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NEW HUMAN PAPOVAVIRUS (B.K.) ISOLATED FROM URINE AFTER RENAL TRANSPLANTATION
1253 mother and the baby are presented in fig. 2. No rubella antibody was detected in the IgM fraction.
NEW HUMAN PAPOVAVIRUS (B.K.) ISOLATED FROM URINE AFTER RENAL TRANSPLANTATION
Discussion
In these three cases a significant rise in rubellaantibody titre was detected in pregnant women who were exposed to children with rubella early in pregnancy but had no history of clinical reactions after exposure. The first question posed by a significant rise in titre is what type of antibody response it represents: a primary response during a subclinical infection, or a secondary response in a naturally immune subject ? In case 1 the answer was obvious as rubella immunity was checked some months before the beginning of the pregnancy. This is unusual, however; and very often (as in cases 2 and 3) the first serum sample is taken after exposure, and in some cases the time of the exposure is not clearly defined. For these reasons analysis of serum fractions obtained after sucrose density-gradient ultracentrifugation has been recommended2, 3 The results obtained in the three patients clearly show that the specific rise of rubella H.i. antibody was only in the IgG fraction-a response characteristic of reinfection. The second problem is to determine whether the mother’s early secondary (booster-type) response will protect the embryo from infection by the virus. In these three cases reinfection occurred during the first trimester of gestation, and the three infants exhibited no malformations or symptoms which could be attributed to congenital rubella. Moreover, using the same sucrose density-gradient techniques with the infants’ sera, rubella antibodies were present only in the IgG fraction and had the same titre as in the mothers’ sera. Three cases are not enough to conclude that reinfection is without risk to the product of conception, but they reinforce clinical experience. Furthermore, viraemia has never been detected in immune subjects after experimental challenge with wild-type rubella virus. 4,51 It is important to differentiate the types of antibody response when a significant increase in serum H.I. antibody titre has been detected. The sucrose densitygradient ultracentrifugation technique is at present the most reliable method, and we think that this should be applied in all cases of rise in rubella antibody titre without rash before a decision to perform a therapeutic abortion is made. It will be important also to collect similar observations in women with vaccine-induced immunity.
Requests for reprints should be addressed to A. B., Laboratoire S.E.S.E.P., Chateau de Longchamp, 75 Paris 16, France. We thank Dr. M. Badillet, Dr. M. Beaudet, and Dr. J. Renaudet for their cooperation. REFERENCES
1. Boué, A.,
114, 2. 3.
Nicolas, A., Lang,
R. Ann. Inst.
Pasteur, Paris, 1968,
317.
Vesikari, T., Vaheri, 1968, i, 221. Banatvala, J. E., Best, J. M., Bertrand, J., Bowern, A. Br. med. J.
N. A., Hudson, S. M. ibid. 1970, iii, 247. 4. Schiff, G. M., Donath, R., Rotte, T. Am. J. Dis. Child. 1969, 118, 269. 5. Wilkins, J., Leedom, J. M., Portnoy, B., Salvatore, M. A. ibid.
p. 275.
SYLVIA D. GARDNER
ANNE M. FIELD
Virus Reference Laboratory, Central Public Health Laboratory, Colindale Avenue, London N.W.9
DULCIE V. COLEMAN
B. HULME
Department of Histopathology and Cytology and Mary’s Hospital, London W.2
Medical Unit, St.
The isolation of a new papovavirus from the urine of a renal allograft with ureteric obstruction is described. Virus recipient were in the cells lining the ureter by observed particles electron microscopy, and high, rising antibody titres to the virus were demonstrated in the patient’s serum. This virus is not identical with any of the previously described members of the polyoma subgroup and has provisionally been named B.K. virus after the patient.
Summary Summary
Introduction
activated latent virus infections are frequently seen in renal allograft recipients. Most commonly encountered are members of the herpesvirus 2 In 1970 group, particularly cytomegalovirus. 1,2 Spencer and Andersen3 also reported a high incidence of warts in their patients one year or more after PRIMARY
or
transplantation. During a combined cytological and virological investigation of post-allograft patients the urine from one of them was found to contain a large number of virus particles. Morphologically these resembled members of the polyoma subgroup of the papovaviruses. Details of this case and preliminary attempts to identify the virus are reported here. Case-report Patient B. K., a 39-year-old male Sudanese, was admitted to St. Mary’s Hospital, London W.2, on May 17, 1970, with chronic pyelonephritis and advanced renal failure. Urological investigations showed a chronic trigonal cystitis confirmed by microscopical examination of biopsy material. A transplant, using the left kidney and ureter donated by a brother, was undertaken on June 24, 1970; the kidney was placed in the right iliac fossa and the donor ureter was implanted in the bladder. Postoperative progress was uneventful; two mild rejection episodes were treated on the 5th and 110th days after transplantation, and immunosuppression was maintained with prednisone 20 mg. daily and azathioprine 3 mg. per kg. body-weight daily. The patient was readmitted to hospital on Oct. 5, 1970, with pain over the renal graft and anuria. This persisted for 16 hours and was followed by a brisk diuresis. The creatinine clearance fell during this period from 100 ml. per minute to 9 ml. per minute. Anuria recurred two days later and an intravenous pyelogram showed evidence of a distal ureteric obstruction. Exploration revealed fibrosis and ischaemia of the ureter extending from the bladder to 5 cm. below the uretero-pelvic junction, and the distal 2 cm. of the ureter was surrounded by dense granulation tissue. The donor ureter was divided 2 cm. below the uretero-pelvic junction, and ureter, granulation tissue, and adjacent bladder were excised; a uretero-ureteric anastomosis was undertaken utilising the recipient’s right ureter.
1254 TABLE I-LABORATORY FINDINGS ON
10 URINE SAMPLES
* N.D. = not done. M.S.U. mid-stream urine. Cytomegalovirus isolated. (-1-)=after subculture to Vero cells. C.S.U. = catheter specimen of urine. Date of transplant: June 24, 1970. Date of operation for ureteric obstruction: Oct. 10, 1970. =
TABLE II-ANTIBODY TO B.K. VIRUS IN SERA FROM PATIENT AND DONOR
*
Reciprocal of serum dilution. htmagglutination inhibition.
H.I.
=
c.F.
=
I.E.M.
complement fixation. =
immune electron
microscopy.
Postoperative progress was uneventful and on May 5, 1971, the serum-creatinine was 0-9 mg. per 100 ml. and the creatinine clearance was 78 ml. per minute. Materials and Methods
Immune electron
microscopy (l..M.).-C;rude virus
sus-
pension derived from infected tissue-cultures as described was diluted according to its virus-particle content. This antigen was mixed using 0-1ml. volumes with equal volumes of unheated sera diluted in phosphate-buffered saline (P.B.s.). The mixtures were incubated for 60 minutes at room temperature, then overnight at 4°C. 1-5 ml. cold P.B.S. was added before centrifuging at 12,000 r.p.m. for 60 minutes. Pellet contents were negatively stained as above. Detection of antibody molecules coating virus particles and causing aggregation of the particles into clumps indicated the presence of viral antibody in the serum dilution used. The highest serum dilution producing this effect was taken as the end-point, and the antibody titre was expressed as a reciprocal of this dilution. Thin sections.-Cells pelleted at low speed from urine samples and 1 mm. cube pieces of ureter (placed whole in formalin for histology) were fixed in 1 % glutaraldehyde in cacodylate buffer and postfixed in 1% osmium tetroxide. Following dehydration they were embedded in an eponaraldite mixtures. Thin sections were stained with uranyl acetate and lead citrate.
Clinical Specimens Urine and serum samples from the patient B. K. and the donor were collected on the dates shown in tables i and 11.
Viruses and Antisera Polyoma virus and antiserum and a strain of SV 40 virus were kindly supplied by Dr. L. V. Crawford (Imperial Cancer Research Fund), human common wart serum by Mrs. J. D. Almeida (Royal Postgraduate Medical School, London W.12), and SV40 antiserum by Dr. D. Magrath (Medical Research Council Laboratories, Hampstead).
Cytological Methods 10 ml. samples of urine were filtered through a millipore membrane with an average pore diameter of 5 (1.. Cells retained by the membrane were stained by haematoxylin and eosin and examined by light microscopy.
Electron Microscopy Urines.-3 ml. volumes of clarified urine were centrifuged at 18,000 r.p.m. for 60 minutes. The pellets were resuspended in approximately 0-11 ml. distilled water, then mixed with an equal volume of 2% sodium-silicotungstate negative stain at pH 7-0. A drop was applied to a type 400 formvar-carbon-coated specimen grid’and examined in an AEI EM6B electron microscope at an instrumental magnification of 60,000. Tissue-culture.-Infected cells were scraped from the glass into the culture medium. The suspended cells were disrupted by treatment for 4 minutes in an ultrasonic bath, were centrifuged at 18,000 r.p.m. for 60 minutes, and the pellet contents were negatively stained as above.
Virological Methods Virus isolation.-Urine
samples
were
centrifuged at 2500 deposit
r.p.m. for 10 minutes and the supernatant and
inoculated independently into tissue-cultures. Virus isolation was attempted in monolayer cultures of primary rhesus monkey kidney (M.K.), human embryo lung fibroblast (H.E.L.), and a continuous line of African green monkey kidney (Vero)5 cell cultures. The M.K. cultures were incubated rolling at 33 °C and the H.E.L. and Vero cultures were incubated stationary at 37 °C. Cultures were examined twice weekly and maintained with weekly fluid were
changes.
Complement fixation (C.F.).-Vero cultures were inoculated with B.K. virus and incubated until the cytopathic effect was well advanced, which took about 28 days. After alternate cycles of freezing and thawing the infected cells were harvested and used as antigen. Uninfected cultures treated in the same way were used as control antigens. Complement-fixation tests were performed in W.H.O. perspex plates using 0-025 ml. volumes, 3 units of complement, and overnight fixation at 4°C. Haemagglutination inhibition (H.l.).-Agglutination tests done at 4°C using 0-5% washed human 0 cells. 0-25 ml. volumes of a 0-5% suspension of cells were added to equal volumes of doubling dilutions of infected culture fluids. The highest dilution of virus suspension showing complete hsmagglutination was taken as representing 1 hmmagglutinating unit. All sera used were treated overnight with broth-culture filtrates of Vibrio cholerre, inactiwere
vated
at
56 °C for 1 hour and then absorbed with packed
1255
Fig. la-B.K. virus particles from urine. ( x 180,000.)
human 0 cells. Doubling dilutions of these sera were made in W.H.O. perspex plates using 0-25 ml. volumes. An equal volume of antigen containing 4 ha:magglutinating units was added to each dilution. The mixtures were allowed to react at room temperature for 1 hour, and then 0’25 ml. volumes of 0-5% human 0 cells were added to each well and the plates placed at 4°C for about 1 hour. The highest serum dilution which completely inhibited hsmagglutination was taken as the end-point and the antibody titre was expressed as a reciprocal of this dilution.
Results
The cytology, electron isolation results on ten urine in table i.
microscopy, samples are
and virus summarised
Cytology On examination by light microscopy prepared membranes were found to be packed with inclusionbearing epithelial cells. The majority of these cells were mononuclear and contained a single round basophilic intranuclear inclusion separated from the nuclear membrane by a clearly defined halo. These inclusion-bearing cells were present in the first urine examined but gradually diminished in numbers in the subsequent samples and were absent from a urine taken 16 days after operation.
Fig. It)—B.K. virus particles (V) in nuclei (N) of epithelial cells lining the lumen (L) of the donor ureter. ( x 6000.) (Insert: virus particles in outlined area. [ x 22,500.])
revealed large numbers of similar virus particles in nuclei and scattered in the degenerate cytoplasm. Intact cells without virus were also present. Virus Isolation A cytopathic effect was first observed in M.K. cells 18 days after the cultures were inoculated with urine collected on Oct. 6, 1970. Subculture of cells and supernatant fluid to further M.K. cultures failed to cause any changes in the monolayers and only small numbers of virus particles were seen by electron microscopy, in contrast to the large numbers observed
Electron Microscopy Examination of urines showed
large numbers of
virus particles with average diameter 43-6 nm (fig. la). The particles were morphologically identical with members of the polyoma subgroup of the papovavirus group. In later samples increasing numbers of these particles were coated with a substance which resembled antibody. The virus grown in tissue-culture was morphologically identical with the virus seen in the urines. Thin sections of the donor ureter showed virus Particles in the nuclei of many epithelial cells bordering the lumen (fig. lb). Sections of the cells in urine
Fig. 2-Cytopathic effect caused by B.K. virus: first Vero cell
monolayer 109 days after inoculation.
passage in
1256
in the first-passage material. After further passage in M.K. cell cultures no virus particles appeared. Results on subsequent urine samples are shown in table I. Following urine inoculation, cytopathic changes were slow in developing in Vero cell cultures and were not apparent for 1-3 months. Such changes consisted in a darkening of the cytoplasm and the appearance of scattered granular round cells on the surface of the monolayer (fig. 2). The cytopathic effect increased very slowly and was seldom complete before a further 28 days. At this stage marked clumping of the cells was seen with destruction of the monolayer. Supernatant fluids from cultures showing these changes agglutinated human 0 and guineapig cells to high A virus resembling the papovaviruses was titre. demonstrated in the cell cultures by electron microscopy. Examination of many control cultures treated No cytopathic effect in H.E.L. cell-cultures inoculated with the first three urine samples, and virus particles could not be demonstrated by electron microscopy. However, cytomegalovirus was isolated from later specimens
similarly was uniformly negative.
was seen
(table i). The virus, provisionally named B.K. after the patient, has been satisfactorily propagated in series in Vero cell-cultures, but even on passage cytopathic changes are generally inapparent for at least 14 days. Antibody Response in Patient and Donor The results of c.F., H.I., and I.E.M. tests on serum samples from the patient B. K. and the donor are shown in table n. Low levels of antibody were demonstrated in the patient’s serum before transplantation, and the titres were unchanged in a serum taken on Sept. 4, 1970, 5 weeks before the operation for ureteric obstruction. A greater than fourfold increase in antibody occurred during the period when the transplanted ureter became obstructed and B.K. virus was excreted. Antibody titres continued to rise for the next 3 months. Low levels of antibody were also demonstrated in a single serum sample taken from the donor 4 months after kidney transplantation.
Serological Reactions with Members of the Papovavirus Group Viruses and antisera were available from some members of the papovavirus group. Reactions between these viruses and the patient’s serum and B.K. virus and papovavirus antisera were studied in preliminary attempts to identify B.K. virus (table ill). There were no cross-reactions between B.K. virus and antisera to polyoma or human common-wart virus. Reciprocal tests were also negative. A possible antigenic relationship between B.K. and SV 40 viruses was detected. By immune electron microscopy homologous antibody titres were high. In the reciprocal tests a weak reaction occurred only at low serum dilutions. Thus two SV 40 antisera showed very low antibody titres (neat and 1/5) to B.K. virus compared with the high homologous SV 40 antibody titres (1/80 and 1/320). The SV4o antisera gave similar results when tested with three strains of SV41 The patient’s convalescent serum reacted virus. with SV4o virus only when undiluted, whereas weakly the homologous B.K. antibody titre in this serum was
TABLE III-SEROLOGICAL REACTIONS WITH MEMBERS OF THE PAPOVAVIRUS GROUP
*
Reciprocal of serum dilution. done. A.C. anticomplementary.
N.D. = not
=
1/320. A cross-reaction between B.K. virus and SV40 antisera was also seen by hsemagglutination-inhibition. Since SV40 virus has no hxmagglutinin the reciprocal tests could not be made. Discussion
The
6
is divided, mainly accordsize of the the virus ing particles, into two subgroups. Members of the papilloma subgroup, which includes human common-wart virus, have particles 52-55 nm. in diameter and those of the polyoma subgroup have particles 40-45 nm. in diameter. B.K. virus particles have the typical morphology of members of the papovavirus group both in negative stain and in thin-section preparations. The size of the particles, 43-6 nm. diameter, indicates that the virus belongs to the polyoma subgroup. Viruses of this subgroup already isolated and studied are polyoma, B SV40,9 K,10 and rabbit kidney vacuolating (R.K.v.)." The papovaviruses naturally infect various animals and characteristically cause chronic and latent infections. Some members of the group have been shown to be oncogenic, although in the polyoma subgroup tumours rarely occur spontaneously in the natural host. One member of the papovavirus group known to infect man is the common-wart virus. B.K. virus is smaller than wart virus and does not appear to be related to it antigenically. Also wart virus has not been grown in cell-culture in vitro. SV4e virus naturally infects rhesus and cynomolgus monkeys,9 and various vaccines containing the virus have accidentally and experimentally been given to man.12,13 Antibody to SV40 has been demonstrated in persons in close contact with monkeys.l4 So far no clinical illness has been associated with these infections. SV 40 virus grows rapidly in tissue-culture, whereas B.K. virus grows extremely slowly. No htmaggludnins have been described for SV 40 virus, and three strains of SV 40 virus used in this study failed to hasmagglutinate human 0 cells. B.K. virus, on the other hand, agglutinates human 0 and guineapig red cells at 4°C. The serological results (table m) suggest that B.K. and SV 40 viruses are not identical or even closely related but they may share some antigens. Throughout this investigation every precaution was to
papovavirus
group
1257
taken to avoid possible contamination of B.K. virus Continuous monitoring of the with SV4o virus. rhesus monkey-kidney-cell cultures used in the laboratory failed to demonstrate any latent infection with this virus. Vero cells have been used in many previous investigations and have never been shown to be infected with SV40 virus. Also no virus particles have been All work with seen in thin sections of these cells. known strains of SV 40 virus was done in a different
CULTIVATION OF PAPOVA-LIKE VIRUS FROM HUMAN BRAIN WITH PROGRESSIVE MULTIFOCAL LEUCOENCEPHALOPATHY BILLIE L. PADGETT GABRIELE M. ZURHEIN
Departments of Medical Microbiology and Pathology, University of Wisconsin Medical School, Madison, Wisconsin 53706
room.
Polyoma virus which causes a natural infection of mice agglutinates human 0 and guineapig red cells at 4°C, but no serological cross-reaction between this virus and B.K. could be demonstrated. K virus infects mice and R.K.V. virus occurs as a latent infection of cottontail rabbits. The biological properties previously described for these two viruses do not correspond with those of B.K. virus, but serological cross-reactions have yet to be investigated. A possible fifth member of the polyoma subgroup has been described in man. Papovavirus particles were seen in sections of brain tissue from patients with progressive multifocal leucoencephalopathy.11 Since this virus has never been grown in cell cultures its relationship with other members of the group is unknown. Particles resembling papovaviruses have also been demonstrated by electron microscopy in a continuous cell-culture derived from a human nephroblastoma 16 and brain-cell cultures from cases of subacute
sclerosing panencephalitis.17
B.K. appears to be a new papovavirus with properties similar but not identical to previously isolated members of the polyoma subgroup. In the case presented the serological evidence suggests that both the recipient and donor had been infected with B.K. virus before the renal transplantation. It seems likely, therefore, that the infection described was due to activation of virus latent either in the patient or in the transplanted kidney and ureter. The ischasmic changes present in the ureter were probably secondary to rejection, and it is possible that the necrosis resulting from this provided a suitable site for virus multiplication; indeed virus particles were seen in large numbers in epithelial cells lining the lumen of the donor ureter. The shedding of large numbers of these cells may have contributed to the ureteric obstruction. Virus continued to be excreted for at least 1 month following removal of the donor ureter, which suggests that the damaged ureter was not the only site of virus multiplication. Immunowas a factor in suppression probably establishing the a normal humoral infection; nevertheless, antibody response was demonstrated and the virus gradually disappeared from the urine. We are grateful to Prof. K. A. Porter (St. Mary’s Hospital, London W.2) for providing the formalin-fixed ureter for electron microscopy; to Mr. A. A. Porter for preparing the electron micrographs and for excellent technical assistance; and to Mr. W. Clifford for fig. 2. Requests for reprints should be addressed to S. D. G. REFERENCES 1.
Rifkind, D., Goodman, N., Hill,
R. B. Ann. intern. Med.
66, 1116. 2.
Crarghead, J. E., Hanshaw, J. B., Carpenter, C. B. J. 1967, 201,
Am. med. Ass.
725.
References continued at foot of next
1967,
column
DUARD L. WALKER ROBERT J. ECKROADE
BERT H. DESSEL Veterans Administration Hospital, Wood, Wisconsin 53193
papova-like virus has been cultivated case of progressive multifocal leucoencephalopathy (P.M.L.) complicating Hodgkin’s disease. The virus was isolated by inoculation of primary cultures of human fetal glial (P.H.F.G.) A
SSummary um ary from the brain of a
cells with ropsy.
extracts
made from brain obtained at
nec-
Electron-microscopic examination of sections of
brain and of infected P.H.F.G. cultures revealed cells with nuclei containing virions, often in crystalline array, which are similar in size to members of the polyomaSV40 subgroup of papovaviruses. Similar virions were observed in phosphotungstic acid (P.T.A.) stained extracts of the P.M.L. brain and of infected P.H.F.G. cultures. Initial studies on the cell susceptibility and antigenicity of this virus suggest that it is a new
papovavirus. Introduction
PROGRESSIVE multifbcal leucoencephalopathy (P.M.L.) is a rare demyelinating disease of man. 1-3 The suggestion that this disease is a result of viral infectionwas strengthened when electron-microscopic studies 4,5 revealed papovavirus-like particles in nuclei of oligodendrocytes in demyelinated areas of the brain. Later, virions similar in size and shape to those of the polyomaSV 40 subgroup of papovaviruses were observed in preparations from formalin-fixed P.M.L. brains stained with Many phosphotungstic acid (P.T.A.).6s attempts 3,7,8 to demonstrate some biological activity by these particles have been unsuccessful. We report here the cultivation of a virus, resembling members of the polyoma-SV 40 subgroup of papovaviruses, directly from the brain of a case of P.M.L.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Spencer, E. S., Andersen, H. K. Br. med. J. 1970, iii, 251. Mollenhauer, H. H. Stain Technol. 1964, 39, 111. Yasamura, Y., Kawakita, Y. Nippon Rinsho, 1963, 21, 1201. Melnick, J. L. Science, 1962, 135, 1128. Crawford, L. V., Crawford, E. M. Virology, 1963, 21, 258. Stewart, S. E., Eddy, B. E., Gochenour, A. M., Borgese, N. G., Grubbs, G. E. ibid. 1957, 3, 380. Sweet, B. H., Hilleman, M. R. Proc. Soc. exp. Biol. Med. 1960, 105, 420. Kilham, L. Science, 1952, 116, 391.
Hartley, J. W., Rowe, W. P. ibid. 1964, 143, 258. Magrath, D. I., Russell, K., Tobin, J. O’H. Br. med. J. 1961, ii, 287. Morris, J. A., Johnson, K. M., Aulisio, C. G., Chanock, R. M., Knight, V. Proc. Soc. exp. Biol. Med. 1961, 108, 56. 14. Shah, K. V. ibid. 1966, 121, 303. 15. Zu Rhein, G. M., Chou Shi-Ming. Science, 1965, 148, 1477. 16. Smith, J. W., Pinkel, D., Dabrowski, S. Cancer, 1969, 24, 527. 17. Koprowski, H., Barbanti-Brodano, G., Katz, M. Nature, 1970, 225, 1045.
NEW HUMAN PAPOVAVIRUS (B.K.) ISOLATED FROM URINE AFTER RENAL TRANSPLANTATION
Discussion
In these three cases a significant rise in rubellaantibody titre was detected in pregnant women who were exposed to children with rubella early in pregnancy but had no history of clinical reactions after exposure. The first question posed by a significant rise in titre is what type of antibody response it represents: a primary response during a subclinical infection, or a secondary response in a naturally immune subject ? In case 1 the answer was obvious as rubella immunity was checked some months before the beginning of the pregnancy. This is unusual, however; and very often (as in cases 2 and 3) the first serum sample is taken after exposure, and in some cases the time of the exposure is not clearly defined. For these reasons analysis of serum fractions obtained after sucrose density-gradient ultracentrifugation has been recommended2, 3 The results obtained in the three patients clearly show that the specific rise of rubella H.i. antibody was only in the IgG fraction-a response characteristic of reinfection. The second problem is to determine whether the mother’s early secondary (booster-type) response will protect the embryo from infection by the virus. In these three cases reinfection occurred during the first trimester of gestation, and the three infants exhibited no malformations or symptoms which could be attributed to congenital rubella. Moreover, using the same sucrose density-gradient techniques with the infants’ sera, rubella antibodies were present only in the IgG fraction and had the same titre as in the mothers’ sera. Three cases are not enough to conclude that reinfection is without risk to the product of conception, but they reinforce clinical experience. Furthermore, viraemia has never been detected in immune subjects after experimental challenge with wild-type rubella virus. 4,51 It is important to differentiate the types of antibody response when a significant increase in serum H.I. antibody titre has been detected. The sucrose densitygradient ultracentrifugation technique is at present the most reliable method, and we think that this should be applied in all cases of rise in rubella antibody titre without rash before a decision to perform a therapeutic abortion is made. It will be important also to collect similar observations in women with vaccine-induced immunity.
Requests for reprints should be addressed to A. B., Laboratoire S.E.S.E.P., Chateau de Longchamp, 75 Paris 16, France. We thank Dr. M. Badillet, Dr. M. Beaudet, and Dr. J. Renaudet for their cooperation. REFERENCES
1. Boué, A.,
114, 2. 3.
Nicolas, A., Lang,
R. Ann. Inst.
Pasteur, Paris, 1968,
317.
Vesikari, T., Vaheri, 1968, i, 221. Banatvala, J. E., Best, J. M., Bertrand, J., Bowern, A. Br. med. J.
N. A., Hudson, S. M. ibid. 1970, iii, 247. 4. Schiff, G. M., Donath, R., Rotte, T. Am. J. Dis. Child. 1969, 118, 269. 5. Wilkins, J., Leedom, J. M., Portnoy, B., Salvatore, M. A. ibid.
p. 275.
SYLVIA D. GARDNER
ANNE M. FIELD
Virus Reference Laboratory, Central Public Health Laboratory, Colindale Avenue, London N.W.9
DULCIE V. COLEMAN
B. HULME
Department of Histopathology and Cytology and Mary’s Hospital, London W.2
Medical Unit, St.
The isolation of a new papovavirus from the urine of a renal allograft with ureteric obstruction is described. Virus recipient were in the cells lining the ureter by observed particles electron microscopy, and high, rising antibody titres to the virus were demonstrated in the patient’s serum. This virus is not identical with any of the previously described members of the polyoma subgroup and has provisionally been named B.K. virus after the patient.
Summary Summary
Introduction
activated latent virus infections are frequently seen in renal allograft recipients. Most commonly encountered are members of the herpesvirus 2 In 1970 group, particularly cytomegalovirus. 1,2 Spencer and Andersen3 also reported a high incidence of warts in their patients one year or more after PRIMARY
or
transplantation. During a combined cytological and virological investigation of post-allograft patients the urine from one of them was found to contain a large number of virus particles. Morphologically these resembled members of the polyoma subgroup of the papovaviruses. Details of this case and preliminary attempts to identify the virus are reported here. Case-report Patient B. K., a 39-year-old male Sudanese, was admitted to St. Mary’s Hospital, London W.2, on May 17, 1970, with chronic pyelonephritis and advanced renal failure. Urological investigations showed a chronic trigonal cystitis confirmed by microscopical examination of biopsy material. A transplant, using the left kidney and ureter donated by a brother, was undertaken on June 24, 1970; the kidney was placed in the right iliac fossa and the donor ureter was implanted in the bladder. Postoperative progress was uneventful; two mild rejection episodes were treated on the 5th and 110th days after transplantation, and immunosuppression was maintained with prednisone 20 mg. daily and azathioprine 3 mg. per kg. body-weight daily. The patient was readmitted to hospital on Oct. 5, 1970, with pain over the renal graft and anuria. This persisted for 16 hours and was followed by a brisk diuresis. The creatinine clearance fell during this period from 100 ml. per minute to 9 ml. per minute. Anuria recurred two days later and an intravenous pyelogram showed evidence of a distal ureteric obstruction. Exploration revealed fibrosis and ischaemia of the ureter extending from the bladder to 5 cm. below the uretero-pelvic junction, and the distal 2 cm. of the ureter was surrounded by dense granulation tissue. The donor ureter was divided 2 cm. below the uretero-pelvic junction, and ureter, granulation tissue, and adjacent bladder were excised; a uretero-ureteric anastomosis was undertaken utilising the recipient’s right ureter.
1254 TABLE I-LABORATORY FINDINGS ON
10 URINE SAMPLES
* N.D. = not done. M.S.U. mid-stream urine. Cytomegalovirus isolated. (-1-)=after subculture to Vero cells. C.S.U. = catheter specimen of urine. Date of transplant: June 24, 1970. Date of operation for ureteric obstruction: Oct. 10, 1970. =
TABLE II-ANTIBODY TO B.K. VIRUS IN SERA FROM PATIENT AND DONOR
*
Reciprocal of serum dilution. htmagglutination inhibition.
H.I.
=
c.F.
=
I.E.M.
complement fixation. =
immune electron
microscopy.
Postoperative progress was uneventful and on May 5, 1971, the serum-creatinine was 0-9 mg. per 100 ml. and the creatinine clearance was 78 ml. per minute. Materials and Methods
Immune electron
microscopy (l..M.).-C;rude virus
sus-
pension derived from infected tissue-cultures as described was diluted according to its virus-particle content. This antigen was mixed using 0-1ml. volumes with equal volumes of unheated sera diluted in phosphate-buffered saline (P.B.s.). The mixtures were incubated for 60 minutes at room temperature, then overnight at 4°C. 1-5 ml. cold P.B.S. was added before centrifuging at 12,000 r.p.m. for 60 minutes. Pellet contents were negatively stained as above. Detection of antibody molecules coating virus particles and causing aggregation of the particles into clumps indicated the presence of viral antibody in the serum dilution used. The highest serum dilution producing this effect was taken as the end-point, and the antibody titre was expressed as a reciprocal of this dilution. Thin sections.-Cells pelleted at low speed from urine samples and 1 mm. cube pieces of ureter (placed whole in formalin for histology) were fixed in 1 % glutaraldehyde in cacodylate buffer and postfixed in 1% osmium tetroxide. Following dehydration they were embedded in an eponaraldite mixtures. Thin sections were stained with uranyl acetate and lead citrate.
Clinical Specimens Urine and serum samples from the patient B. K. and the donor were collected on the dates shown in tables i and 11.
Viruses and Antisera Polyoma virus and antiserum and a strain of SV 40 virus were kindly supplied by Dr. L. V. Crawford (Imperial Cancer Research Fund), human common wart serum by Mrs. J. D. Almeida (Royal Postgraduate Medical School, London W.12), and SV40 antiserum by Dr. D. Magrath (Medical Research Council Laboratories, Hampstead).
Cytological Methods 10 ml. samples of urine were filtered through a millipore membrane with an average pore diameter of 5 (1.. Cells retained by the membrane were stained by haematoxylin and eosin and examined by light microscopy.
Electron Microscopy Urines.-3 ml. volumes of clarified urine were centrifuged at 18,000 r.p.m. for 60 minutes. The pellets were resuspended in approximately 0-11 ml. distilled water, then mixed with an equal volume of 2% sodium-silicotungstate negative stain at pH 7-0. A drop was applied to a type 400 formvar-carbon-coated specimen grid’and examined in an AEI EM6B electron microscope at an instrumental magnification of 60,000. Tissue-culture.-Infected cells were scraped from the glass into the culture medium. The suspended cells were disrupted by treatment for 4 minutes in an ultrasonic bath, were centrifuged at 18,000 r.p.m. for 60 minutes, and the pellet contents were negatively stained as above.
Virological Methods Virus isolation.-Urine
samples
were
centrifuged at 2500 deposit
r.p.m. for 10 minutes and the supernatant and
inoculated independently into tissue-cultures. Virus isolation was attempted in monolayer cultures of primary rhesus monkey kidney (M.K.), human embryo lung fibroblast (H.E.L.), and a continuous line of African green monkey kidney (Vero)5 cell cultures. The M.K. cultures were incubated rolling at 33 °C and the H.E.L. and Vero cultures were incubated stationary at 37 °C. Cultures were examined twice weekly and maintained with weekly fluid were
changes.
Complement fixation (C.F.).-Vero cultures were inoculated with B.K. virus and incubated until the cytopathic effect was well advanced, which took about 28 days. After alternate cycles of freezing and thawing the infected cells were harvested and used as antigen. Uninfected cultures treated in the same way were used as control antigens. Complement-fixation tests were performed in W.H.O. perspex plates using 0-025 ml. volumes, 3 units of complement, and overnight fixation at 4°C. Haemagglutination inhibition (H.l.).-Agglutination tests done at 4°C using 0-5% washed human 0 cells. 0-25 ml. volumes of a 0-5% suspension of cells were added to equal volumes of doubling dilutions of infected culture fluids. The highest dilution of virus suspension showing complete hsmagglutination was taken as representing 1 hmmagglutinating unit. All sera used were treated overnight with broth-culture filtrates of Vibrio cholerre, inactiwere
vated
at
56 °C for 1 hour and then absorbed with packed
1255
Fig. la-B.K. virus particles from urine. ( x 180,000.)
human 0 cells. Doubling dilutions of these sera were made in W.H.O. perspex plates using 0-25 ml. volumes. An equal volume of antigen containing 4 ha:magglutinating units was added to each dilution. The mixtures were allowed to react at room temperature for 1 hour, and then 0’25 ml. volumes of 0-5% human 0 cells were added to each well and the plates placed at 4°C for about 1 hour. The highest serum dilution which completely inhibited hsmagglutination was taken as the end-point and the antibody titre was expressed as a reciprocal of this dilution.
Results
The cytology, electron isolation results on ten urine in table i.
microscopy, samples are
and virus summarised
Cytology On examination by light microscopy prepared membranes were found to be packed with inclusionbearing epithelial cells. The majority of these cells were mononuclear and contained a single round basophilic intranuclear inclusion separated from the nuclear membrane by a clearly defined halo. These inclusion-bearing cells were present in the first urine examined but gradually diminished in numbers in the subsequent samples and were absent from a urine taken 16 days after operation.
Fig. It)—B.K. virus particles (V) in nuclei (N) of epithelial cells lining the lumen (L) of the donor ureter. ( x 6000.) (Insert: virus particles in outlined area. [ x 22,500.])
revealed large numbers of similar virus particles in nuclei and scattered in the degenerate cytoplasm. Intact cells without virus were also present. Virus Isolation A cytopathic effect was first observed in M.K. cells 18 days after the cultures were inoculated with urine collected on Oct. 6, 1970. Subculture of cells and supernatant fluid to further M.K. cultures failed to cause any changes in the monolayers and only small numbers of virus particles were seen by electron microscopy, in contrast to the large numbers observed
Electron Microscopy Examination of urines showed
large numbers of
virus particles with average diameter 43-6 nm (fig. la). The particles were morphologically identical with members of the polyoma subgroup of the papovavirus group. In later samples increasing numbers of these particles were coated with a substance which resembled antibody. The virus grown in tissue-culture was morphologically identical with the virus seen in the urines. Thin sections of the donor ureter showed virus Particles in the nuclei of many epithelial cells bordering the lumen (fig. lb). Sections of the cells in urine
Fig. 2-Cytopathic effect caused by B.K. virus: first Vero cell
monolayer 109 days after inoculation.
passage in
1256
in the first-passage material. After further passage in M.K. cell cultures no virus particles appeared. Results on subsequent urine samples are shown in table I. Following urine inoculation, cytopathic changes were slow in developing in Vero cell cultures and were not apparent for 1-3 months. Such changes consisted in a darkening of the cytoplasm and the appearance of scattered granular round cells on the surface of the monolayer (fig. 2). The cytopathic effect increased very slowly and was seldom complete before a further 28 days. At this stage marked clumping of the cells was seen with destruction of the monolayer. Supernatant fluids from cultures showing these changes agglutinated human 0 and guineapig cells to high A virus resembling the papovaviruses was titre. demonstrated in the cell cultures by electron microscopy. Examination of many control cultures treated No cytopathic effect in H.E.L. cell-cultures inoculated with the first three urine samples, and virus particles could not be demonstrated by electron microscopy. However, cytomegalovirus was isolated from later specimens
similarly was uniformly negative.
was seen
(table i). The virus, provisionally named B.K. after the patient, has been satisfactorily propagated in series in Vero cell-cultures, but even on passage cytopathic changes are generally inapparent for at least 14 days. Antibody Response in Patient and Donor The results of c.F., H.I., and I.E.M. tests on serum samples from the patient B. K. and the donor are shown in table n. Low levels of antibody were demonstrated in the patient’s serum before transplantation, and the titres were unchanged in a serum taken on Sept. 4, 1970, 5 weeks before the operation for ureteric obstruction. A greater than fourfold increase in antibody occurred during the period when the transplanted ureter became obstructed and B.K. virus was excreted. Antibody titres continued to rise for the next 3 months. Low levels of antibody were also demonstrated in a single serum sample taken from the donor 4 months after kidney transplantation.
Serological Reactions with Members of the Papovavirus Group Viruses and antisera were available from some members of the papovavirus group. Reactions between these viruses and the patient’s serum and B.K. virus and papovavirus antisera were studied in preliminary attempts to identify B.K. virus (table ill). There were no cross-reactions between B.K. virus and antisera to polyoma or human common-wart virus. Reciprocal tests were also negative. A possible antigenic relationship between B.K. and SV 40 viruses was detected. By immune electron microscopy homologous antibody titres were high. In the reciprocal tests a weak reaction occurred only at low serum dilutions. Thus two SV 40 antisera showed very low antibody titres (neat and 1/5) to B.K. virus compared with the high homologous SV 40 antibody titres (1/80 and 1/320). The SV4o antisera gave similar results when tested with three strains of SV41 The patient’s convalescent serum reacted virus. with SV4o virus only when undiluted, whereas weakly the homologous B.K. antibody titre in this serum was
TABLE III-SEROLOGICAL REACTIONS WITH MEMBERS OF THE PAPOVAVIRUS GROUP
*
Reciprocal of serum dilution. done. A.C. anticomplementary.
N.D. = not
=
1/320. A cross-reaction between B.K. virus and SV40 antisera was also seen by hsemagglutination-inhibition. Since SV40 virus has no hxmagglutinin the reciprocal tests could not be made. Discussion
The
6
is divided, mainly accordsize of the the virus ing particles, into two subgroups. Members of the papilloma subgroup, which includes human common-wart virus, have particles 52-55 nm. in diameter and those of the polyoma subgroup have particles 40-45 nm. in diameter. B.K. virus particles have the typical morphology of members of the papovavirus group both in negative stain and in thin-section preparations. The size of the particles, 43-6 nm. diameter, indicates that the virus belongs to the polyoma subgroup. Viruses of this subgroup already isolated and studied are polyoma, B SV40,9 K,10 and rabbit kidney vacuolating (R.K.v.)." The papovaviruses naturally infect various animals and characteristically cause chronic and latent infections. Some members of the group have been shown to be oncogenic, although in the polyoma subgroup tumours rarely occur spontaneously in the natural host. One member of the papovavirus group known to infect man is the common-wart virus. B.K. virus is smaller than wart virus and does not appear to be related to it antigenically. Also wart virus has not been grown in cell-culture in vitro. SV4e virus naturally infects rhesus and cynomolgus monkeys,9 and various vaccines containing the virus have accidentally and experimentally been given to man.12,13 Antibody to SV40 has been demonstrated in persons in close contact with monkeys.l4 So far no clinical illness has been associated with these infections. SV 40 virus grows rapidly in tissue-culture, whereas B.K. virus grows extremely slowly. No htmaggludnins have been described for SV 40 virus, and three strains of SV 40 virus used in this study failed to hasmagglutinate human 0 cells. B.K. virus, on the other hand, agglutinates human 0 and guineapig red cells at 4°C. The serological results (table m) suggest that B.K. and SV 40 viruses are not identical or even closely related but they may share some antigens. Throughout this investigation every precaution was to
papovavirus
group
1257
taken to avoid possible contamination of B.K. virus Continuous monitoring of the with SV4o virus. rhesus monkey-kidney-cell cultures used in the laboratory failed to demonstrate any latent infection with this virus. Vero cells have been used in many previous investigations and have never been shown to be infected with SV40 virus. Also no virus particles have been All work with seen in thin sections of these cells. known strains of SV 40 virus was done in a different
CULTIVATION OF PAPOVA-LIKE VIRUS FROM HUMAN BRAIN WITH PROGRESSIVE MULTIFOCAL LEUCOENCEPHALOPATHY BILLIE L. PADGETT GABRIELE M. ZURHEIN
Departments of Medical Microbiology and Pathology, University of Wisconsin Medical School, Madison, Wisconsin 53706
room.
Polyoma virus which causes a natural infection of mice agglutinates human 0 and guineapig red cells at 4°C, but no serological cross-reaction between this virus and B.K. could be demonstrated. K virus infects mice and R.K.V. virus occurs as a latent infection of cottontail rabbits. The biological properties previously described for these two viruses do not correspond with those of B.K. virus, but serological cross-reactions have yet to be investigated. A possible fifth member of the polyoma subgroup has been described in man. Papovavirus particles were seen in sections of brain tissue from patients with progressive multifocal leucoencephalopathy.11 Since this virus has never been grown in cell cultures its relationship with other members of the group is unknown. Particles resembling papovaviruses have also been demonstrated by electron microscopy in a continuous cell-culture derived from a human nephroblastoma 16 and brain-cell cultures from cases of subacute
sclerosing panencephalitis.17
B.K. appears to be a new papovavirus with properties similar but not identical to previously isolated members of the polyoma subgroup. In the case presented the serological evidence suggests that both the recipient and donor had been infected with B.K. virus before the renal transplantation. It seems likely, therefore, that the infection described was due to activation of virus latent either in the patient or in the transplanted kidney and ureter. The ischasmic changes present in the ureter were probably secondary to rejection, and it is possible that the necrosis resulting from this provided a suitable site for virus multiplication; indeed virus particles were seen in large numbers in epithelial cells lining the lumen of the donor ureter. The shedding of large numbers of these cells may have contributed to the ureteric obstruction. Virus continued to be excreted for at least 1 month following removal of the donor ureter, which suggests that the damaged ureter was not the only site of virus multiplication. Immunowas a factor in suppression probably establishing the a normal humoral infection; nevertheless, antibody response was demonstrated and the virus gradually disappeared from the urine. We are grateful to Prof. K. A. Porter (St. Mary’s Hospital, London W.2) for providing the formalin-fixed ureter for electron microscopy; to Mr. A. A. Porter for preparing the electron micrographs and for excellent technical assistance; and to Mr. W. Clifford for fig. 2. Requests for reprints should be addressed to S. D. G. REFERENCES 1.
Rifkind, D., Goodman, N., Hill,
R. B. Ann. intern. Med.
66, 1116. 2.
Crarghead, J. E., Hanshaw, J. B., Carpenter, C. B. J. 1967, 201,
Am. med. Ass.
725.
References continued at foot of next
1967,
column
DUARD L. WALKER ROBERT J. ECKROADE
BERT H. DESSEL Veterans Administration Hospital, Wood, Wisconsin 53193
papova-like virus has been cultivated case of progressive multifocal leucoencephalopathy (P.M.L.) complicating Hodgkin’s disease. The virus was isolated by inoculation of primary cultures of human fetal glial (P.H.F.G.) A
SSummary um ary from the brain of a
cells with ropsy.
extracts
made from brain obtained at
nec-
Electron-microscopic examination of sections of
brain and of infected P.H.F.G. cultures revealed cells with nuclei containing virions, often in crystalline array, which are similar in size to members of the polyomaSV40 subgroup of papovaviruses. Similar virions were observed in phosphotungstic acid (P.T.A.) stained extracts of the P.M.L. brain and of infected P.H.F.G. cultures. Initial studies on the cell susceptibility and antigenicity of this virus suggest that it is a new
papovavirus. Introduction
PROGRESSIVE multifbcal leucoencephalopathy (P.M.L.) is a rare demyelinating disease of man. 1-3 The suggestion that this disease is a result of viral infectionwas strengthened when electron-microscopic studies 4,5 revealed papovavirus-like particles in nuclei of oligodendrocytes in demyelinated areas of the brain. Later, virions similar in size and shape to those of the polyomaSV 40 subgroup of papovaviruses were observed in preparations from formalin-fixed P.M.L. brains stained with Many phosphotungstic acid (P.T.A.).6s attempts 3,7,8 to demonstrate some biological activity by these particles have been unsuccessful. We report here the cultivation of a virus, resembling members of the polyoma-SV 40 subgroup of papovaviruses, directly from the brain of a case of P.M.L.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Spencer, E. S., Andersen, H. K. Br. med. J. 1970, iii, 251. Mollenhauer, H. H. Stain Technol. 1964, 39, 111. Yasamura, Y., Kawakita, Y. Nippon Rinsho, 1963, 21, 1201. Melnick, J. L. Science, 1962, 135, 1128. Crawford, L. V., Crawford, E. M. Virology, 1963, 21, 258. Stewart, S. E., Eddy, B. E., Gochenour, A. M., Borgese, N. G., Grubbs, G. E. ibid. 1957, 3, 380. Sweet, B. H., Hilleman, M. R. Proc. Soc. exp. Biol. Med. 1960, 105, 420. Kilham, L. Science, 1952, 116, 391.
Hartley, J. W., Rowe, W. P. ibid. 1964, 143, 258. Magrath, D. I., Russell, K., Tobin, J. O’H. Br. med. J. 1961, ii, 287. Morris, J. A., Johnson, K. M., Aulisio, C. G., Chanock, R. M., Knight, V. Proc. Soc. exp. Biol. Med. 1961, 108, 56. 14. Shah, K. V. ibid. 1966, 121, 303. 15. Zu Rhein, G. M., Chou Shi-Ming. Science, 1965, 148, 1477. 16. Smith, J. W., Pinkel, D., Dabrowski, S. Cancer, 1969, 24, 527. 17. Koprowski, H., Barbanti-Brodano, G., Katz, M. Nature, 1970, 225, 1045.