Distribution of polyoma virus in the organs of newborn Wistar rats after subcutaneous inoculation

Zbl. Bakt. Hyg. A 258, 327-336 (1984)

Distribution of Polyoma Virus in the Organs of Newborn Wistar Rats After Subcutaneous Inoculation WILLEM VERHAGEN, GERHARD STAUCH, EZZATOLLAH MOHT ASCHEM, and HARALD CHORITZ Aus dem Institut fur Virologie und Seuchenhygiene (Direktor: Prof. Dr. J. Drescher) und dem Pathologischen lnstitut (Direktor: Prof. Dr. A. Georgii) der Medizinischen Hochschule Hannover

With 1 Figure· Received March 17, 1984 . Accepted June 29, 1984

Summary Polyoma virus of the Stuart Eddy strain was tritium-labelled by cultivating the virus in phosphate free Hepes buffered medium in presence of tritium labelled nucleotide triphosphates. The distribution of labelled virus in the organs and in the blood of newborn Wistar rats was examined at graded time intervals from two hours until 40 days after inoculation. The highest amounts of virus per organ was refound in the liver and the highest amount per mg organ weight in the spleen. The virus concentration in most organs exceeded by far the concentration found in the blood, indicating adsorption of virus in these organs. It was found that the distribution of tritium in the target organs for tumor appearance (kidneys and brain) did not differ basically from that in the non-target organs. As a consequence, the distribution of virus in the organs does not explain the fact that the kidneys and the brain are the predilection sites for tumor induction. Tritium labelling did not measurably influence the oncogenicity, infectivity and hemagglutinating activity of the virus. Introduction Tumor induction by polyoma virus in immunocompetent rats is characterized by the following two findings: 1. The spectrum of tumors recorded is narrow, since kidney and brain tumors are nearly exclusively found (5, 6, 7). 2. There exists a critical time of tumor induction, since inoculation of animals within 4 days after birth is a prerequisite for tumor formation (15, 17). The spectrum of tumors and the length of the tumor induction period can be enlarged by the use of immunosuppressed animals (e. g. thymectomized rats or animals treated with cyclosporin A). In addition, the same is true for intrafetal application of the virus (10,12,13,14,15,16,17). At present, it is unknown why the kidneys and the brain are the predilection sites for polyoma virus induced rat tumors. One possible explanation is to assume that these

328

W. Verhagen, G. Stauch, E. Mohtaschem, and H. Choritz

organs have a higher content of viral receptors than do non-target organs, resulting in the presence of a higher concentration of virus within the critical time of tumor induction in these target organs. Therefore, the experiments described in this paper were designed in order to examine the time course of distribution of polyoma virus in various organs. For this purpose, newborn Wistar rats were inoculated with tritium labelled polyoma virus and the concentration of virus was determined in the various organs at graded time intervals. Since polyoma virus does not measurably replicate in newborn rats (1), the levels of tritium found in the organs can be considered to reflect the concentration of virus present. It was found in control experiments that tritium labelled virus did not differ in oncogenicity and antigenicity from unlabelled virus.

Material and Methods Analytical grade chemicals were used throughout.

Virus The polyoma virus strain SE was used. The virus was propagated in secondary mouse cell cultures as described by Desselberger et al. (4).

Tritium labelling of polyoma virus The first steps of virus propagation were identical as described by Desselberger et al. (4). After 24 h the infected cells were washed with phosphate free hepes buffered basal medium Eagle (BME) with Earle's salts (Seromed, Munich) in order to remove the phosphate. The cells were then incubated with this medium supplied with 2 % heat inactivated (30' 56°C) donor calf serum. 100 IV penicillin and 100 ~g streptomycin per ml were also added to avoid bacterial contamination. After two hours, tritium labelled nucleotide triphosphates (NEN, Dreieich, Germany) were added in equal amounts yielding a total activity of 20 ~Ci/ml. Cells were incubated for 12 days. After freezing of cells at -26°C, the virus was harvested.

Harvest of virus After thawing of the cells the labelled and the unlabelled viruses were harvested according to Desselberger et al. (4). In brief: Cells and cellular debris were sedimented for one hour at 4000 rpm in a 3350 rotor in a Heraeus Minifuge II at 4°C. The pellet was redissolved in one-tenth of the initial volume receptor destroying enzyme (cholera filtrate, Philips Duphar, NL) and incubated at 3JOC in a water bath for 18 h. Subsequently, the cell debris was pelleted by centrifugation. The virus containing supernatant was further purifed by one cycle of differential centrifugation, in a 60 Ti Rotor (Beckman) at 35,000 rpm and 4°C during 2 h. The resuspended virus was stored at -26°C in 2 ml ponions.

CsCI-density gradient centrifugation In order to test the degree of tritium labelling of the polyoma virus, 1.7 ml of a cold saturated CsCI solution was added to 3.4 ml of the virus suspension. After mixing (initial density 1.31 g/cm l ) the virus was banded by centrifugation in a Beckman SW 50.1 rotor during 20 h at 4°C and 37,000 rpm (159,000 g).

Distribution of Polyoma Virus

329

ctJm X 10' HA

X

10' 100 - - - - Hemagglutinin - - - - Radioactivlty['H] ---tJuoyanl densltylglml]

Fig. 1. CsCI - density gradient of tritium labelled polyoma virus. Hemagglutinin (.-.-.-.-.-.-.-.-.) Radioactivity (- - - - - - - - -) Buoyant density ( ) For unlabelled normally grown polyoma virus an identical hemagglutinin pattern was obtained. Fractionation was done with a fractionator developed in our institute to give a constant drop volume. Fractions of 6 droplets were made and investigated for their hemagglutinin and tritium content. Hemagglutination- and hemagglutination inhibition test Tests were carried out as described by Desselberger et al. (4). Animal experiments Pregnant Wistar rats were purchased from the Zentralinstitut fur Versuchstierkunde, Hannover, and held under standard conditions.

0.49

± Standard deviation

0.54+

453.4 + 414.6

0.40+

332.5 +

7.0 + + 1.8 a 27.8 + + 66.7 + 402.9 172.6 + 48.4 + + 20.2 49.5 + + 18.6 9.5 + + 10.3 13.8 + + 65.6 d 3.9 + n.d.

2.9 51.9 270.8 33.3 17.2 15.0 62.3

0.11

92.8

6.8 14.8 82.1 31.7 21.7 6.1 9.1 3.6

:+

+ + + + + + + 4.5 4.7 76.0 14.6 16.0 3.2 19.6 3.0

0.78+ 0.10

654.7+81.8

11.6 38.5 397.7 72.2 84.5 15.9 30.7 3.6

29.8 + 9.6 274.0 + 36.6 679.6 + 253.8 240.4 + 91.6 362.0 + 55.8 86.6 -.; 28.6 87.0 + 62.4 1.3+ 2.5

C

26.4 + 32.4 291.8 e"± 49.8 455.4 + 170.0 256.2 + 73.4 402.4 + 31.6 ~ + 17 .6 167.6 + 93.2 2Q.6+ 25.1

19.8 229.4 446.0 159.4 349.6 59.2 88.8 2.7

40 days

+ 12.0 2.6 + 3.0 + 53.4 221.6 + 78.8 + 191.0 92.6 + 54.0 + 28.4 26.6 :; 10.2 + 34.2 143.0 + 38.6 + 10.2 47.8 + 27.2 + 53.6 27.6 + 36.2 :; 4.6 n-:-d. d

192 hrs

1.42+ 0.37

2.09+ 0.35

2.02+

0.26

1.61+

0.25

0.67± 0.13

1194.8 + 314.2 1760.7 + 286.5 1702.0 + 220.1 1374.9 + 210.8 56 1 • 8 ± 1 13 • 0

16.3 + 7.3 167.4 + 76.9 567.4 + 285.0 206.9 + 86.6 144.2 + 44.7 26.4 + 7.7 43.5 + 22.0 22.7+ 38.3

Counts (dpm) per organ found at different time (hours) after inoculation 24 hrs 48 hrs 96 hrs 6 hrs 12 hrs 2 hrs

b Standard deviation of the sum of counts was obtained by calculation of VL(s.d.)2 (Kleczkowski,9). Calculated by multiplication with reciprocal value of efficiency of counting (0.36) and 42.7 (1 cpm = 42.7 TCID so ) n.d. = not determined e Maximal values were indicated by underlining

a

TCID~O (xl0 )

equivalen c

Sum b

Thymus Lungs Liver Spleen Kidneys Parotis Brain Blood

Organ

Table 1. Distribution of tritium labelled polyomavirus in newborn Wistar rats as measured by counting the radioactivity in the organs.

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Distribution of Polyoma Virus

331

Newborn rats were inoculated subcutaneously (dorsal route) within 24 h after birth with 0.3 ml virus suspension using a tuberculin syringe (Brunswick, 501-LG) equipped with a thin needle (Acufirm, LL 23) to avoid reflux of the inoculated material and hemorrhages. For the investigation of the distribution of the labelled virus groups of at least 7 animals per time interval were killed at graded time intervals after inoculation and dissected as described below. The animals were observed daily and the number of living animals was recorded. Animals which showed paralysis and other signs of neurological damage were killed with ether. Prior to autopsy, blood of the animals was obtained by cardiac puncture. Routinely, brain, parotis, heart, lungs, thymus, spleen, liver and kidneys were taken. Obvious macro-pathological changes of the organs were recorded. Subsequently, the organs were fixated in 4 % phosphate buffered formaldehyde for histologic examination and measurement of tritium content. At day 40 the experiment was terminated. Surviving animals were sacrified as described above.

Measurement of tritium content The amount of labelling of the virus within the gradient was measured as follows: To 10 !-tl of each fraction 500 !-tl Soluene 350 (Packard) was added. After 30 min incubation at room temperature, 20 ml of a counting mixture containing 0.3 g POPOP and 5 g PPO per liter toluene (Merck) was added. Counting took place in a liquid scintillation counter, type A 2425 (Packard). The polyoma virus, purified by differential centrifugation and used for the animal experiments was measured for its tritium content, in like manner. The tritium content of the organs was assayed as follows: After fixation with formaldehyde, the organs were divided into two equal parts. One part was taken for the histological examination and the other part was placed in Eppendorf reaction vessels and dehydrated by means of a L2 freeze drier equipment (WKF). After drying the samples were placed in combustion cones (# 506 5913, Packard) and weighed on a Sartorius P 2004 MP balance. The samples were then combusted in a Sample Oxydizer 306 (Packard) to CO 2 and tritiated H 2 0. The combustion chamber was then rinsed with lumagel (Baker Chemicals) and the mixture was pumped into counting vials and counted. The counting efficiency was 50 % for the aqueous samples and 36 % for the combusted samples.

Histology 3-4 !-tm thick sections of the organs were prepared with a Jung microtome. After staining with hematoxylin - eosin, the sections were observed with a Leitz Ortholux microscope equipped with a Orthomat unit for micrographs.

Results

Comparison of labelled and unlabelled polyoma virus The yields of labelled and unlabelled virus expressed in terms of infectivity and hemagglutination titers were found not to differ measurably (data not shown). The specificity of labelling of virus was tested by measuring the HA and tritium content after CsCI-gradient centrifugation.

0.07

0.04

0.03

0.14

n.d. a

Spleen

Kidneys

Parotis

Brain

Blood

0.03

0.15

0.15

0.52

0.08

0.01

0.05

0.02

0.13

0.11

0.61

0.1)6

0.02

12

0.02

0.04

0.02

0.12

0.17

0.47

0.14

0.01

24

0.001

0.05

0.05

0.21

0.14

0.39

0.16

0.02

48

a

0.76

0.32

2.53

6.59

14.87

2.30

4.93

2.71

n.d. a

Lungs

Liver

Spleen

Kidneys

Parotis

Brain

Blood

4.06

6.14

18.8

3.86

1.54

4.09

2.07

Thymus

6

2

Organ

Not determined

0.01

0.10

0.05

0.24

0.15

0.27

0.17

0.02

96

0.89

1.44

2.37

0.81

5.29

5.11

1.45

2.37

1.77 0.08

2.37 8.52

11.51

1.29 0.81

n.d.

0.28

0.72

8.12 11.28

5.73

0.64

6.81 13.43

0.47

2.87

0.51

960

n.d.

0.05

0.09

0.25

0.05

0.16

0.39

0.005

960

2.92

3.59 4.03

3.49

4.46

192

5.12

96

0.002

0.07

0.04

0.26

0.12

0.33

0.17

0.01

192

14.53

26.65

22.32 6.83

6.32

6.25

7.06

48

5.13

4.75

5.54

24

6.50

16.45

5.02

1.55

4.36

12

Counts (dpm) per mg organ dry weight at indicated time (hours) after inoculation

Table 3. Distribution of tritium labelled polyomavirus in newborn Wistar rats expressed in terms of counts per mg organ dry weight

Not determined

0.01

0.60

Liver

a

0.04

0.11

Lungs

0.02

0.01

Thymus

6

2

Organ

Counts (dpm) found per organ/sum of counts per organs examined at indicated times (hours) after inoculation

Table 2. Distribution of tritium labelled polyomavirus in newborn Wistar rats expressed in terms of fractions of total radioactivity refound

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Distribution of Polyoma Virus

333

As can be seen from Fig. 1 the tritium peak coincided with the peak of the hemagglutination acticity of the full particles with a buoyant density of 1.3190 g/cm 3 , indicating that the DNA of polyoma virus has been labelled. The virus was further purified by ultracentrifugation during 2 h at 35,000 rpm and 4°C in a Beckman 60 Ti rotor and L 5-65 Beckman ultracentrifuge under sterile conditions. The unlabelled virus was centrifuged likewise. The hemagglutinin content, infectivity titer and labelling were measured. Again, no significant differences in the titers of labelled and unlabelled virus were recorded. The oncogenicity of labelled and unlabelled polyoma virus was tested comparatively and it was found that the ratios of tumors of the brain (hemangiomas) and kidneys (fibrosarcomas) induced by the labelled and unlabelled polyoma virus did not differ significantly (P < 0.05) (data not shown). Furthermore, the HI antibody response to labelled and unlabelled virus did not differ significantly (P < 0.05). Therefore, the conclusion was reached that the labelling with tritium of the DNA of polyoma virus did not measurably influence its oncogenic and immunogenic potency. Distribution of tritium labelled polyoma virus in the organs Newborn Wistar rats were inoculated with 0.75 x 10 6 TCID 50 (= 17547 3H counts) per animal and the tritium content in various organs and in the blood was recorded at graded time intervals up to 40 days after inoculation. As controls, a group of rats was treated in like manner, using unlabelled virus. The 3H counts per organ found are listed in Table 1. Note that the sum of counts refound in the organs tested reached a peak within 48 to 96 h after inoculation and amounted maximally to 27.9 per cent of the radioactivity inoculated per animal. This finding suggests that adsorption of virus onto the organs was completed within 48 to 96 h after inoculation and declined thereafter. Furthermore, the conclusion is reached that the vast majority of the virus inoculated was not refound in the organs tested. The rest of the virus was possibly eliminated and or remained at the injection site or in organs not examined. However, the organs tested represented roughly 12 per cent of the total body weight and contained maximally 27.9 per cent of the radioactivity inoculated. This finding warrants the conclusion that the other organs and tissues contained on an average less virus than did the organs examined. In the various organs tested and in the blood, the peaks of tritium refound were also recorded 48 to 96 h after inoculation. Thereafter, the tritium levels declined with the exception of that of the lungs where relatively high levels were still recorded 40 days after inoculation. Table 2 gives the fraction of tritium found per organ relative to the sum of tritium found in all organs tested at the indicated time intervals. Note that the majority of tritium refound (60 to 47 per cent) was recorded in the liver between 2 and 24 h after inoculation and that the values declined thereafter. By contrast, relatively low values were found in the kidneys until 24 h after inoculation and relatively high levels thereafter. The fraction of virus refound in the blood were much smaller than the respective values found in most organs (e. g. liver, kidneys, spleen, lungs), indicating the virus was adsorbed in these organs. 22 Zhl. Bakt. Hyg. A 258/2-3

334

W.Verhagen, G.Stauch, E.Mohtaschem, and H.Choritz

The relative portion of tritium found in the lungs reached a peak level 40 days after inoculation. Relatively constant values were recorded for the rest of the organs examined. Table 3 gives the ratios of tritium per mg dry weight of the organs. It is obvious that the spleen showed the highest values followed by the kidneys and the parotis. The highest value in the liver was recorded 2 h after inoculation and a second peak 48 hours after inoculation. 13 kidney tumors obtained 40 days after inoculation of animals were tested for tritium content and an average value of 0.41 counts per mg dry weight of tumor was found. By contrast, tumor-free portions of the kidneys contained on an average 4.33 counts per mg. Assuming that the counts found in the tumors represented the virus that induced the tumors, the corresponding virus concentration is estimated to be 48.7 TCID so . In the organs of the control animals inoculated with unlabelled virus, no tritium was detected.

Discussion The experiments described in this paper were designed in order to examine whether the distribution of polyoma virus in the organs of neonatally inoculated rats could explain the finding that in immunocompetent animals tumors appear in the kidneys and in the brain, only. The determination of the virus concentration in the organs was carried out on the basis of measuring their tritium content. Therefore, it may be questioned whether the ratio of tritium to virus was constant in the organs compared. Obviously, replication of virus or its disintegration at different rates in the organs could yield erroneous results. Replication of polyoma virus in rats can be excluded (1). On the other hand, the following results suggest that disintegration of virus could occur in the liver more rapidly than in other organs: Campbell et al. (3) examined the spread of Mengo virus in mice and found that large amounts of infectious virus could be recovered from most organs with the exception of the liver. Brunner et al. (2) found in mice inoculated with labelled VSV and NDV and Jahrling and Gorelkin (8) in hamsters inoculated with labelled VEE virus that the vast majority of labelled virus was refound in the liver. These findings suggest that virus is degraded in the liver more rapidly than in other organs. If this were true for polyoma virus, this would not affect the comparison between the target organs (kidneys and brain) and the non-target organs with the exception of the liver. Furthermore, it should be recalled that it was found that labelling did not measurably influence the oncogenicity of polyoma virus. When comparing the tritium content of the various organs examined, no differences in favour of the target organs of tumor formation were found: The highest virus concentration per mg were found in the spleen and the highest virus concentration per organ in the liver. This finding indicates that the spread of virus in the organs alone cannot explain that the kidneys and the brain are the predilection sites for tumor induction. The existence of target organs could be explained by assuming that they are the only organs possessing cells which can be successfully transformed by polyoma virus. An-

Distribution of Polyoma Virus

335

other explanation would be to assume that the immunologic surveillance operates more effectively in the non-target than in the target organs. The finding that the spectrum of tumors can be enlarged when inoculating immunosuppressed rats with polyoma virus could support this interpretation (14, 15, 16, 17). Since polyoma virus suspensions contain empry bodies and pseudoviria in addition to the full bodies (11), it could be further assumed that the full bodies are the only transforming particles and are selectively stored in the target organs. Experiments to elucidate these questions are in progress. Acknowledgements. The authors thank Prof. Dr. J. Drescher for the helpful discussion and gratefully acknowledge the competent help of Mrs. C. Ellguth and Mrs. B. Thierkopfin preparing this manuscript.

References

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336

W. Verhagen, G. Stauch, E. Mohtaschem, and H. Choritz

14. Stauch, G., H. Ostertag, and A. Georgii: Tumorinduktion bei Feten im Uterus von Ratten durch Polyomavirus. Verh. Dtsch. Ges. Path. 63 (1979) 527 15. Stauch, G., G. Schmidt und A. Georgii: Unterschiede des Tumorspektrums durch H-SE Polyomavirus in Abhangigkeit von der perinatalen Entwicklung bei der Ratte. Verh. Dtsch. Ges. Path. 65 (1981) 321-324 16. Stauch, G., A. Nagel, W. Verhagen, and A. Georgii: Influence of cyclosporin A® on tumor induction in the Wistar rat after application of polyoma virus. Scientific Proceedings Second Symposion of the Section of Experimental Cancer Research (SEK) of the German Cancer Society. ]. Cancer Res. Chn. Oneal. 105 (1983) A 49 17. Vanderputte, M., P. Denys, R. Leyten, and P. de Somer: The oncogenic activity of the polyoma virus in thymectomized rats. Life Sci. 2 (1963) 475--478

Dr. Willem Verhagen, Institut fur Virologie und Seuchenhygiene der Medizinischen Hochschule, Konstanty-Gutschow-Str. 8, D-3000 Hannover 61