The Start Of A Collaboration

Gabriele Zu Rhein clearly wanted to see this virus cultivated and characterized. She had demonstrated its presence in oligodendrocytes in PML brain tissue. The logical next step was to cultivate and identify it in order to study its role in nervous system disease. She wanted to have a part in that process, but she did not have the virology laboratory or virology background to do this, so she began prodding me to take it on in a collaborative project. I had a virology laboratory in the Department of Medical Microbiology in the same building but one floor below the Department of Pathology and Gabriele's electron microscope suite. Gabriele and I had co-authored a paper in 1956 concerned with a virus infection, but had not worked together in the interim. I had been studying persistent, chronic viral infections in cell cultures and animals and also was in the middle of a study of myxoma and fibroma virus infections. I had no experience with polyomaviruses, but I had some experience with papilloma-viruses, the other genus of the papovavirus family. In that project, like many other virologists, I had tried and failed to cultivate human papillomavirus in cell cultures, so I had experienced the frustrations of trying to cultivate a very fastidious and difficult virus.

In my consideration of an effort to try to cultivate the virus of PML, an important person was Billie Padgett (Fig. 3.1). Billie was an experienced virologist who had done her Ph.D. thesis research in my laboratory working with influenza virus. She continued in my laboratory as a postdoctoral fellow studying myxoma and fibroma viruses in both cell cultures and rabbits before going

Figure3.1. Billie Padgett, Duard Walkerand Gabriele Zu Rhein at Dr. Walker's retirement party in 1988 (Left to right). Courtesy of Gabriele Zu Rhein.

to Canberra, Australia, to work with Frank Fenner for 2 years. She returned to Madison, Wisconsin, in January 1967 to continue work with myxoma and fibroma viruses. Fortunately, she was willing to make a switch to work on PML, and, as the project developed, it was her patience and tenacity in learning how to get good cultures of fetal spongioblasts that led to the successful cultivation of JC virus. Since neither Billie nor I had experience with polyomaviruses, we were starting out as novices in that area. It would have been easy to obtain mouse polyoma virus and SV40 to study their cultivation, but we did not want to contaminate our laboratory with them. These are hardy viruses, and SV40, in particular, is notorious for contaminating a laboratory and persisting and appearing in subsequent experiments. Although our lack of experience with polyomaviruses might seem to have been a disadvantage, we did not view it as a serious problem, and we knew we would have one major advantage in our work. That was because this was to be a collaborative project with Gabriele, who would provide essential expertise in neuropathology and electron microscopy, and her prominence in the PML field would make it reasonably likely that we could obtain needed virus-containing tissue. Certainly, a serious problem facing anyone setting out to isolate the virus of PML was the paucity of virus-containing tissue available for study. PML was an uncommon disease, and locating terminal patients under circumstances where an autopsy could be performed and fresh or frozen tissue obtained was not likely to be achieved easily or quickly. As it turned out, Gabriele's recognition among pathologists and neuropathologists was essential to our obtaining good tissue.

Gabriele alerted pathologists and neurologists about our need for virus-containing tissue from cases of PML in sufficient quantity to allow significant work. We expected to have to wait a while for tissue, but we certainly hoped that the wait would not be as long as it turned out to be. It was nearly a year before a case appeared.

On March 9, 1967, Gabriele received a letter from Dr. Aurea del Rosario, a hematology resident at the Veterans Administration Hospital in East Orange, NJ. There was a patient in the VA Hospital who had Hodgkin's disease and the clinical features of PML. She had read Gabriele's paper on polyoma-like particles in PML. She was seeking help in confirming the diagnosis, but also offered any specimens that could be useful to Gabriele's research. Gabriele kept in very close touch with Dr. del Rosario by mail and telephone during the surprisingly long course of this patient's terminal illness. It was not until January 21, 1968, that Gabriele received word that the patient had died and that an autopsy would be performed the next day. Gabriele gathered appropriate containers, dry ice, and tissue culture medium and flew to New Jersey on the morning of January 22. She and the pathology resident removed the brain and sliced it immediately. Characteristic lesions were found in both hemispheres. Tissue was selected to go into culture medium, some was frozen, and some went into appropriate fixatives. Gabriele was soon on her way back to the airport and she was back in Madison very late that evening.

There is a later interesting addendum to this tissue procurement expedition. Three or four years later I was asked to discuss JC virus at a conference in New Jersey. During the discussion I mentioned, rather casually, that our first supply of fresh PML tissue had come from New Jersey. The Director of Public Health for the State of New Jersey happened to be in attendance and became quite agitated and incensed that he had not been informed about the case and that the tissue had been allowed to leave New Jersey rather than going to his laboratory. He had wanted to try cultivating the virus.

After obtaining the New Jersey tissue, we had enough virus-containing tissue to begin serious work. We began what turned into a long and frustrating series of efforts to cultivate the virus from the diseased tissue. We tried explants of the fresh brain tissue, but after many weeks of coaxing and coddling only a few fibroblasts and astrocytes grew. We made 10% extracts of diseased brain tissue and clarified it by centrifugation and then used the supernatant fluid to inoculate cell cultures. A similar extract of normal human brain tissue was used on control cultures. Monolayer cultures of primary and secondary human embryonic kidney cells, Hep-2 cells, and human fibroblasts were inoculated. None of these cell cultures showed any evidence of being infected even after weeks of cultivation and medium changes and subculturing. Mice, newborn mice, and hamsters were inoculated by the intracerebral and peritoneal routes and were observed for many months without showing signs of disease or tumor. Guinea pigs and rabbits were inoculated, guinea pigs intraperitoneally and rabbits intravenously to raise antibody against the virus. In this last effort we had a small measure of success. Using frozen sections of New Jersey brain tissue and the indirect method of immunofluorescent staining we found some guinea pig and rabbit sera that reacted at a low level with cell nuclei in the PML tissue. However, antibodies against human tissue were also present, and these interfered greatly. We had to absorb the antihuman antibodies out of the serum with dried human brain powder. This helped, but the antisera were very weak.

Billie began trying to cultivate primary human fetal glial (PHFG) cells. At the 1965 meeting of the American Association of Neuropathologists Gabriele had heard H.M. Shein describe a method for cultivating human fetal astrocytes and spongioblasts in dispersed cell cultures. He also described his use of these cultures to study the effects of SV40 infection on astrocytes and spongioblasts. Such cultures sounded very promising, but there were serious problems in producing them. Human fetal brain tissue was not easily obtained, and good cell cultures derived from the tissue were not easily produced. There was no commercial source, so they had to be produced in our laboratory from tissue obtained from local hospitals. We had gotten our fetal tissue supply problems worked out and Billie was making progress, but the cultures were still mainly what Shein considered to be astrocytes. Although we inoculated PHFG cell cultures with New Jersey brain tissue extract, they gave no indication of infection that we could recognize at that time.

Ten percent extracts of degenerating PML brain tissue tended to have a toxic effect on most cell cultures, and diluting away from that toxic effect obviously reduced the inoculum size. Therefore we began trying to purify and concentrate the virus to escape the toxicity and to obtain antigen of sufficient potency to develop antisera in rabbits and guinea pigs. The lipids in white matter presented a real challenge. How could we free the virus from that mass of myelin lipid and cell membranes? It became a matter of trial and error and using any clues available from the work of other investigators. Many techniques were tried, including genetron extraction and centrifuging to a pellet through 5-20% sucrose, but we eventually settled on one that started with homogenizing the tissue in a mortar or a blender. It was then sonicated and treated with sodium deoxycholate and trypsin and subjected to differential centrifugation. Supernatant fluid from a final low-speed centrifugation was diluted to the equivalent of a 10% tissue extract in buffered saline. Extracts of diseased tissue and normal human brain tissue prepared in this way were used in subsequent animal inoculations and for inoculating cell cultures. We referred to these extracts as semipurified because the virus certainly was far from really purified.

During the early phases of our PML project our financial support was from two National Institutes of Health (NIH) grants that were for research on persistent viral infections but were not directly aimed at PML. We needed additional funds and ones designated for research on the viral agent of PML. Fortunately, at about this time the concept of "slow virus infections'' and particularly "slow virus infections of the central nervous system'' was attracting attention. The Icelandic virologist Bjorn Sigurdsson had introduced the terms in his research on rida, scrapie, visna, and maedi of sheep because of the long incubation periods and slow progression of these diseases. Carleton Gajdusek used the terms in his work on kuru and Creutzfeldt-Jakob disease, and Gajdusek and others began including PML among the slow virus infections of the central nervous system. This gave PML some prominence and "pizzazz'' and made it more likely that we could gain support.

However, in 1968 we were already beyond those halcyon days of NIH when research funds were easily obtained. Funding had already tightened up, so it seemed rather unlikely that a proposal to study just the viral etiology of PML would be well received, particularly because we did not yet have even a suggestion of success in cultivating the PML virus. NIH study sections had developed a preference for supporting "a sure thing,'' that is, a project already nearly accomplished, rather than risking money on an uncertainty. And we were a real uncertainty.

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