(C)2005 by Richard Katz
From 1971 until 1975 I worked in Ed Penhoet's laboratory as a virologist. I was a graduate student, Dr Penhoet's first graduate student in fact. I worked with VSV (vesicular stomatitis virus) and flu (influenza virus).
When I abruptly left the lab, in 1975 or so, I was working on neither of those. I had been working with what Dr John Holland, Dr Penhoet's mentor in virology and hence my mentor, called an "adventitious agent." Something had appeared, unsummoned, in certain bottles of tissue culture cells that Dr Milman was growing on the sixth floor. This "something" had the power to make confluent monolayers of animal cells look very very sick.
Usually you only see that appearance of cytopathic effect in tissue culture cells before they die. These cells in Milman's lab on the sixth floor didn't die; well, eventually, they would, I guess, like John Maynard Keynes said, "In the long run we're all dead." But no, tissue culture cells are forever. They're immortal. Regular tissue culture cells you get from, say, chopping up a monkey liver and trypsinizing it, to separate the monkey liver into monkey liver cells, live for a couple months, maybe sixty generations. Then they die. But in the lab, one used "immortalized" tissue culture cell "lines", cells that had been "transformed." Generation after generation, they would divide every day or so to make a nice even lawn of cells -- a confluent monolayer -- on the sides of their bottles lying flat in their incubator, forever.
This agent, whatever it was, was just something I noticed one day in several bottles of CHO cells -- Chinese hamster ovary cells -- in the Milman lab on the sixth floor.
I didn't think much of it, these sick cells in somebody else's lab. Dr Holland's no-nonsense advice had always been, "Don't fuck around with any adventitious agents. Bleach'em down the sink." And so whenever a bottle of tissue culture cells in the Holland lab went sour, you'd just squirt a bunch of bleach on them and toss them down the drain. This was a while ago; nowadays they probably do something to make them even deader.
But somehow the sick cells in the bottles on the sixth floor recovered. I looked at one of those bottles, with a microscope, a day or two after seeing those sick cells, and I didn't see any sick cells. All I saw was an almost confluent monolayer of healthy looking cells. I checked out the initials on the bottles, to see whose they were. They were Angela Longo's.
Angela was a girl my age, a fellow grad student, working in the Milman lab. The Milman lab and the Penhoet lab shared a tissue culture facility. So if the Milman lab had this plague, maybe we had it, or we would get it. Maybe not; in fact, theoretically not, because the way you organize tissue culture is to have a one way street for anything leaving the tissue culture facility. Ed and I had set all that up; after all, for quite a while it was just me and Ed.
I found Angela and she seemed unaware that she had a problem. Yeah, she said, the cells in the bottle looked a little funny; but, "I passaged them," she said, "and they're fine." She had gotten them growing again; she had split them, given them some room, so they started dividing; and, apparently, made them not look sick any more.
Whatever that meant, physiologically, it was interesting to me.
"Don't fuck around with adventitious agents," I could hear John Holland saying in my ear. So I borrowed a bottle of Angela's tissue culture cells; split them one for four; grew up a bunch of them; freeze-thawed them, centrifuged appropriately to get a reasonably pure preparation of whatever (if any) virus they had (it had to be a virus); and took some to the electron microscope technician who worked down the hall.
The EM person took beautiful micrographs for me. Every virologist I showed them to said immediately, "Good pictures. Some kind of herpesvirus. Where'd you get it?" And I'd tell them the story, and they'd lose interest, somewhere along about the part about confluent monolayers. But for some reason, those electron micrographs of a herpesvirus -- some cousin of shingles and meningitis and recurrent genital sores -- were stunning. I think the only reason anybody ever went on to invent anything more advanced than the transmission electron microscope was so they could image all the things that the transmission electron microscope can't see -- not big enough, not enough contrast, not in vivo, whatever. But these virions were extremely photogenic in Alice's machine. They were beautiful. And, they spoke eloquently for themselves. These virions were evidently present in sufficient numbers that if you just took a picture of where they were living, you'd be assured of seeing a few of them. That's like driving around town, looking for, say, loaded tractor-trailers. If you never come across a single loaded truck, you are probably in a No Trucks zone. You're NOT on the far right lane of the freeway.
Let me get back to influenza for a second: I didn't want to "investigate" influenza, I wanted to cure it. Somewhere out there, there's a penicillin for the flu. Penicillin gets in there and ruins bacterial cell wall synthesis. Cell walls are bacteria's weak spot, the bacterial Achilles heel. Humans don't have cell walls; we have cell membranes; so penicillin kills bacteria, but not us. Five hundred million years of bacterial gangrene, and now, since the Forties, we win.
I had done a few experiments at the Holland lab in San Diego in between going to the beach and having a really interesting love affair. It appeared to me, that infection of confluent monolayers of mouse L cells with VSV resulted in markedly less yield of virus, than when I infected non-confluent, rapidly growing monolayers of cells. You got lots of virus and deadly infections in the bottles of "young" cells, but not much happened to the "old" cells. That seemed pretty cool. Something in that bottle of old cells over there was taking that badassed virus down a peg. Seemed like one should be able to take it down all the way and then pin that sumbitch to the MAT.
Now bear with me, I'm going to have to weave together these threads about infecting confluent monolayers on the one hand; and the sick cell/healthy cell thing on the other. I told you that I did experiments infecting cells growing in bottles with killer viruses. Virus, actually; I only used vesicular stomatitis virus (VSV) for these experiments. I never got to do any others, like flu. I ran into some problems with reproducibility, when I went back to Berkeley, and got stuck at step one, for four years. Damn! I hate to even think about that. Four years! When I finally cured the irreproducibility, I quit, but not because ... more on that in a minute.
So along comes this adventitious agent, this contaminant, this unknown unwelcome intruder into the orderly confines of my laboratory. And it seemed to fill the bill. It was manifesting a differential cytopathic effect on growing versus nongrowing cells, and it was REVERSIBLE! You could, if you chose to do so, study it.
I did, indeed, choose to do so.
So there I was, day after day, night after night, in Room 220 of the Biochemistry Building at the University of California at Berkeley, the number one ranked Department of Biochemistry in the United States of America, culturing these obviously contaminated cells and harvesting the frozen/thawed lysate of their many many offspring. A true mad scientist.
Let's back up. Let's talk about this Milman character, the guy with the lab on the sixth floor. Dr Gregory Milman was a young assistant professor, as young as any other assistant professor, a biochemist on his first real job. He held the same rank as Ed, my advisor. I could have picked to go work with Milman. I could have picked to go work with any of the Professors. I already had two published papers, one with only my own name on it; and its followup paper, with my name plus the name of a truly world famous biophysicist at the University of Pennsylvania, right along with mine, the Imprimatur of the Best, and both papers published in first class journals, too. The Berkeley Department of Biochemistry was one luminary after another, legendary biochemists: Horace Barker, discoverer of Vitamin B12, the structure of cyanocobalamine, and the true deserver of Melvin Calvin's Nobel prize for elucidating the pathway of carbon in photosynthesis, after World War II; Esmond E. Snell, pyridoxal phosphate, Vitamin B6; Bruce Ames, the histidine operon and the Ames Test; running down the list to Dan Koshland, the foremost biopolitician and the Editor of Science magazine, voice of the American Association for the Advancement of Science; and finally down to Charles Dekker, a man who years before had authoritatively announced that DNA was not, indeed could NOT be, the genetic material. Fossils, perhaps, these Professors; but as a group, they were remarkable men. And they were all men, every man jack of them; and recently, they had reasoned, collectively, that the Department needed young men -- kind of like parthenogenetic spawning -- like Ed, men who knew a thing or two about DNA, and even RNA. And, of course, proteins. Ed was first class materiel, to be sure; he had done his doctoral thesis on proteins, on aldolase, isoenzymes of aldolase, a fellow who surely had his biochemical head squarely on his shoulders.
Ed panned out. Ed not only made full Professor, Ed went sideways and cofounded what became the biggest pure-play biotechnology business on Earth, and hence rewarded his mentors with endowed chairs, sponsored lectureships, and high paying jobs for lots of good people from Berkeley.
There were no Nobelists in the Biochemistry Department crew, which was kind of unusual for Berkeley. The Molecular Biology and Virus Department had, or had had, a few. Later on the Molecular Biologists subsumed the Biochemistry Department. Kairy Mullis was a Biochemistry graduate student of approximately the same vintage as I was, and he got a Nobel Prize later for inventing the Polymerase Chain Reaction, whilst he was working as a technician (!) at Cetus Corporation in Emeryville. Ed's company bought Cetus; before Kairy got his Prize, he did get a ten thousand dollar tip from Cetus for his "suggestion", then he got fired. Or so I have heard.
Milman just wasn't cut out to be like Ed. Let it go at that.
But Milman's wife, now she was a real piece of work. As far as she was concerned, her husband and his lab were right up there with Jonas Salk and the whole Salk Institute. That's a good thing. That's the good news. The bad news turned out to be that her husband, and her husband's lab, was trying to be relevant, like Ed was doing. They wanted to grow animal cells -- tissue culture cells -- instead of the biochemist's stock in trade of bacteria or yeast or plants or what have you. And it was well known, even to the United States Government, that animal cells in tissue culture, biological material derived from mice and rats and cows and monkeys and dogs and yes, even humans, were not quite normal, and more than likely harbored etiologic agents. In other words, everywhere you looked in her husband's lab, and in our lab, were those yellow and purple stickers that shrieked BIOHAZARD. This was a long time ago, the Nineteen Seventies; and even way way back then, even the Government knew that this stuff might be bad for you, even while it was going to cure every disease the Reader's Digest had ever highlighted.
Mrs Milman got pregnant. One afternoon she comes walking down the hall on the sixth floor, ripe as a watermelon, and I accosted her thusly: "Mrs Milman, maybe you shouldn't go in there."
To which she responded, and quite haughtily I might add, "This is my Husband's laboratory."
I only responded with "Yes, Ma'am, I know that, but these signs are no mere formality, and it says right here, No Pregnant Women." She slammed the door on her way in; I wasn't headed in there anyway.
So there I was, not a month or two later, late at night, toiling away in Ed's lab, centrifuging a nice big batch of my new friend the mystery agent, the Virus from the Dark Lagoon, the Herpesvirus that would cure ... you name it, I had as grand a delusion as the next guy, believe me. Right then I was right up there with Louis Pasteur.
The centrifuge spun slowly to a stop and I retrieved my plastic tube of the putative virus fraction, a creamy impacted very palpable smear in the bottom of the tube. How could you really have so much of something so small, smaller even than what one called micro-organisms, too small to really be alive; so much you could, with the naked eye, simply see it?
Boom! You're in a truckstop.
As I lifted the tube from the centrifuge rotor, my fellow grad student, Chris Reading, a fine fellow who even co-owned a boat with me that we used to go out fishing on in San Francisco Bay, said, suddenly, "You heard about Milman's kid, didn't you?"
"Nine days after birth."
"Undiagnosed infection of the central nervous system."
I labelled the tube, put the plastic tube in a rack, cleaned up my lab bench, emptied my desk, put the tube and the rack in the deep freeze, said goodbye and walked out. Ain't been back.
Now let me tell you about the flu.
You've read in the paper, about how the flu mutates. So you think you can relate to that. But flu virus doesn't even have DNA. It's way different from whatever you're thinking of. But that's not really the scary thing. What's scary is what it looks like in the electron microscope. . Every other virus, the little submicroscopic virions are like peas in a pod, like polio here. Not flu. Flu virions are all different. They look like sausages. But they look like sausages made by unhinged people in a sausage factory that makes no sense at all.
Check out the photos, the electron micrographs.
Flu -- bird flu, any flu -- flu is different.
Notes on "Flu" --
Right after I finished writing the story, I went halfway up on Nicholl Knob by the reservoir with a friend, sat down on a stump, and read the story aloud. My dog Scrabble, the greyhound, came up behind me as I read, parked his snout about two or three inches right behind my ear, and listened intently to the entire thing, start to finish, standing so close to me that I heard every breath that he took.
The dog was listening with his nose.
Damnedest thing I've ever seen.
Richard Katz 20 Belvedere Avenue, Point Richmond, California 94801 telephone 510 236 1865 richard808 AT gmail dot com