Identification Of A New Virus

In the late 1960s and early 1970s much work was being done to determine the epidemiology of cytomegalovirus (CMV), first isolated a decade earlier, and

Polyomavirus
Figure 4.3. Dr. Sylvia Gardner. Courtesy of Mr. J. Gibson.

its transmission in donated blood and organs. A collaboration was set up between Dr. Dulcie Coleman in the Department of Histopathology and Cytology at St. Mary's Hospital and Sylvia Gardner at Colindale to investigate, both cytologically and virologically, the presence of CMV infection in renal transplant patients.

On October 7, 1970, a midstream urine sample, collected a day earlier, arrived in the laboratory from a Sudanese patient who had received a renal transplant from his brother on June 24, 1970. A phone call the same day from St. Mary's Hospital informed us that this urine contained many inclusion-bearing cells, and so electron microscopy examination would be well worthwhile. Two days later, Anne Field saw very large numbers of papovavirus particles in the high-speed urine pellet and suggested that they were particles of common wart virus (the only human papovavirus known at the time). It was queried whether the patient had genital warts; a report earlier that year (Spencer and Andersen, 1970) had described a high incidence of warts after renal transplantation. Ultrathin sectioning of the cells in a subsequent urine sample collected on October 12 from the same patient showed many virus particles within enlarged cell nuclei.

On the day of receipt, the first urine was inoculated into tubes of both secondary rhesus monkey kidney (MK) cells and human embryo lung fibro-

Monkey Kidney

blasts (HEL) and observed every 3 or 4 days for a cytopathic effect (CPE). On day 19 a CPE appeared in the inoculated MK cell cultures, and the culture fluid was found to contain papovavirus particles. The fluid from uninoculated cells of the same batch was also examined as it was known that SV40, a monkey polyomavirus, was sometimes a contaminant in these cells; however, the controls were negative. In contrast, no CPE was seen in the HEL cells up to 27 days after inoculation. Vero cells were also inoculated on October 12, and on day 37 a CPE consisting of rounded cells was clearly visible.

The isolate was subsequently passaged in MK and Vero cells but appeared to die out in the MK cultures, and by the third passage CPE was no longer detectable even after 47 days in culture. Papovavirus was subsequently isolated from many more urine samples from this patient and others, but was always slow growing in culture, requiring immense patience. Virus harvested from the second passage in Vero cells was used as an antigen in complement fixation tests, and a seroconversion to the agent was demonstrated in the patient at the time the virus was isolated. The agent was named BK, the patient's initials, and the original isolate is known as the Gardner strain.

Measurement of the virus particles (45 nm diameter) showed the isolate to be a polyomavirus, as distinct from a papillomavirus, and work was undertaken to distinguish it from the only known polyomaviruses at the time: mouse polyoma, K virus of mice, monkey SV40, and rabbit kidney vacuolating agent. In early 1971 a range of biologic properties was investigated to characterize the new agent. It was found to agglutinate erythrocytes from different animal species, including humans, which immediately distinguished it from SV40, and enabled a hemagglutination inhibition (HI) test to be developed to add to the standard complement fixation test. Seroprevalence studies were later undertaken in various populations. The tumorigenic property of BKV in hamsters was also established early on.

After the virus was isolated and shown not to be human papillomavirus or SV40, it was thought that it might be the virus that had been described in association with the rare neurologic disease progressive multifocal leucoen-cephalopathy (PML) but that had thus far not been grown. Sylvia Gardner, therefore, wrote to laboratories in the United States that had published cases of PML and requested some PML brain tissue from them (hence the rumors heard by Duard Walker in Wisconsin!). No brain tissue was forthcoming, but we were later able to study patients in the United Kingdom with PML.

After our manuscript was submitted to The Lancet, the editors informed us that they had received another paper from the United States on the isolation of a human polyomavirus. Both papers had been accepted and would be published together.

Following the publication of the two original papers, it was obviously important to find out as soon as possible whether BK and JC viruses were related or even identical, and immediate collaboration was set up across "the pond'' between Sylvia Gardner, Duard Walker, Billie Padgett, and Gabriele Zu Rhein. Viruses were exchanged, and it was established that, indeed, the isolates were two new human polyomaviruses, related to each other and also to SV40.

The original BKV paper was identified in 1989 as a "Citation Classic'' by the Institute for Scientific Information.

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