumn of 1918, at the end of World War I, and actually I’m sure it was a key factor in having the War end in November.  

40% of young, healthy, well-fed, 18 to 25-year-old strapping American GIs dropped dead of flu in 1918Troops in training camps, or in trenches, or on troop ships are always highly susceptible to respiratory diseases anyway, and here was a virus that spread extremely well but also had a high propensity to kill people right at that age, so its impact on the military was enormous.  The US entered World War I very late compared to the European combatant countries, so there were far fewer US military casualties -- about 100,000 in total in World War I.  But of those, over 40,000 died of influenza.  So 40% of people who were young, healthy, well-fed, 18 to 25-year-old strapping American GIs dropped dead of flu in 1918. Which is an absolutely unbelievable number, when you think about that!

The search for a positive case

We came close to thinking that we should abandon the projectAnyway, it turns out that the Armed Forces Institute of Pathology had autopsies on about 100 such cases.  The retrieval system was so excellent that we were able to do key word searches for 'influenza pneumonia', find the cases, retrieve them.  The cases showed up on my desk…There they were, having not been touched in 80 years!  And then we tried to develop techniques to see if we could tease out tiny fragments of the virus’s Genome The complete package of genetic material for a living thing, organised in chromosomes. A copy of the genome is found in every cell. from these tissues.  It took about a year before we found a positive case, or worked out a technique that would allow us to do that.  We just kept getting negative and negative results.  We came close to thinking that maybe we should abandon the project, that it just wasn’t going to work and wasn’t worth the effort.  But it was just such a keenly interesting project that we wanted to keep going.  And the more we read about this virus and the outbreak and the devastating impact it had, the more committed we became (we meaning Ann Reid and I) to get the project to work.  And so it ended up finally working – we found one positive case out of about 70 that we had looked at…



Initially, I was struck by the way that case looked under the microscope. It was actually quite an interesting case: it was a soldier who had developed influenza pneumonia and was hospitalized.  But what was recognized clinically is that he had developed a pneumonia of his left lung, [and the right lung was seemingly normal]. Now it’s quite common to have pneumonia of just one lung, not necessarily both.  And at the time of autopsy that was confirmed – he had massive bacterial pneumonia of his left lung that was fatal, and his right lung was almost completely normal.  But what was there -- and it was not actually noted at the time of autopsy -- but what was there if you looked at the sections quite carefully were little tiny areas of very acute inflammation around the terminal bronchioles in that lung that were characteristic of very early phases of the influenza viral replication.  

And so what I think happened was that you actually had an asynchrony in the course of disease in that he got influenza infection, and then had a bacterial pneumonia in his left lung which overtook the viral infection and killed him, but the influenza virus infection of his right lung was somehow delayed by several days.  So when he died it left a snapshot of the very earliest stages of the virus infecting the lung.  It was a very subtle change, and it took me a while to look at enough autopsies to get a sense of that.  Influenza does not have characteristic changes that allow you to be confident, to just look under the microscope and say, “This is definitely influenza.”  You can sort of suspect it, but you can’t be certain.  But there was something about that case that just struck me as an excellent example, and so once I’d identified it, we extracted RNA, did our test and, boom! We found influenza virus RNA!

SA:  And how excited were you when you found it?

JT:  Very! [We laugh]  Very, very, very excited!  You know, nobody knew anything about that virus.  The amount of material that we had was vanishingly tiny, and the quality of the genetic material, the virus, was horrible.  So it was a rather daunting task to go from the first part of the project, which was sort of “Can we find a positive case?” to the sudden and horrible realization “Damn, we’ve found a positive case!  Now what do we do?” [Laughs]  Now we actually had to try to do large-scale sequencing of the virus to try to work out what it was.  


I was quite worried that this case was not adequate for that – there was literally just not enough material.  So we put a moratorium on using that material to sequence more of the virus while we looked for additional cases.  I found another case of a person who had died, eerily, on the very same day in September 1918, but in a different army camp, that also ended up being positive.  So now we had two cases.  And then I got a reprieve from an unexpected source, though also a pathologist, Johan Hultin, a really amazing man who's in his mid-80s now in San Francisco.  

SECTION 5

Alaskan permafrost holds vital evidence

Johan Hultin was a Swedish immigrant, who was at med school in Stockholm and had come to the United States to interrupt his medical studies to do a PhD.  He ended up in the University of Iowa in the microbiology department, and one amazing thing sort of led to another for him.  A person had visited the lab and given a seminar on influenza, and had mentioned the importance of the 1918 outbreak (this was in about 1949 or 1950) and said that unfortunately we would never know anything about this virus because the only way to find it would be to recover it from someone who had died of flu and had then been frozen.  Light bulbs went off in Johan’s mind.  He found out that there were a number of small immigrant villages in Alaska that had suffered devastating outbreaks of this flu, where the victims were then buried in permafrost, in the ice.  So he went up to Alaska, talked to people and found out where things were, and ended up proposing a project in which they would exhume the bodies frozen in the ice of people who'd died of influenza and try to recover the virus from that.

They went up to Alaska and dug up a number of bodies and brought back lung tissue and tried to recover live virusThey actually did this project in 1951: they went up to Alaska and dug up a number of bodies and brought back lung tissue to the laboratory and tried to recover live virus.

SA:  In the 1950s?!

JT:  In 1951.  They knew what viruses were by then. Influenza viruses had been discovered in the 1930s, so they knew how to culture influenza viruses.  Electron microscopes, of course, had been invented; they could see them, so they knew what influenza viruses were.  But you know, when you think about it, there was no ‘containment’ [such as specially designed labs that protect the scientists from contamination as well as ensuring the organisms don’t get into the environment], so had they actually recovered the virus I’m not sure what they would have done with it!  I’m not sure that they thought that one through.  But they were unable to recover it.  

Nothing biological survives freezing and thawingIt makes sense -- the virus is very fragile, and while you say that the bodies were buried permanently in the ice, 'permafrost' is actually a misnomer.  There are actually continual cycles, where the temperature kind of goes just above and just below freezing.  And with biological material, freezing and thawing is the very worst thing you can do, because ice crystals form and poke holes in membranes of cells, and it causes all sorts of damage.  So, basically, nothing biological survives freezing and thawing.  That makes sense.  

We published our initial findings, just little tiny fragments of sequence of the virus from our pathology material from the AFIP, in Science magazine in 1997. And Johan Hultin, who was in his 70s, read this article and wrote me a letter, explained this whole story.  He actually sent me photocopies from Crosby’s book where it’s mentioned and so on.

SA:  And you hadn’t known about Hultin at that stage?

JT:  No, no.  I mean I’d read Crosby’s book, but it hadn’t really registered. So he wrote me a letter, sent his CV to show that he was a respectable pathologist and so on, that he wasn’t a kook!  And he volunteered to go back and do this again.

SA:  You didn’t look at the old material that he’d collected?

JT:  He didn’t have it!  Here’s the amazing thing -- he worked with a microbiologist and a pathologist; the frozen material that they had in the 50s was kept in their freezers for a while, but then as it was not positive it was not thought to be of any use, and it was thrown out. There was formalin-fixed pathology material made from it, which was kept, and the pathologist at the University of Iowa then moved to other institutions and kept that material with him.  It turns out that that material was kept till the early 1990s – '92 or '93, I think -- when it was then disposed of….just a couple of years before we started this project in 1995!  We started the flu project in 1995 and had a publication in 1997.  So really very bad timing.

So Johan proposed to go back and exhume bodies again and get more frozen material that we could now do molecular analysis on.  Of course no molecular analysis was possible in 1951 – the structure of DNA stands for deoxyribonucleic acid.  This is the material inside the nucleus of the cells of living organisms that carries genetic information (see also RNA). wasn’t even determined till 1953.  We agreed that that would be an interesting thing to do.  He funded the expedition entirely on his own.  He went back and people in that community, some still remembered him.  They had been children and they were now the elders in this community, remembering him 45 years later.  He got permission to do an exhumation and he sent us material.

SA:  You didn’t go with him?

It became obvious within about a week of receiving the material that it was going to be positiveJT:  No, I did not. I didn’t feel I could add anything, that he really had this expertise.  So he went and did it independently and sent us material.  And it became obvious within about a week of receiving the material that it was going to be positive.  He had lung tissue from four cases; three of them were negative and one was really positive.  And we had lots and lots of material.  So now we had three positive cases.

SA:  So was that really thrilling when you got the stuff from Hultin?

JT:  It was absolutely thrilling.  Absolutely amazingly thrilling!

Using the frozen material it became possible to sequence the entire genome of the virusSo what we did was we ended up SequencingThe process by which the exact arrangement of the units of information on a specific stretch of DNA, or a gene, are determined. the haemagglutinin gene, the sort of main gene of the virus, from all three of the cases.  And what we found, amazingly, was that they were basically identical, one to the other -- that out of 1700 bases in this gene, these three cases differed from each other by only one nucleotide.  So we knew that this was really the pandemic virus; there was no question that this was the virus.  And using the frozen material it became possible to sequence the entire genome of the virus.  

It was still a very slow and difficult processBut it was still a very slow and difficult process.  It took an enormous amount of effort on our part, from 1997 through early 2005, to fully sequence the Genome The complete package of genetic material for a living thing, organised in chromosomes. A copy of the genome is found in every cell. of the virus.  One of the things we concluded from this study was that this was a sort of bird flu virus that adapted somehow to humans.  And one thing that happened in parallel with this was that our publication -- the first publication of the 1918 virus -- occurred in March 1997 at the exact moment that a three-year old child in Hong Kong got infected with an H5N1 bird flu virus and died.  

This was the first time that there was evidence that a bird virus could actually infect a human and cause disease. The two stories -- the 1918 and the H5N1 bird flu story – have kind of intertwined with each other This was the first time that there was evidence that a bird virus could actually infect a human and cause disease.  Before that it was thought that a bird virus infected birds and just couldn’t infect humans.  There was that bird flu outbreak in Hong Kong, where 18 people got infected, six of whom died, and so there was this enormous upsurge of interest in influenza.  People were worried that what the H5N1 virus was doing was paralleling what 1918 did – causing high mortality in young people.  And so the two stories -- the 1918 and the H5N1 bird flu story – have kind of intertwined with each other over the last ten years.

“Flu just kind of took over my life”

While all this was going on I was still running a molecular diagnostics laboratory doing mundane pathology tests -- LeukaemiaCancer of the white blood cells, which are a vital component of the immune system (see also lymphoma).s and liver biopsies for hepatitis and whatever else.  And it got to the point where we had such a large laboratory that, the year before I left, we were receiving upwards of 20,000 cases a year of different kinds of things.  So it was really quite a huge, factory operation.

SA:  Your service work?

JT:  Yes, the service work.

SA:  So you were just doing the flu project in whatever time you had available?

Flu just kind of took over my life!JT:  Right.  It was actually a huge amount of work, but I was on service perhaps a third of the time. Then I had administrative duties because I was chair of this molecular group, and then I had my research time – I had research projects on breast cancer, on dolphin viruses, on T-cell development, and influenza.  It just got impossibly too much.  So breast cancer was the first to go, then the dolphin viruses had to go, and I sort of clung on to the T-cell development project for a while as my old interest, until flu just sort of rudely pushed it off the plate.  And flu just kind of took over my life!  It was certainly something I never expected – it just sort of happened.  It was this ridiculous idea that came out of reading that John Dalton eyeball paper.  It’s just the way things happen! [We both laugh]

SA:  Okay, so you’d sequenced the virus, you got this great string of letters -- then what?  What can you read from the sequence?

A virus is nothing but a package of genes inside some proteins. So whether it’s alive or not is kind of debatable. I think of viruses as alive because they are very clever little beasts!JT:  Right.  The answer is we actually have extremely little understanding of going from that string of letters to knowing what it means.  A virus is nothing but a package of genes inside some proteins, okay?  So whether it’s alive or not is kind of debatable.  It’s either a kind of a complex chemical or a very simple life form.  Personally I think of viruses as alive because they are – I’m being very anthropomorphic here – they are very clever little beasts!  So I definitely think of viruses as living and sort of my adversaries.

So, we have the sequence now.  We know what impact it had on human beings in 1918.  We know that this string of letters produced a virus that killed 40 million people.  Somewhere in there are changes that make that virus different from other influenza viruses that don’t do that.  So there are two fundamental questions to ask about 1918 that can be expanded more broadly, and those are: it looks like the virus moved from birds to humans, so what changes are necessary, what mutations have to occur, for a bird virus, which normally cannot infect a human being, to become transmissible in humans?  Not just infect one individual human, but become transmissible from one person to another, which is necessary for it to cause an outbreak.  The current bird flu virus infects individuals – so if you are exposed to chickens who have the virus, or are dying of the virus, or to dead poultry that you are handling and who are infected, you can get infected and you can die, but you cannot transmit the virus to your brother…luckily!  (Or if you do so, only with great difficulty.) So the saving grace for us, with this highly lethal virus now, is that it is not transmissible.  It hasn’t ‘learnt’ to be transmissible, whatever that means.  But 1918 did.

We don’t know the answersOkay, so one of the questions we’re trying to answer is how is it that a pandemic virus like 1918 gets into people?  What changes are necessary to allow it to go from an animal source into people?  Did it have to go through some other animal in between?  Did it have to adapt first to a pig and then to a human?  Or did it go directly into a human?  We don’t know the answers, but we’re trying to use the sequences to ask questions about that problem.

The extraordinary W-shaped mortality curve

Why did 1918 kill so many people?  And why did it specifically kill young adults?Then we have the second question, which is: why did 1918 kill so many people?  And why did it specifically kill young adults?

SA:  Did older adults and elderly people actually have a lower mortality rate than usual?

JT:  Yes.  Let me find you a picture that will help you… [He brings up a graphic on his computer screen for me.]  Okay, so this is a graph plot of people who died of influenza by age.  Normally, when you look at influenza deaths, what you get is roughly a ‘U’ shaped curve – so, infants who don’t have a good immune system yet, people who are less than one, are very prone to die of flu, and the elderly whose immune systems are thought generally not to be working so well die, but everyone in between, while they get the flu, they recover.  So you get this sort of ‘U’ shaped curve.  What happened in 1918 is you still had high mortality in infants and the elderly, but you had this sort of peak of mortality in between that had never been seen before, and it peaks in people who are around 25 or so.  
 
SA:  Extraordinary!

We think this means is that people older than about 35 or so had some immunity to the 1918 virus JT:  It really was extraordinary.  But there is a subtle, but I think quite important, point here which is that the mortality in the elderly was actually lower in 1918 than for ordinary flu of the decade before.  And what we think this means is that people older than about 35 or so had some immunity to the 1918 virus that protected them. And what that means is that there must have been an influenza virus of a similar composition that was circulating in the mid-1800s.  There was an influenza pandemic in 1890, and there was one around 1850, so what we think is that the 1850 pandemic was also of the same sub-type as 1918 and so people who were alive then had some protection already. So that could account for why it is down here [among the elderly], but it doesn’t account for why there is such low mortality here…  [He points at the graph on his computer again.]

SA:  The 5-14 age band?

JT:  Yes, why do children not die?  I don’t have the answer for you yet, but we’re trying to figure this out.

SA:  That’s the big question at the moment is it?

It now is possible, through the wizardry of modern molecular biology, to go from a sequence to actually making the virusJT:  That’s the big question.  And one of the possibilities that’s being explored is that the 1918 virus induced a very strong inflammatory response, in that people whose immune systems were the most robust actually did the worst, and that that could account for this.  Now we have some animal data, my colleagues and I.  We’ve sequenced the virus, and it now is possible, through the wizardry of modern molecular biology, to go from a sequence to actually making the virus.  So the 1918 virus has been made again and put into animals in these ‘high containment’ labs.  It’s been done now in two places – at the Centers for Disease Control in Atlanta, with my colleague Terry Tumpey, as well as by investigators in a high containment lab in Winnipeg, Canada, who have put the 1918 virus into macaques.

SA:  Is it very difficult to go from the letters to actually creating the proteins?

JT:  It actually isn’t that difficult now.  It’s amazing - techniques have been invented that allow you to make, in a sense, influenza viruses from cloned genes.  It’s a technique called ‘reverse genetics’ that a number of investigators kind of simultaneously invented.  It was a natural progression from the way things were going, but Drs. George Brownlee at Oxford, Peter Palese at Mount Sinai, and Yoshi Kawaoka in Wisconsin kind of all independently worked on this.   

It’s not the virus that kills you but your own body’s immune responseSo one idea about the 1918 and the H5N1 virus, with their high mortality, is that they produce this marked inflammatory response.  And it’s not the virus that kills you but your own body’s immune response against the virus -- your immune system responds so vigorously against the virus that your lungs are secondarily damaged.  It’s sort of an interesting hypothesis, and there are some data to support it.  But to me it doesn’t explain this picture [indicating again the graphic].  So I think it's still mysterious, and I’m working hard to try to figure this out, but it’s a difficult problem.  Because what we have is the virus, but we do not have serum samples from people of these different age groups collected just before 1918, so we don’t know what their AntibodyA protein produced by the body's immune system that recognises and attacks foreign substances. status is like.  And we don’t know anything about the influenza viruses that circulated in humans before 1918, and I think that’s quite important.

“A very rare combination of circumstances?”

Could it be that 1918 really was a very rare combination of circumstances? We still can’t explain the most fundamental questionAnd so I wonder if there’s something about the exposure history of this group [the elderly] in 1918 based on prior exposure to other influenza viruses when they were younger, and that there was an odd immune response in this population [the 15-35 year olds] to the virus.  And that while the virus was a virulent one, in and of itself, it wasn’t the killer virus.  It was this horrible combination of infecting the wrong people at the wrong time, in a way that might be so unique and unusual that it might never occur again.  And so could it be that the reason 1918 stands out as a completely unique event in history is that it really was a very rare combination of circumstances?  We still can’t answer that question definitively.  And so, unfortunately, after all this work – after more than 10 years of my life, and others' too, to find the virus, sequence the virus, make it, study models – we still can’t explain the most fundamental question of why 1918 behaved the way it did.  But that’s what we’re trying to do…

SA:  Are there hypotheses?  I mean, do you know what those people’s immune systems were like?  Can you read that from their cells?

I don’t want to admit defeat – but it is certainly possible that we might never be able to explain thisJT:  No.  It’s an interesting question.  We might be able to do something like that -- that is, we might be able to look at the expression of immune-related genes in their tissues, for example -- from the autopsy tissues.  But I think we’re going to be limited in what we can and cannot do.  We can only get tiny amounts of genetic material from these 90 year old blocks, and so it is a very frustrating problem.  It could very well be – and I don’t want to admit defeat – but it is certainly possible that even having the virus, having the ability to model it in animals, but in the absence of this other information, we might never be able to explain this…Which would be very frustrating! [We laugh]  I would like a nice coherent picture with a happy ending and so on, but that’s still a fundamental problem.

SA:  Fascinating!  So there are all kinds of other things in the picture, variables that you can’t necessarily discover now?  It’s not just the virus itself?

Infection is really a dance between the host and the pathogenthe bacteria or the virus may be identical.  It’s the person that variesJT:  Right.  Right.  And I think this is actually a more realistic approach, because, you know, infection is really a dance between the host and the pathogen.  So, different people react differently to the same infection – some people have a very mild course and some a very serious course, and yet the bacteria or the virus may be identical.  It’s the person that varies.  It could be that they have different genetic backgrounds and so somehow they respond differently; could just be random things, or health-related issues -- one person has heart disease which makes them more likely to have a bad effect, for example.  But on such a large scale as [the 1918 pandemic], you can’t imagine that it’s something like that.  It has to be something sort of common to this age group.  So that’s why I think the most likely thing is something to do with the exposure of this group to something when they were children. Perhaps the 1890 pandemic – perhaps there was something about that that caused an unusual cross-reactivity.  

One of the things we’re trying to do to test this is to look backwards further in time -- can we find any influenza virus isolates from before 1918?  We don’t think such material exists at the AFIP archives, but we do think it might exist elsewhere and I have been collaborating now for a number of years with Professor John Oxford, who is in London.  He has helped me gain access to the morbid anatomy department at the Royal London, and we are actively screening right now cases from their pathology collection for influenza from as early as 1905, up through 1917, and we hope to find some positive cases to help set the stage for how the 1918 virus emerged.

We found a number of influenza-positive cases (I think we have five) from that collection in London from the autumn wave of the 1918 flu. We’ve sequenced the haemagglutinin part of several of those cases, and again they’ve been almost identical to the North American isolates, meaning that one virus entered into the population and then just spread everywhere very rapidly, without having to evolve; it was able to seed itself into the population.  So, you imagine, going from Alaska to London, it’s about 10,000 miles…The sequences isolated from people who died in the same month, November 1918, are absolutely identical, 10,000 miles apart!

SA: Amazing… amazing!

JT:  It is.  But what we’re trying to do is go backward in time, before 1918, and we also want to fill in the gap.  Influenza viruses weren’t isolated until the 1930s, so we don’t know anything about what happened to the flu after 1918, and so we’re trying to screen for cases in the 1920s.  Another reason for that is… [he searches through his computer files again to show me something].  Here we go… This is a picture published in the German literature in 1924, and this fellow graphed influenza deaths in Breslau, which I think is now in Poland but was I guess part of Germany at that time – you get this sort of same ‘W’ [shaped curve], just like our ‘W’ from 1918.  But here he publishes it for the flu season of 1920 and there’s still a bit of mortality in young adults, but by the 1921/22 season, nothing.  So what if there is something in the virus that produced this [unusual mortality in young adults], but that whatever those mutations were they went away, so that by the 1920s it became ordinary flu?   So if we could find, say, the 1923 or 1924 virus and sequence it, any differences between 1918 and the 1920s would lead us to a hypothesis as to why it behaved the way it did.  

This kind of ‘viral archaeology’ is extraordinarily painful!But this kind of ‘viral archaeology’ is extraordinarily painful!  And very slow.  I mean, it’s not like you can just go to the freezer and pull out the virus.  We have to find autopsy cases; we have to sequence these teeny tiny fragments one at a time and put them together.  Now we’re making more progress here than we are with the pre-1918.  We have some cases from the mid-1920s – I don’t have the dates exactly, but 1925/26 – where we are SequencingThe process by which the exact arrangement of the units of information on a specific stretch of DNA, or a gene, are determined. across the genes of the virus with this goal specifically in mind.   You know, there still might be ways in which we can utilise, in a sense, our pathology background to help us get at this problem.  We’re trying to attack it in every way we can think of, but it’s a difficult problem.

SA:  The other thing is, you said that 1918 was also a bird flu – how d’you know that?

JT:  Well because it looks very much like a bird flu in its sequences.  We’ve identified a series of mutations – about 25 mutations or so – that we think distinguish 1918 from typical bird viruses.  Basically those are the only changes in the proteins that distinguish it from bird viruses; there are very few changes.

SA:  So you can see where it jumped the species?

JT:  Yeah.  But we don’t know which of those changes are crucial for this process, whether it's all of them or some sub-set of them.  Now we have to go through and do that kind of test, where we take the 1918 virus and make those mutations back to the sort of typical bird sequence and see if it no longer causes infection in the lab animal, and so on.  I mean, those are the exact kind of experiments we’re proposing to do once our lab is functional. [At the time of the interview, JT had just moved to a brand new lab in NIH, where the high containment section was about to be opened.] The kind of very careful molecular virology where we go back and look at one stage at a time and kind of work this out.  We look at: have the current H5N1 bird flu viruses acquired any of the same changes?  And you find that some of the H5N1 viruses have some of the same mutations found in 1918, suggesting that they may be undergoing some of the same pressures to adapt to humans, but they’ve not yet acquired enough of these changes to make them go.  One of the other goals is to work that out.

Unanswered questions

How do influenza viruses jump around between species, why particularly are 1918 and the current H5 viruses so lethal?So that’s why I say there are these two big questions: one, how do influenza viruses jump around between species to go from birds to pigs to humans to horses to wherever they go?  (Because they’re causing infections in all sorts of different species, and the viruses are just moving everywhere.)  And second: why particularly are 1918 and the current H5 viruses so lethal?  Those are sort of the two questions.  Those two things may or may not be related to each other; I don’t really know yet at this point, but we’re attacking them as two different questions.  So…

SA:  So you’ve got these animal models, what are you doing with them?  Have you looked at birds as well; have you tried to find the 1918 virus in birds before it jumped species?

JT:  We have.  And we have one publication…We found birds preserved in alcohol that were collected around 1915 or so stored in the Natural History Museum of the Smithsonian Institution, here in Washington.  And we got permission to take small samples from these birds – ducks, specifically.  (Ducks were thought to be the natural hosts for influenza viruses.)  And the virus is not a respiratory virus for them, but an intestinal virus, so they actually spread the virus by the oral-fecal route.  So we did little biopsies of their rear ends, their cloacas, and looked for evidence of influenza, and were able to find some tiny fragments of influenza that does look sort of like typical bird flu.  But we didn’t find sequences that were close matches to 1918.

The origin of the 1918 virus is still rather mysterious, just like its mortality pattern.  Is there a species of bird out there that has a virus like the 1918 virus?  We don’t knowThe origin of the 1918 virus is still rather mysterious, just like its mortality pattern.  I don’t know where the 1918 virus originated.  My best hypothesis at the moment from the data we have would be that it’s from a bird-like source, but perhaps a source of virus that we have not yet sampled.  We know that ducks and other birds commonly are infected with flu viruses, but no comprehensive surveillance in wild animals has ever occurred for where viruses are.  And this would be very difficult and very expensive.  These infections in birds cause no symptoms at all, so it’s not like the birds are dropping dead everywhere, like the H5[-infected] swans and things did.  A bird, especially a big bird like a swan, that’s lying there dead is easy to find.  But a bird that has no symptoms at all…it’s hard to know if they’re shedding virus or not.  So you would have to randomly grab birds and do sampling, and it’s very difficult.  But is there a species of bird out there that has a virus like the 1918 virus?  We don’t know.

Far more complex than we ever imagined

SA:  So how do you feel about it at the moment, this whole thing?  I mean, you’ve obviously done absolutely fantastic ground work – but do you feel that it might actually be hitting the buffers at some point because there are too many things you can’t test?

A lot of the ideas and models that have been developed over the last 50 or 60 years are just too simplistic;simple explanations just don’t work.  It’s just an unbelievably complicated and dynamic systemJT:  [Pauses].  What I learn, the more I think about the whole influenza problem, is that it is just a very complex system, and that a lot of the ideas and models that have been developed over the last 50 or 60 years to explain how influenza works are just too simplistic; they just don’t fit the complex data that we have now.  And so we’re trying to take a new look at how all of this happens – how viruses move round different species; how they evolve in bird hosts; how they evolve in humans.  There are lots of other things my lab is doing now that I’m not describing that are not at all related to pathology.  We’re doing complete sequence analysis of thousands of influenza viruses from humans, from birds…We’re looking at how the virus evolves on a yearly basis, trying to figure out: how does it move around?  How come flu seasons cause a winter outbreak in the northern hemisphere, and six months later there’s a flu outbreak in the southern hemisphere, in New Zealand and Australia.  How are the viruses moving around?  Are they the same viruses?  Are they different viruses? We’re trying to tease all this together.  And every time we ask a simple question about flu and start investigating it we realise that the simple explanations just don’t work.  It’s just an unbelievably complicated and dynamic system.  

This is a lifetime’s workObviously there are lots of other people working very hard on all sorts of issues around influenza and I don’t want to claim that what we’re doing is the key to everything.  But the position I’ve taken here in setting up this new laboratory is that it's designed to take a very broad approach in looking at influenza, from surveillance in wild birds, up through clinical trials in humans, and everything in between.  And a kind of long-term approach – not one where I have to get a grant reviewed every three years so that I have to do these little packets [of research], but to think of this as a much longer term process.  Really this is a lifetime’s work, and more than one lifetime of work, just to work on this.  We hope to take the interest that has been generated with the 1918 project and the H5 to help us answer these basic questions.

SECTION 6

A holistic view

SA:  Getting back to the pathology thing – what do you think are the advantages for you, as somebody who has obviously gone very deep into pure science as well, what are the advantages of being a pathologist, having a pathology training?

I strongly recommend that pathologists think more about scienceJT:  I think there is a huge advantage.  I think that people who have training in one discipline of basic science often have a rather narrow set of experiences, and training as a physician in general -- but specifically as a pathologist -- I think gives one a much broader view, a kind of a holistic view of things.  I spend most of my time thinking about nucleic acid sequences – those strings of letters that you mentioned – but I’m constantly trying to put it in the context of the epidemiology, of the clinical picture and so on.  And I think that’s really a valuable thing.  I strongly recommend that people who are pathologists think more about science. I think you’ll find that a lot of pathologists, the vast majority of pathologists, are fully clinical physicians, and are not engaged in research.  But I think that pathology, at its inception in the 19th century, was very vested in basic research as well – [the clinical and scientific roles] really were well-integrated, and then they kind of drifted apart, and I think pathology is one of the specialities that would allow people to do both.  Obviously what you’re doing when you look through the microscope at cells is you’re looking at disease, in a sense, at a cellular level.  So it’s a tremendous opportunity…

SA:  So you say that you see yourself as a physician, do you?

JT:  I do.  I do, and I’m very glad that I’m a pathologist.  One thing I wanted to tell you, an anecdote that I just thought of again, is that Tim O’Leary, my former boss, has several children, and one of his sons came into the office one day (this was years ago when he was maybe seven or eight years old, a very smart little boy), and I was chatting with him for a few minutes.  Somehow the conversation came up that, like his father, I was a pathologist, and so I asked him this question: did he know what a pathologist was?  And he gave me this answer, which I think is just fabulous, he said, “A pathologist is the kind of doctor that figures out where the problem is but then can’t do anything about it”!  [We both laugh]

So there you go, that’s been the sort of definition!  And here we are, 90 years later, trying to figure out what the problem is about 1918, but we can’t do anything about it!  So I thought that was really an excellent definition of pathology…

SA:  That’s beautiful!  But one thing I wanted to ask: how much can one actually see of any of these things.  Okay, so you say that now with the electron microscope you can see viruses – do you actually see a wriggler under the microscope?

JT:  Yeah, yeah…  [He turns to his computer]. Let me try and show you an electron micrograph of 1918…

SA: [Looking at the images on the screen] That’s what it looks like under the microscope?

JT:  Yes.  This is a cartoon representation…So what it is is a bit of membrane that it steals from the cell, and it has its genes as little mini chromosomes, eight separate little strings of genetic material, and then it has these proteins, sort of like spikes that stick up on the outside of it.  And one of them, haemagglutinin, recognizes sugars on the tips of cells that it wants to infect.  So you can actually see, in this high resolution picture, you can actually see the membrane and then you can get the impression of these little spikes sticking out, you can actually see them.

SA:  Yes, and those things inside…?

JT:  Those are the genes, the actual gene segments.  So it’s really quite remarkable that you can actually see it.

SA:  And with a light microscope what would you see?

JT:  Nothing, nothing at all.  No, this is magnified tens of thousands of times.  In a light microscope, you can get perhaps a 1000 x magnification, and this would be something like 40,000 x.  There’s no way you could see this with a light microscope.  Some viruses, when they infect cells, accumulate into kind of a big clump and you can see the clump, not the individual viruses.  The clump actually reflects thousands of viruses.  Herpes viruses, for example, when they replicate they do so in just one place in the nucleus, and when you look at it under a microscope you can see a dot in the cell, a sort of 'viral inclusion' (flu doesn’t do that), but you certainly cannot see an individual virus under a light microscope.  

SA:  So you can actually see the genes -- that is an amazing thing.

JT:  Yeah.  I mean you can see the segments, the strings of them. They’re wrapped in proteins… [Looking in his computer again] Yes, here we go – cellsalive.com – it shows you at 1x magnification, and then at 10 x magnification you can see a hair, right?  At 100 x magnification you can see dust mites.  And here you can begin to see pollen, and a red blood cell, and at 10,000 x magnification you can see bacteria.  So these are pollen, these are red blood cells and these are the size of bacteria.  At 100,000 x magnification these are viruses around a bacterium.  And this [new slide] is a million-fold amplification, so we’re talking really really teeny tiny things.  Viruses vary in size of course, but you need to get beyond 20-30,000-fold magnification to begin to see viruses.  So it’s impossible with a light microscope -- which is obviously why we couldn’t see them back then.

SECTION 7

In the footsteps of Alexander Borodin

SA:  Okay.  So the other big question is: what do you do outside of all this?  If you ever have any time…! [We both laugh]

JT:  Well, what I really want to be when I grow up is a composer, actually!  

SA:  Music?

It seemed more practical to have music as a hobby and science as a profession than the other way aroundJT:  Yes, actually music has been the other key passion of my life.  I was a practical kind of a person, so, having a dual interest in science and music, it seemed more practical to have music as a hobby and science as a profession than the other way around.  It would be kind of hard to be in an orchestra and say, “Can I borrow your lab for the weekend?”  But there’s always a chamber group somewhere you can play with.  I’ve always been very interested in music.  I actively played a number of instruments, mainly wind instruments -- oboe, clarinet, English horn and other things.  I played the piano too, but I’m mainly a woodwind player. I used to be actually a very good oboist, but now I just don’t play.

I would get a score and put it on the stereo and sit there and say, “Now, how did he make that sound?”Since my teenage years I’ve been very interested in composition.  Of course I didn’t have the opportunity to go to conservatory, so I’ve had to do this all through self-study.  But starting as a child, I would get a score to Tchaikovsky’s 5th symphony and put it on the stereo and sit there and say, “Now, how did he make that sound?”  And having played in bands and orchestras and chamber groups for years and years and years, I’ve sort of got a sense of things, and have read books on orchestration, and I'm foolhardy enough to just sit and write music.  I’ve written symphonies and a comic opera and a number of things.  It’s very difficult to get big things played…

SA:  So you were trying to tease out the genetic code of Tchaikovsky’s music, were you?

JT:  Yeah, I guess so!  In the last 15 years or so I’ve concentrated more on chamber music.  Now in relation to working very hard… I’m married and I have two young children at home who are now 10 and 12, so obviously I have practically no time available for music.  But I’m very interested in it, and I hope to do more later, when things settle down in my life.  But I have tried and continue to try to compose.  I’ve had some things played.  I had a string quartet played a couple of times.  Actually, there’s a poster here [he indicates it on his wall] -- I once had a concert at the AFIP where they played one of my string quartets in relation to Alexander Borodin, who is a hero of mine.  

Borodin was a physician who did organic chemistry research and composed on the side as a hobby.  Even though he was one of the Great Five Russian composers, he was not a full-time composer, he was a professor of organic chemistry at the military medical school in St Petersburg.  And there are descriptions of him telling how he would come out of his lab, he'd be busily scribbling his musical scores, and then he'd go back to his lab where he had beakers of things bubbling away.  So he’s sort of a hero of mine -- but he wrote more music than I have!  

SA:  Does your work inform your music, or is music a way of completely clearing your mind of other things?

A lot of people who are interested in science are interested in musicJT:  I don’t know.  I guess a lot of people who are interested in science are interested in music.  Music obviously is very structured, it’s very organised, it has kind of a mathematical basis and so on.  But I think that’s looking for ways to link them, and you know, they’re very different.  But I’m definitely drawn to music.  I listen to music all the time.  Every day I bring in a big stack of music while I’m working.  I’m mainly a classical music person.  By that I mean from kind of medieval music up through modern 20th century classical.  I don’t really listen to pop music or rock music or anything.

SA:  And the things that you have done yourself…?

Composing music is something that I would really love to do moreJT:  I would consider them…They’re clearly sort of tonal, melodic works.  Conservative, 20th century, neo-classical stuff, I guess you would say.  It’s music that has a harmonic structure and is relatively traditional, I guess.  I don’t want to grade my success as a composer… I can say that I’m an “extremely successful unsuccessful composer” – I’ve been very good at being unsuccessful!   But I really like it.  I wish I had more time to do it.  I’ve had an overture played that I was able to conduct when I was in college; I’ve had a couple of movements of a symphony played… I’m drawn to write big things, orchestral scores and things, but since it’s really difficult to get anything played I’ve sort of limited myself to chamber music, because you can occasionally convince four or five people into your living room and run through a piece.  But composing music is something that I would really love to do more.  I really like it.

SA:  So, one final question: your work, your pathology -- what has delving so deeply into the nuts and bolts of life, done to your philosophy of life? The big questions like the meaning of life?  Has it been just science or has it really informed your philosophy?

I fully believe there is a molecular basis for everything we seeJT:  Well that’s a very interesting question.  I guess I would have to say that I have this hope that you can actually find basic mechanisms for how things work, that I strongly believe that -- especially in the biological system, as complex as they are -- things happen for very specific reasons that can be explained, that can be elucidated.  That is, it’s not so complex and stochastic that you could never actually understand the mechanisms.  I’m fully aware of how complicated things are, but I fully believe there is a molecular basis for everything we see and that it could actually be worked out. So I have faith.  That’s sort of what keeps me going; it’s faith that eventually science will be able to explain a lot of things, and that will be enormously helpful in terms of practical applications in medicine, advances in therapy and so on.  I do strongly believe there is a definitive sort of programmatic basis for everything and that we could work it out.

SA:  So does that rule out religious belief?

JT:  No, no it doesn’t rule out religion at all.  I didn’t grow up in a religiously active family, but I grew up with some sort of basic, non-descript Christian faith.  I haven’t gone to church in a long time, but I certainly wouldn’t rule religion out.  I have a lot of respect for people who have a strong faith.

ENDS

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