Journal of Medical Virology 8:143-150 (1981)

The Persistence of Papovavirus BK DNA Sequences in Normal Human Renal Tissue John Heritage, Peter M. Chesters, and Dennis J. McCance

Department of Microbiology, Guy’s Hospital Medical School, London Bridge

Evidence has accumulated indicating that BK virus, following an inapparent primary infection, persists in the renal organs of normal healthy individuals and reactivates upon immunosuppression. Data to support this hypothesis are presented and suggest that BK virus DNA sequences are present at very low levels in the kidneys of more than 50% of the population and that this persistence is localized in several foci within these organs.

Key words: BK DNA, hybridization, normal renal tissue

INTRODUCTION

BK virus (BKV) was first isolated from the urine of a patient who had received a kidney and ureter transplant three months previously [Gardner et al, 19711. Subsequent serological studies showed that BKV was distributed world wide and that antibodies to BKV may be found in nearly 80% of the adult pop- ulation [Brown et al, 1975; Gardner, 1973; Mantyjarvi et al, 1973; Portolani et al, 1974; Rziah et al, 1978a; Shah et al, 19731. Seroconversion occurs during childhood, although no clinical condition has been identified in association with the primary infection. Further studies on renal transplant recipients suggested that over one third had evidence of BKV excretion in the urine during im- munosuppression [Coleman et al, 1973; Kreck et al, 1975; Lecatsas et al, 19731. Reactivation of BKV and detection in urine is also observed in patients whose immune response is impaired by congenital defects, lymphomas, leukemias, and other debilitating diseases [Flower et al, 1977; Reese et al, 1975; Rziah et al, 1978b; Taguchi et al, 19791. The evidence suggested that after primary in- fection BKV persists at very low levels in the renal organs and could reactivate and be detected in urine after immunosuppression of some patients. This has been found with other papovaviruses of animals (D.J. McCance, unpublished

Accepted for publication June 17, 1981. Address reprint requests to Dennis J . McCance, Department of Microbiology, Guy’s Hospital Medical School, London Bridge, SE1 9RT, England.

0146-6615/81/0802-0143SO2.50 0 1981 Alan R. Liss, Inc.

144 Heritage, Chesters, and McCance

data). The low level of persistence is shown by the fact that BKV has never been isolated from normal, healthy people, although it has once been isolated from the urine of a pregnant woman [Coleman et al, 19801.

To gain evidence for the persistence of BKV in renal tissue, its presence in healthy kidneys removed from cadavers has been investigated by DNA-DNA hybridization techniques using 32P-labelled BKV DNA as a probe and, when fresh post mortem tissue has been available, by tissue explants from kidneys.

MATERIALS AND METHODS

Source of Organs

others aged between 55 years and 89 years, none of whom had died of renal disease. Table I gives sex, age, and cause of death of subjects. Tissue was frozen in small pieces over liquid nitrogen and stored at *20°C until used. The time interval between death and storage of the kidney tissue varied from 3 hours to 48 hours.

Kidneys were removed from fourteen subjects, one a child nine days old, the

DNA Extractions DNA was extracted from kidneys using a modification of the method of

Gross-Bellard et al [ 19731. Tissue (0.5 gm) was cut up into small pieces (be- tween 11-30 such pieces per kidney were extracted) and homogenized in 7 ml of buffer A (10 mM Tris/HCl pH 8, IOmM EDTA, 10 mM NaCI, 0.5% W N SDS) with 50 pg/ml proteinase K (fungal: BDH). The resulting homogenate was incubated for 16-24 hours at 37"C, after which it was extracted twice

TABLE 1. Age, Sex, and Cause of Death of Subjects

Cadaver Presence of BKV No. Cause of death Sex Age (years) DNA sequences

~~ ~~~ ~ ~~

1 Coronary thrombosis F 80 + 2 Chronic bronchitis M 69 +

and emphysema Pulmonary embolism M 66 +

4 Chronic bronchitis M 64

5 Coronary thrombosis M 60 7 Myocardial infarction M 62 8 Carcinoma of the M 55 +

9 Aortic Aneurism M 70 1Oa Congenital heart F 9 days

11 Aortic stenosis M 89 + 12 Suffocation M 60 +

158 Cerebral haemorrhage M 60 + 16 Coronary thrombosis M 58 +

-

and emphysema - -

pancreas - -

disease

13 Coronary thrombosis M 59 -

aCadavers from whom both kidneys were obtained.

BK DNA in Normal Human Renal Tissue 145

with 1 volume of phenol saturated With buffer B (500 mM TrislHCl pH 8, 10 mM EDTA, 10 mM NaCl, 0.5% W N SDS). The aqueous phase on each oc- casion was separated by centrifugation at 1,OOOg for 15 minutes at 4"C, and the aqueous phase was then dialysed at room temperature against buffer C (50 mM Tris /HCl pH 8, 10 mM EDTA, 10 mM NaCI) until the absorbance outside the dialysis bags, measured at 270 nm, was less than 0.05 OD units. The solution inside the bag was incubated with 20 p g h l pancreatic ribonuclease (Sigma) for 1 hour at room temperature, followed by 100 pg/ml proteinase K for 2 hours at 37°C after addition of SDS to 0.5%. This was then extracted with 1 volume of phenol (50 parts), chloroform (50 parts), iso-amyl alcohol (1 part) and 8 hydroxyquinoline lgm/liter, saturated with 10 mM Tris/HCl pH 8. After separating the aqueous phase as above, it was dialysed once again against buffer C at room temperature until the abosrbance was less than 0.05 units outside the bag. The high molecular weight DNA was then precipitated with 0.1 volume of sodium acetate (20% W/V) and 1 volume of isopropanol. The precipitate was washed in 75% ethanol, 0.5% W/V sodium acetate and dissolved in TE (10 mM Tris/HCl pH 7.5, 1 mM EDTA). DNA samples were stored at -20°C until used.

Restricted of DNA and Agarose Gel Electrophoresis

solved in the appropriate restriction buffer. Restriction of DNA was achieved using the enzymes Eco RI, Bam HI, and Hpa I1 (Boehringer Mannheim) for 2 hours at 37°C. The reaction was stopped with EDTA and DNA fragments pre- cipitated at -70°C with 0.1 volume of sodium acetate and 2.5 volumes of ethanol. Ten-microgram samples of uncut DNA (not treated with enzymes) were similarly precipitated and all precipitates were dissolved in 10 pl of load- ing buffer (10 mM Tris/HCl pH 7.5, 1 mM EDTA, 50% W N sucrose). Samples were run on a horizontal 0.8% neutral agarose gel (40 mM TrisMC1 pH 7.5, 5 mM sodium acetate, 1 mM EDTA), after which the DNA was depurinated [Wahl et al, 19793 and transferred to nitrocellulose filters by the Southern blot technique [Southern , 19751.

Ten-microgram samples of DNA were precipitated as above and redis-

DNA-DNA Hybridization

washed for 2 hours at 68°C in 6 x SSC (1 x SSC is 0.15 M sodium chloride and 0.15 M sodium citrate), 0.2% ficoll, 0.2% polyvinylpyrrolidone, and 0.2% bovine serum albumin. The filters were then transferred to the hybridization solution which contained 6 x SSC, 0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, 400 pg ml single-stranded salmon sperm DNA, 0.1% SDS, and the nick translated 32P-labelled BKV DNA [Rigby et al, 19771 (specific activity, 2x 108 cpm/pg of DNA) and hybridized at 68°C for 48 hours. The BKV DNA used for nick translation was cloned at the Bam H1 site into the plasmid pBR322 and was a gift of Dr. P.M. Howley, NIH. After 48 hours, the filters were washed six times at room temperature and once at 68°C in 0.3% SSC and 0.1% SDS and then exposed to Fuji X-ray film for 7 days.

The nitrocellulose filters were baked at 80°C under vacuum and then

146 Heritage, Chesters, and McCance

Viral DNA For nick translation the pBR322 cloned BKV DNA was used. Recon-

structions contained either the cloned BKV DNA or form I BKV DNA purified by the method of Seif et al [1979].

Explant Tissue Culture and lmmunofluorescent Staining for Viral Antigens Tissue was minced finely and pieces of approximately 1 mm3 were placed

on coverslips in a multiwell tissue culture plate. The pieces were covered with growth medium comprising Eagle’s MEM (Wellcome Research Laboratories) with 15% foetal calf serum (Gibco, Europe). Explants were placed in 5% COP at 37°C and the medium changed daily until cells were observed to be growing out, and thereafter twice weekly.

Cells were fixed in acetone for 10 minutes, and then treated with mouse antiserum to BKV for 15 minutes at 37°C. After washing twice for 10 minutes each in phosphate-buffered saline (PBSA), fluorescein isothiocyanate- conjugated goat anti-mouse serum was applied and cells incubated at 37°C for 15 minutes. This was removed with two 10-minute washes of PBSA, and slides were mounted in 90% glycerol in PBSA.

RESULTS

Hybridization of BKV DNA With DNA Extracted From Kidneys

molecule found in the BKV virion, form I1 is a circular form produced by a nick in one strand of the DNA, and finally form I11 which is a linear molecule produced by a cut in both strands of the DNA. The restriction enzyme Eco R1 cuts the BKV genome at one site producing a linear molecule (form 111, fig. 1, lane a). Restriction of pBR322 cloned BKV genome with Bam H1 also produces a linear molecule (fig. 1, lane 8).

Investigations of uncut or Eco-RJ-cut kidney DNA has detected BKV DNA sequences in kidneys removed from eight of the fourteen cadavers tested (Table 1). Not all samples from each kidney contained viral DNA sequences. Any inconsistencies in the technique were largely excluded since each sample was tested at least three times before being considered negative for the pres- ence of BKV DNA sequences. The kidney from cadaver 1 yields BKV DNA sequences in 46% of the 30 samples tested (Fig. 1, lanes e and f), while se- quences were found in slightly more than half of the l l samples extracted from cadaver 8, and about 90% of the 26 samples extracted from cadaver 16 have been found to contain BKV DNA sequences (Fig. 1, lanes b-d). BKV DNA sequences have also been detected in 5-10% of 64 DNA samples extracted from the kidneys of cadavers 2, 3, 11, 12, and 15. Both kidneys from cadaver 3 contained BKV DNA. Where it was possible to separate cortex and medulla, both were tested separately, and in tissue taken from cadaver 11 BKV DNA has been detected in both regions at approximately the same frequency. All samples contained BKV DNA in a range of < 1 to 5 genome equivalents per cell as judged by the comparison of band intensities between the reconstruction and test samples. Figure 1 shows representative samples of BKV DNA in the kidneys of cadavers 1 and 16, where the levels were <1 (lanes b and c) and

BKV DNA can have three configurations, form I is a supercoiled DNA

BK DNA in Normal Human Renal Tissue 147

Fig. 1. Examples of BKV DNA on nitrocellulose filters from kidneys of two cadavers, 1 and 16. Lane a is a reconstruction where 10 pg of high molecular weight kidney DNA from cadaver 4 (negative for presence of BKV genome) and 50 pg BKV DNA were restricted with the enzyme Eco R1 (approximately 5 genome equivalentskidney DNA). Lanes b-d contain Eco-RI-restricted high molecular weight DNA from three pieces of kidney from cadaver 16, and lanes e and f con- tained Eco-R1-restricted high molecular weight kidney DNA from two pieces of kidney from cadaver 1. Lane g contains a marker of linear BKV DNA produced by restnction of the plasmid pBR 322 containing BKV DNA with Bam HI.

approximately 5 (lanes e and f) genome equivalents. Two other restriction en- zymes known to cut BKV DNA at one site and produce a linear molecule were used and Figure 2 shows high molecule weight DNA from a piece of kidney from cadaver 16 cut with Bam H1 and Hpa I1 in addition to Eco R1 (Fig. 2e, f, and d). The extra bands in the reconstructions in Figure 2, lanes b and c were due to incomplete restrictions of BKV DNA producing a mixture of form I (bottom band), form 111 (top band), and the plasmid pBR322 the middle band in lane (c). The BKV DNA sequences detected in the uncut DNA samples ran on the gel to the same distance as BKV marker DNA indicating that viral DNA does not appear to be integrated into the host DNA (data not shown). Host DNA was also extracted from brain tissue of these cadavers by the same method, but no BKV DNA sequences were found in any of the DNA samples taken either from white or grey matter.

1 and 5. Over a period of time up to 47 days, cells growing out from the explants were stained for the presence of BKV antigens. Cultures from both cadaver kidneys were negative even though the kidney of cadaver 1 contained BKV DNA sequences by DNA-DNA hybridization tests.

Explant cultures were successfully set up from only two cadaver kidneys,

148 Heritage, Chesters, and McCance

Fig. 2. Examples of BKV DNA on nitrocellulose filters from the kidney of cadaver 16. Lane a is a marker of linear BKV DNA produced by restriction of prototype BKV DNA with Eco RI. Lanes b and c contain reconstructions where 10 pg of high molecular weight kidney DNA from cadaver 4 (negative for presence of BKV genome) and 50 pg of BKV DNA (b) and pBR322-cloned BKV DNA (c) were restricted with enzymes Hpa I I (b) and Bam HI (c). Lanes d-f contain Eco Rf , Barn HI, and Hpa If (respectively) restriction of high molecular weight DNA from a piece of kidney from cadaver 16.

DISCUSSION

BKV is a ubiquitous virus and approximately 80% of the population in England have antibodies to this virus [Gardner, 19771. This virus is thought to persist in renal tissue in an unknown percentage of seropositive people as sug- gested by its reactivation and detection in the urine of immunosuppressed pa- tients. To investigate its persistence in renal tissue, its presence has been sought in healthy organs by DNA-DNA hybridization techniques. This method permitted the study of many more organs and a larger portion of each kidney than would be possible using techniques such as explant cultures. Also this technique is very sensitive detecting papovavirus DNA at levels of 0.1 genome equivalents per cell (R. Kamen, personal communication), and usable quantities of DNA may be extracted from kidneys up to 48 hours post mortem (J. Heri- tage and P.M. Chesters, unpublished data).

Using DNA-DNA hybridization, 57% of the cadavers in this study (8 of 14) harboured BKV genomes in their kidneys. Serological studies have indi-

BK DNA in Normal Human Renal Tissue 149

cated that BKV persists in 20% of normal healthy people as measured by the presence of BKV-specific IgM antibody [Flower et al, 1977; D.J. McCance, unpublished data]. In one of these studies [Flower et al, 19771, IgM antibody against BKV was detectable for at least three years. Our results suggest that within a kidney the virus persists in small foci distributed throughout the cortex and medullary regions of the kidney. BKV DNA does not appear to be inte- grated into host kidney DNA as free BKV DNA was detected in uncut sam- ples. At the moment, it is not known if the BKV DNA detected is the same as the prototype BKV isolated by Gardner et a1 [ 197 I].

In situ hybridization is in progress on kidney sections to try and detect which cells harbour the virus and in what quantities. Further studies are being carried out using molecular hybridization techniques to look for the presence of BKV DNA sequences in ureters of normal individuals since BKV particles have been detected and associated with ureteric stenosis in renal transplant pa- tients [Gardner et d , 1971; Coleman et al, 19783.

ACKNOWLEDGMENTS

We would like to acknowledge the generous gift from Dr. P.M. Howley (NIH, Bethesda) of pBR 322-cloned BKV genome and the help given by Pro- fessor A.K. Mant and Dr. K.A.P. Lee (Guy’s Hospital) in obtaining tissue from cadavers and the cause of death.

This work was done with the aid of a Medical Research Council Grant No. G979/108/5.

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