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Detection of parvovirus DNA in human serum using biotinylated RNA hybridisation probes
Journal of Virological Methods, 19 (1988) 279-288 Elsevier
279
JVM 00700
Detection of parvovirus DNA in human serum using biotinylated RNA hybridisation probes D .A. Cunningham ‘, J.R. Pattison
and R.K. Craig1
‘Medical Molecular Biology Unit, Department of Biochemistry, University College and Middlesex School of Medicine, London, U.K.; =Department of Medical Microbiology, School of Clinical Sciences, University College and Middlesex School of Medicine, London, U.K. (Accepted
21 December
1987)
Summary Methods were established for the detection of parvovirus DNA in human serum using single-stranded RNA probes. The sensitivity of detection of virus using 32Pradiolabelled RNA versus non-radiolabelled biotinylated RNA probes using a streptavidin-polyalkaline phosphatase detection system was compared. Virus was detected using 32P-labelled and biotinylated RNA probes at serum dilutions of 10m3, equivalent to approx. 3 pg of viral DNA. Using biotinylated RNA probes and a dot-blot system, diagnosis of numerous serum samples could be performed within 8 h of receipt of samples, using an RNA probe which was synthesised and stored at -20°C for up to 12 months without loss of sensitivity. Our work demonstrates the potential of biotinylated RNA probes in the routine analysis of viral sequences in serum. Parvovirus; detection
Nucleic
acid hybridisation;
Human
serum;
Biotinylated
RNA;
Rapid
Introduction The human parvovirus, originally found in human serum by Cossart et al. in 1975, is a single-stranded DNA virus which packages approximately equal numbers of positive or negative DNA strands (Summers et al., 1983). It grows in rapidly di-
Correspondence to: Dr. D.A. Cunningham, Medical Molecular Biology Unit, Department of Biochemistry, University College and Middlesex School of Medicine, The Windeyer Building, Cleveland Street, London WlP 6DB, U.K. 0166-0934/88/$03.50
0
1988 Elsevier
Science
Publishers
B.V.
(Biomedical
Division)
280
viding cells (Anderson and Pattison, 1984) and has been associated with febrile illness (Shneerson et al, 1980), the aplastic crisis of sickle cell disease and hereditary spherocytosis (Serjeant et al., 1981; Kelleher et al., 1983), and erythema infectiosum (fifth disease) (Anderson et al., 1983). Human parvovirus has been shown to cross the placenta, eliciting an immune response in the fetus. Animal parvoviruses induce fetal abnormalities and there is some evidence that human parvovirus may harm the fetus (Knott et al., 1984; Mortimer et al., 1985). As human parvovirus cannot be grown in tissue culture, methods of detection rely on counterimmune electrophoresis, electron microscopy or, more commonly, radioimmunoassay (Cohen et al., 1983) or nucleic acid hybridisation using a cloned DNA probe (Anderson et al., 1985; Clewley, 1984). Dot hybridisation assays using s2P-radiolabelled hybridisation probes which are rapid and sensitive have been described previously (Anderson et al., 1985; Clewley, 1984). However, these probes have several disadvantages for routine laboratory diagnosis. The isotope presents a safety hazard, is relatively expensive, and the probe has a short shelf-life (t = 14.3 days). Potentially such problems may be overcome using non-radioactive modifying groups which, after introduction into nucleic acid are stable, and can be detected using sensitive enzyme based assays. The most commonly used modifying group is the vitamin biotin, which can be introduced into nucleic acid enzymically, using nucleotide derivatives (Langer et al., 1981). The affinity of biotin (Kdiss = 10-i”) for the protein avidin (from eggwhite) or streptavidin (from Streptomyces avidinii) is then exploited in the detection of biotinylated probes (Leary et al., 1983). The avidin group can be complexed to enzymes, which produce a specific calorimetric reaction on incubation with dye solutions. We describe here a method for the detection of parvovirus in serum using a biotin based non-radioactive detection system, and compare the speed and sensitivity of the method with “‘P-radiolabelled hybridisation systems in current use.
Materials
and Methods
Materials [a-“*P]CTP (800 C’/1 mmol) was obtained from New England Nuclear (Boston, MA, U.S.A.). Allylamine-UTP, caproylamido-biotin-N-hydroxysuccinimide ester (CAB-NHS) and polyalkaline phosphatase were purchased from Bethesda Research Laboratories (Paisley, Scotland). Streptavidin was purchased from Amersham International plc, (Amersham, U.K.). Nitroblue tetrazolium (NBT) and 5bromo-4-chloro-3-indolyl phosphate (BCIP) were from Sigma (Poole, Dorset, U.K.). Restriction endonucleases and T7 RNA polymerase were from Boehringer Corporation Ltd. (Lewes, U.K.). Schleicher and Schuell nitrocellulose filter sheets were from Anderman and Company (Surrey, U.K.) while GeneScreen Plus was from New England Nuclear (Boston MA, U.S.A.). The cloning vector pGEM 1 was from P & S Biochemicals (Liverpool, U.K.). All other materials were from sources described elsewhere (Allison et al., 1981).
281
Preparation of RNA probes The plasmid PVTMI has been described previously (Anderson et al., 1985). The 700 bp parvovirus DNA insert was excised with the restriction enzyme PstI and subcloned into the PstI digested and phosphatased vector PGEMl (Promega Biotee, Madison, WI). Radiolabelled single-stranded RNA probes were prepared from Hind111 linearised plasmid by incorporation of [cx-“‘P]CTP into transcripts from the T7 promoter (Melton et al., 1984). Similarly, biotinylated RNA probes were prepared by incorporation of allylamine-UTP (Langer et al., 1981) into transcripts from the T7 promoter and subsequent chemical biotinylation with the biotin ester caproylamido-biotin-N-hydroxysuccinimide ester (CAB-NHS), according to the manufacturers instructions. Trace radiolabelling with [a-‘SS]GTP was used to quantitate RNA synthesis. Dot hybridisation assays Several modifications were made to the method described by Anderson et al. (1985). Aliquots (5 ~1) of serum, and lo-fold dilutions thereof in 2~ SSC, were spotted onto a dry nitrocellulose filter, allowed to dry and baked for 2 h under vacuum at 80°C. Specimens on the filter were then alkali denatured by floating on 0.1 M NaOH containing 1 M NaCI. Samples were then neutralized by floating on 0.1 M Tris-HCl (pH 7.4) containing 1 M NaCl as described by Mason et al. (1982). Filters were then immersed in 2 x SSC containing 0.1% SDS (w/v) and 400 kg/ml proteinase K, and incubated with shaking for 60 min at 37°C. Proteinase K was then removed by three 5-min rinses in 2~ SSC, prior to prehybridisation. Identification of sequences using biotinylated RNA probes When filters were to be hybridised with biotinylated RNA probes, prehybridisation was for 2 h in 5x SSC containing 50% (v/v) formamide, 1% (w/v) SDS, 1 M NaCl, 10% (w/v) dextran sulphate, and 100 kg/ml single-stranded herring testis DNA. Hybridisation was in the same buffer except that formamide was reduced in concentration to 45% (v/v), and the biotinylated probe was present at a concentration of 200 ngiml of hybridisation buffer. Hybridisation was performed in sealed polypropylene bags with 100 ~1 hybridisation buffer present per cm2 of nitrocellulose, for 2-16 h at 42°C:. After hybridisation, filters were washed in 2~ SSC containing 0.1% (w/v) SDS for 30 min at room temperature with three buffer changes, then in 0.2~ SSC containing 0.1% (w/v) SDS for 30 min at room temperature with three buffer changes, and finally in 0.2~ SSC containing 0.1% (w/v) SDS at 55’C for 45 min with three buffer changes. In order to remove non-specific binding sites and so reduce background when the hybrids were visualized by development with a streptavidin-polyalkaline phosphatase detection system, the filters were blocked in 0.1 M Tris-HCl (pH 7.5) containing 0.15 M NaCl and 3% (w/v) bovine serum albumin, at 65°C for 60 min. Filters were then incubated in 0.1 M Tris-HCl (pH 7.5) containing 0.15 M NaCl and 500 ngiml streptavidin for 10 min at room ‘emperaLure with gentle agitation, then washed in the incubation buffer for 30 min with three buffer changes. The filters were then incubated in 0.1 M TrisHCl (pH 7.5) containing 0.15 M NaCl and 1 pg/ml biotinylated polyalkaline phos-
282
phatase for 10 min with shaking, then washed in this buffer for 30 min with three buffer changes, and finally washed in developing buffer which comprised 0.1 M Tris-HCl (pH 7.5) containing 0.1 M NaCl and 50 mM MgCl, for 10 min all at room temperature. The volume of streptavidin and biotinylated polyalkaline phosphatase applied was 7.5 m1/100cm2 filter. Filter-bound alkaline phosphatase was visualized by application to the filter of a solution (75 pl/cm2) comprising 4.4 ~1 of a freshly prepared stock solution of NBT (75 mg/ml) in 70% (v/v) dimethyl formamide and 3.3 ~1 of a freshly prepared stock solution of BCIP (50 mgiml) in 100% dimethyl formamide in 1 ml of developing buffer. A faint purple colour developed over a period of l-2 h. Identification of sequences using a ‘2P-radiolabelled RNA probe When filters were to be hybridised with a-32P-radiolabelled RNA probes prehybridisation was for 2 h and hybridisation for 2-16 h, at 52°C in 5~ SSCl20 mM sodium phosphate (pH 6.8) containing 60% (v/v) formamide, 5x Denhardt’s, 1% (w/v) SDS, 7% (w/v) dextran sulphate, 100 kg/ml single-stranded herring testis DNA, 100 kg/ml Escherichia .coli tRNA and 10 pgiml poly(A) (Krumlauf et al., 1987). For hybridisation, filters were incubated in the prehybridisation buffer containing 2X lo6 cpm 32P-labelled RNA probe per ml of buffer. After hybridisation, filters were washed in 2~ SSC containing 0.1% (w/v) SDS for 30 min with three buffer changes, then incubated with RNase A (20 kg/ml) in 2x SSC for 30 min, washed in 0.2x SSC containing 0.1% (w/v) SDS for 30 min with three buffer changes, and finally washed in 0.2~ SSC, 0.1% (w/v) SDS at 65°C for 45 min with three buffer changes. All washes were at room temperature unless otherwise stated. The regions of hybridisation were then visualized by autoradiography using Kodak X-Omat S film. Southern blotting Serum samples (5 ~1) were diluted in 2~ SSC to a final volume of 100 ~1, then digested with proteinase K (400 kg/ml) at 37°C for 3 h, and purified by sequential extraction with phenol/chloroform (1: 1)) chloroform, and diethyl ether. After ethanol precipitation each purified specimen was electrophoresed on a 1% (w/v) neutral agarose gel, and blotted onto a GeneScreen Plus membrane using a procedure modified from Southern (1975), as described in the GeneScreen Plus manual. Filters were probed with a single-stranded RNA probe (specific activity 2 x 10s cpm/pg). Hybridisation and wash conditions were as described for dot hybridisation assays.
Results Synthesis of single-stranded RNA hybridisation probes We have recloned the 700 bp PstI parvovirus DNA fragment from pVTM1 into the PstI site of the polylinker of the vector pGEM 1. This allows RNA to be tran-
283
scribed from either + or - viral DNA strand, using the SP6 or T7 promoter (Fig. la). In this instance to generate RNA transcripts, the plasmid (pGEM lpv) was linearized using the restriction enzyme Hind111 (for transcription with T7 RNA polymerase) or BamHI (for transcription with SP6 RNA polymerase), to ensure that a virus specific probe was synthesized (Fig. la). As the virus packages both positive and negative strands in approximately equal amounts, either promoter can be used to generate a single-stranded RNA hybridisation probe. The T7 RNA polymerase was the enzyme of choice, as its transcription efficiency and stability was found to be higher than that of the SP6 polymerase. Agarose gel electrophoresis of restricted plasmid, before and after transcription with T7 RNA polymerase demonstrates the presence of a discrete RNA transcript (Fig. la).
1
2
3
Fig. l(a) Preparation of labelled RNA. Plasmid pGEM-pV was linearized with the restriction enzyme HindIII. Probes prepared by transcription from the T7 RNA promoter were electrophoresed on a 1% agarose gel (lane 2). The sample in lane 1 was treated with DNase. Lane 3 contained plasmid DNA.
Fig. l(b) Sensitivity of the dot hybridisation assay. 5 ~1 of infected serum and serial dilutions thereof, were spotted onto nitrocellulose. Using a ‘ZP-labelled RNA probe (spec. act. 2 x lo* cpmipg of DNA) (A), and a biotinylated RNA probe (200 @ml of hybridisation buffer) (B), virus was detected at serum dilutions of lo-‘, equivalent to approx. 3 pg of DNA, in this specimen.
284
Comparison of biotinylated and 32P-labelled RNA probes for the detection of parvovirus sequences
The sensitivities of detection of parvovirus DNA in a reference positive serum (serum AI), were compared using “2P-labelled and biotinylated RNA probes. The reference positive serum has been found to contain 600 ng viral DNA/ml, as described by Anderson et al. (1985). Samples (5 ~1) were applied directly onto the filter, denatured in situ and digested with proteinase K. We found that it was essential that the filters be treated with proteinase K before hybridisation in order to eliminate non-specific interaction of serum with the streptavidin-alkaline phosphate detection system. The results (Fig. lb), demonstrate that whether “P or biotin was used as the detection system, virus could be detected at serum dilutions of lo-“, equivalent to approx. 3 pg of DNA, in this specimen. However, the faint signal at a serum dilution of lo-“, clearly visible on the autoradiograph, did not reproduce when photographed. This result was obtained after (a) a 16-h autoradiographic exposure for 32P, or (b) a 2-h incubation with developing dyes for biotin-streptavidin-alkaline phosphatase. However, a IO-fold increase in sensitivity could be obtained for “2P-labelled probe by the use of longer (72 h) autoradiographic exposure times (data not shown). Rapid, routine analysis of parvovirus sequences in serum
The results described above validate the use and relative sensitivity of biotinylated RNA probes, but involves a procedure which covers 2 days from start to finish. We have now carefully calibrated each step and find that hybridisation times can be reduced from overnight (16 h) to 2 h without loss of sensitivity. Shorter hybridisation time permits us to develop a methodology by which parvovirus DNA can be detected within 8 h of receipt of the serum sample. As a demonstration of the methodology 20 serum samples were tested blind by dot-blotting using {a) biotinylated RNA probe at a concentration of 200 ngiml of hybridisation buffer, and (b) a s2P-labelled RNA probe (specific activity 2 X 10’ dpmipg) in the knowledge that 8 serum samples were from patients with haemolytic anaemia, or hereditary spherocytosis, who had suffered an aplastic crisis, and that the remainder were negative sera from healthy pregnant females who attended an antenatal clinic. The results (Fig. 2) show that parvovirus DNA was detected in all eight specimens using the s2P-labelied RNA probes, but that one specimen which contained viral DNA detectable using a “2P-labelled RNA probe, was not detected in the non-radioactive system. To verify that the observed hybridisation was to viral DNA, in particular in the sample where there was a discrepancy in data, DNA was isolated from two virus positive sera (samples 5 and 15). from the serum in which there was some disparity between the tests (sample 16), and from three control virus-negative sera (samples 7, 12 and 13). Samples were subjected to agarose gel electrophoresis and blotted on to GeneScreen plus and then probed with a “2P-labelled RNA transcript. As shown in Fig. 3, sequences of approx 5.5 kb and 2.2 kb in size, respectively, hybridised to the ‘*P-labelled probe, in the tracks corresponding to the virus-positive samples. The 5.5 kb band probably corresponds to the reannealed viral genome,
285
Fig. 2. Screening of 20 sera for parvovirus DNA; comparison of radioactive and non-radioactive probes. Duplicate filters were prepared on which 5 pi of neat serum and a IO-fold dilution of each sample were spotted. One filter was probed with (A) a “‘P-labelled RNA probe (spec. act. 2x 108 cpm/~g) and the other was probed with (B) a biotinylated RNA probe at a concentration of 200 nglml hybridisation buffer.
Fig. 3. An autoradiograph of a Southern blot of viral DNA. Viral DNA was isolated from 5 pl each of serum samples 5, 15 and 16, which were positive on a 1% neutral agarose gel, blotted onto GeneScreen Plus and probed with a 32P-labelled RNA probe (spec. act. 2x 10” cpmipg) (lanes 1, 5 and 6). 5 pl of each of serum samples 7, 12 and 13 were treated in a similar manner as negative controls (lanes 2, 3 and 4). The bars indicate the positions of the A Hind111 size marker fragments 23 130,9419,6557,4371, 2322 and 2028. respectively.
286
migrating as a duplex, whereas the smaller band is likely to be the single-stranded native virus. Similar banding patterns of duplex and single-stranded virus have previously been described by Cotmore and Tattersall (1984). No viral DNA was present in the three control serum samples analysed whilst the serum which gave conflicting results, produced a low molecular weight smear suggesting that the viral DNA was degraded (Fig. 3).
Discussion Previous studies have shown that DNA hybridisation can be used in the identification of parvovirus in human serum (Anderson et al., 1985; Clewley, 1984). As the virus cannot be grown in tissue culture and other methods of diagnosis have a requirement for larger quantities of serum, DNA hybridization is replacing other methods of diagnosis. We have described some modifications to the dot hybridisation assay which enable us to reduce the time scale of the assay from a 2-3 day This time scale could conprocess to one day (8 h), without loss of sensitivity. ceivably be reduced still further by the use of an even shorter hybridisation time, and the use of nylon membranes to which nucleic acids can be rapidly crosslinked using ultraviolet light (Church and Gilbert, 1984) as opposed to the baking step used for nitrocellulose. Moreover, within this time scale, the sensitivity of biotinylated RNA probes is comparable to “2P-labelled RNA probes (Fig. lb). Results obtained from the screening of 20 clinical specimens using both dot hybridisation assays were in agreement, with one exception, in which the viral DNA subsequently proved to be degraded. However, the sensitivity of ‘2P-labelled probes could be increased with longer autoradiographic exposures, up to 72 h, detecting viral DNA serum dilutions of 10s4 (0.3 pg). Viral DNA could not be detected at this dilution using biotinylated RNA probes. The use of RNA probes eliminates the need for the time-consuming separation of contaminating plasmid sequences from the probe. Furthermore, biotinylated RNA probes can be generated in large quantities, with ease. We favour the use of single-stranded RNA probes since RNA transcripts can readily be generated in large amounts from strong RNA polymerase promoters. For example, a single unlabelled RNA transcription reaction has yielded up to 20 f_r,gof RNA, from 1 kg of plasmid DNA. Moreover, the plasmid template DNA may then be selectively eliminated using deoxyribonuclease. RNA/DNA hybrids are also more stable than DNA/DNA hybrids. Replacement of radiolabelled probes with biotinylated probes eliminates many of the disadvantages associated with dot hybridisation assays. Biotinylated probes can be stored at -20°C for up to 1 year, without effect on probe size, ability to hybridise, or sensitivity of detection. Thus there is a potential for bulk synthesis of probe and subsequent distribution to other diagnostic laboratories throughout the world, thus standardizing probes used in different laboratories. Testing could then be performed with a minimum of facilities, since the diagnosis of parvovirus infections is particularly important in areas which have a high
287
incidence of sickle cell anaemia where infection is likely to result in aplastic crisis. In such situations, a simple kit which included the biotinylated probe would be of value. With the streptavidin-alkaline phosphatase detection system the sensitivity of detection is dependent on the reagent used. Other workers (Chan et al., 1985) have found streptavidin purchased from Amersham International to be more sensitive than that purchased from other manufacturers. We also favour the use of streptavidin from Amersham, as maximum sensitivity was achieved using this reagent in preliminary experiments, as compared to streptavidin, or a streptavidin-alkaline phosphatase conjugate, purchased from other sources (results not shown). The non-specific development of a background colour on the filter sometimes occurs when biotinylated probes are used. The use of freshly prepared buffer and dye solutions and the careful handling of the filter usually reduce background. Thus, although biotinylated probes have many major advantages for routine use considerable care must be exercised with these systems. However, careful choice of reagents should result in improved sensitivity, and so enable us to detect 100% of virus-positive specimens. Clearly, hybridisation assays such as this, using non-radiolabelled RNA probes, will have a role to play in routine diagnostic medical microbiology. DNA hybridisation assays using radiolabelled hybridisation probes have been described for the Epstein-Barr virus, the herpes simplex virus, the hepatitis B virus (for review see Caskey, 1987; Highfield and Dougan, 1985), although some systems require DNA extraction from clinical samples before hybridisation. Most other systems rely on 32P with long autoradiographic exposure times (12 h to 1 week). Our results demonstrate that, using non-radiolabelled RNA probes, assays may be performed over 8 h. As this diagnostic test is easily amenable to automation it may, in the future, be possible to apply it to the routine screening of large numbers of serum samples. Moreover, the technique is applicable for the detection of any foreign nucleic acid sequence (RNA or DNA) in serum, provided that the appropriate hybridisation probe is available.
Acknowledgements We thank the Middlesex Hospital Medical School Special Trustees for supporting this work. Dr. M.J. Anderson for helpful advice and discussion and for making available the sera and plasmid PVTMl, and Mrs. U. Ayliffe for technical help with the latter. References Allison, J., Hall, L., MacIntyre, I. and Craig, R.K. (1981) The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein. Biochem. J. 199, 725-731. Anderson, M.J. and Pattison, J.R. (1984) The human parvovirus: brief review. Arch. Virol. 82, 137-148. Anderson, M. J., Jones S.E., Fisher-Hoch, S.P., Lewis, E., Hall, SM., Bartlett, C.L.R., Cohen, B.J.,
288 Mortimer, P.P. and Pereira, M.S. (1983) The human parvovirus, the cause of erythema infectiosum (fifth disease)? Lancet i, 1378. Anderson, M.J., Jones, S.E. and Minson, A.C. (1985) Diagnosis of human parvovirus infection by dot blot hybridization using cloned viral DNA. J. Med. Virol. 15, 163-172. Caskey, C.T. (1987) Disease diagnosis by recombinant DNA methods. Science 236, 1223-1229. Chan, V.T.W., Fleming, K.A. and McGee, J.O.D. (1985) Detection of subpicogram quantities of specific DNA sequences on blot hybridization with biotinylated probes. Nucleic Acids Res. 13, 8083-8091. Church, G.M. and Gilbert. W. (1984) Genomic sequencing. Biochemistry 81, 1991-1995. Clewley, J.P. (1984) Biochemical characterization of a human parvovirus. J. Gen. Virol. 65. 241-245. Cohen, B.J., Mortimer, P.P. and Pereira, M.S. (1983) Diagnostic assays with monoclonal antibodies for the human serum parvovirus-like virus (SPLV). J. Hyg. (Camb.) 91, 113-130. Cotmore, S.F. and Tattersall, P (1984) Characterization and molecular cloning of a human parvovirus genome. Science 226, 1161-1165. Highfield, P.E. and Dougan, G. (1986) DNA probes for microbial diagnosis. Med. Lab. Sci. 42.352-360. Kelleher, J.F., Luton, N.L.C., Mortimer, P.P. and Kamimura. T. (1983) The human serum ‘parvovirus’: a specific cause of aplastic crisis in hereditary spherocytosis. J. Paediatr. 102, 72@722. Knott, P.D., Welply, G.A.C. and Anderson, M.J. (1984) Serologically proved intrauterine infection with parvovirus. Br. Med. J. 289, 1660. Krumlauf, R.. Holland, P.N., McVey, J. and Hogan. B.L. (1987) Developmental and spatial patterns of expression of the mouse homeobox gene Hox 2.1. Development 99, 603-617. Langer, P.R., Waldrop, A.A. and Ward, D.C. (1981) Enzymatic synthesis of biotin-labelled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633-6637. Leary, J.J., Brigati, D.J. and Ward, D.C. (1983) Rapid and sensitive colourimetric method for visualizing biotin on nitrocellulose: Bioblots. Proc. Natl. Acad. Sci. USA 80, 4045-4049. Mason, W.S., Aldrich. C., Summers, J. and Taylor, J.M. (1982) Asymmetric replication of duck hepatitis B virus DNA in liver cells: free minus strand DNA. Proc. Natl. Acad. Sci. USA 79, 39974001. Melton, D.A., Kreig, P.A., Rebergliat. M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridisation probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12. 2036-2056. Mortimer. P.P., Cohen. B.J.. Buckley, M.M., Cradock-Watson, J.E., Ridehalgh, M.K.S., Burkhardt, F. and Schilt. U. (1985) Human parvovirus and the fetus. Lancet i. 1012. Serjeant. G.R., Mason, K., Topley, J.M., Serjeant. B.E., Pattison, J.R., Jones, S.E. and Mohamed, R. (1981) Outbreak of aplastic crisis in sickle cell anaemia associated with parvovirus-like agent. Lancet i. 595-597 Shneerson, J.M., Mortimer, P.P. and Vandervelde. E.M. (1980) Febrile illness due to a parvovirus. Br. Med. J. 280, 1580. Southern, E. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. Summers, J., Jones, S.E. and Anderson, M.J. (1983) Characterization of the agent of erythrocyte aplasia permits its classification as a human parvovirus. J. Gen. Virol. 64. 2567-2532.
279
JVM 00700
Detection of parvovirus DNA in human serum using biotinylated RNA hybridisation probes D .A. Cunningham ‘, J.R. Pattison
and R.K. Craig1
‘Medical Molecular Biology Unit, Department of Biochemistry, University College and Middlesex School of Medicine, London, U.K.; =Department of Medical Microbiology, School of Clinical Sciences, University College and Middlesex School of Medicine, London, U.K. (Accepted
21 December
1987)
Summary Methods were established for the detection of parvovirus DNA in human serum using single-stranded RNA probes. The sensitivity of detection of virus using 32Pradiolabelled RNA versus non-radiolabelled biotinylated RNA probes using a streptavidin-polyalkaline phosphatase detection system was compared. Virus was detected using 32P-labelled and biotinylated RNA probes at serum dilutions of 10m3, equivalent to approx. 3 pg of viral DNA. Using biotinylated RNA probes and a dot-blot system, diagnosis of numerous serum samples could be performed within 8 h of receipt of samples, using an RNA probe which was synthesised and stored at -20°C for up to 12 months without loss of sensitivity. Our work demonstrates the potential of biotinylated RNA probes in the routine analysis of viral sequences in serum. Parvovirus; detection
Nucleic
acid hybridisation;
Human
serum;
Biotinylated
RNA;
Rapid
Introduction The human parvovirus, originally found in human serum by Cossart et al. in 1975, is a single-stranded DNA virus which packages approximately equal numbers of positive or negative DNA strands (Summers et al., 1983). It grows in rapidly di-
Correspondence to: Dr. D.A. Cunningham, Medical Molecular Biology Unit, Department of Biochemistry, University College and Middlesex School of Medicine, The Windeyer Building, Cleveland Street, London WlP 6DB, U.K. 0166-0934/88/$03.50
0
1988 Elsevier
Science
Publishers
B.V.
(Biomedical
Division)
280
viding cells (Anderson and Pattison, 1984) and has been associated with febrile illness (Shneerson et al, 1980), the aplastic crisis of sickle cell disease and hereditary spherocytosis (Serjeant et al., 1981; Kelleher et al., 1983), and erythema infectiosum (fifth disease) (Anderson et al., 1983). Human parvovirus has been shown to cross the placenta, eliciting an immune response in the fetus. Animal parvoviruses induce fetal abnormalities and there is some evidence that human parvovirus may harm the fetus (Knott et al., 1984; Mortimer et al., 1985). As human parvovirus cannot be grown in tissue culture, methods of detection rely on counterimmune electrophoresis, electron microscopy or, more commonly, radioimmunoassay (Cohen et al., 1983) or nucleic acid hybridisation using a cloned DNA probe (Anderson et al., 1985; Clewley, 1984). Dot hybridisation assays using s2P-radiolabelled hybridisation probes which are rapid and sensitive have been described previously (Anderson et al., 1985; Clewley, 1984). However, these probes have several disadvantages for routine laboratory diagnosis. The isotope presents a safety hazard, is relatively expensive, and the probe has a short shelf-life (t = 14.3 days). Potentially such problems may be overcome using non-radioactive modifying groups which, after introduction into nucleic acid are stable, and can be detected using sensitive enzyme based assays. The most commonly used modifying group is the vitamin biotin, which can be introduced into nucleic acid enzymically, using nucleotide derivatives (Langer et al., 1981). The affinity of biotin (Kdiss = 10-i”) for the protein avidin (from eggwhite) or streptavidin (from Streptomyces avidinii) is then exploited in the detection of biotinylated probes (Leary et al., 1983). The avidin group can be complexed to enzymes, which produce a specific calorimetric reaction on incubation with dye solutions. We describe here a method for the detection of parvovirus in serum using a biotin based non-radioactive detection system, and compare the speed and sensitivity of the method with “‘P-radiolabelled hybridisation systems in current use.
Materials
and Methods
Materials [a-“*P]CTP (800 C’/1 mmol) was obtained from New England Nuclear (Boston, MA, U.S.A.). Allylamine-UTP, caproylamido-biotin-N-hydroxysuccinimide ester (CAB-NHS) and polyalkaline phosphatase were purchased from Bethesda Research Laboratories (Paisley, Scotland). Streptavidin was purchased from Amersham International plc, (Amersham, U.K.). Nitroblue tetrazolium (NBT) and 5bromo-4-chloro-3-indolyl phosphate (BCIP) were from Sigma (Poole, Dorset, U.K.). Restriction endonucleases and T7 RNA polymerase were from Boehringer Corporation Ltd. (Lewes, U.K.). Schleicher and Schuell nitrocellulose filter sheets were from Anderman and Company (Surrey, U.K.) while GeneScreen Plus was from New England Nuclear (Boston MA, U.S.A.). The cloning vector pGEM 1 was from P & S Biochemicals (Liverpool, U.K.). All other materials were from sources described elsewhere (Allison et al., 1981).
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Preparation of RNA probes The plasmid PVTMI has been described previously (Anderson et al., 1985). The 700 bp parvovirus DNA insert was excised with the restriction enzyme PstI and subcloned into the PstI digested and phosphatased vector PGEMl (Promega Biotee, Madison, WI). Radiolabelled single-stranded RNA probes were prepared from Hind111 linearised plasmid by incorporation of [cx-“‘P]CTP into transcripts from the T7 promoter (Melton et al., 1984). Similarly, biotinylated RNA probes were prepared by incorporation of allylamine-UTP (Langer et al., 1981) into transcripts from the T7 promoter and subsequent chemical biotinylation with the biotin ester caproylamido-biotin-N-hydroxysuccinimide ester (CAB-NHS), according to the manufacturers instructions. Trace radiolabelling with [a-‘SS]GTP was used to quantitate RNA synthesis. Dot hybridisation assays Several modifications were made to the method described by Anderson et al. (1985). Aliquots (5 ~1) of serum, and lo-fold dilutions thereof in 2~ SSC, were spotted onto a dry nitrocellulose filter, allowed to dry and baked for 2 h under vacuum at 80°C. Specimens on the filter were then alkali denatured by floating on 0.1 M NaOH containing 1 M NaCI. Samples were then neutralized by floating on 0.1 M Tris-HCl (pH 7.4) containing 1 M NaCl as described by Mason et al. (1982). Filters were then immersed in 2 x SSC containing 0.1% SDS (w/v) and 400 kg/ml proteinase K, and incubated with shaking for 60 min at 37°C. Proteinase K was then removed by three 5-min rinses in 2~ SSC, prior to prehybridisation. Identification of sequences using biotinylated RNA probes When filters were to be hybridised with biotinylated RNA probes, prehybridisation was for 2 h in 5x SSC containing 50% (v/v) formamide, 1% (w/v) SDS, 1 M NaCl, 10% (w/v) dextran sulphate, and 100 kg/ml single-stranded herring testis DNA. Hybridisation was in the same buffer except that formamide was reduced in concentration to 45% (v/v), and the biotinylated probe was present at a concentration of 200 ngiml of hybridisation buffer. Hybridisation was performed in sealed polypropylene bags with 100 ~1 hybridisation buffer present per cm2 of nitrocellulose, for 2-16 h at 42°C:. After hybridisation, filters were washed in 2~ SSC containing 0.1% (w/v) SDS for 30 min at room temperature with three buffer changes, then in 0.2~ SSC containing 0.1% (w/v) SDS for 30 min at room temperature with three buffer changes, and finally in 0.2~ SSC containing 0.1% (w/v) SDS at 55’C for 45 min with three buffer changes. In order to remove non-specific binding sites and so reduce background when the hybrids were visualized by development with a streptavidin-polyalkaline phosphatase detection system, the filters were blocked in 0.1 M Tris-HCl (pH 7.5) containing 0.15 M NaCl and 3% (w/v) bovine serum albumin, at 65°C for 60 min. Filters were then incubated in 0.1 M Tris-HCl (pH 7.5) containing 0.15 M NaCl and 500 ngiml streptavidin for 10 min at room ‘emperaLure with gentle agitation, then washed in the incubation buffer for 30 min with three buffer changes. The filters were then incubated in 0.1 M TrisHCl (pH 7.5) containing 0.15 M NaCl and 1 pg/ml biotinylated polyalkaline phos-
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phatase for 10 min with shaking, then washed in this buffer for 30 min with three buffer changes, and finally washed in developing buffer which comprised 0.1 M Tris-HCl (pH 7.5) containing 0.1 M NaCl and 50 mM MgCl, for 10 min all at room temperature. The volume of streptavidin and biotinylated polyalkaline phosphatase applied was 7.5 m1/100cm2 filter. Filter-bound alkaline phosphatase was visualized by application to the filter of a solution (75 pl/cm2) comprising 4.4 ~1 of a freshly prepared stock solution of NBT (75 mg/ml) in 70% (v/v) dimethyl formamide and 3.3 ~1 of a freshly prepared stock solution of BCIP (50 mgiml) in 100% dimethyl formamide in 1 ml of developing buffer. A faint purple colour developed over a period of l-2 h. Identification of sequences using a ‘2P-radiolabelled RNA probe When filters were to be hybridised with a-32P-radiolabelled RNA probes prehybridisation was for 2 h and hybridisation for 2-16 h, at 52°C in 5~ SSCl20 mM sodium phosphate (pH 6.8) containing 60% (v/v) formamide, 5x Denhardt’s, 1% (w/v) SDS, 7% (w/v) dextran sulphate, 100 kg/ml single-stranded herring testis DNA, 100 kg/ml Escherichia .coli tRNA and 10 pgiml poly(A) (Krumlauf et al., 1987). For hybridisation, filters were incubated in the prehybridisation buffer containing 2X lo6 cpm 32P-labelled RNA probe per ml of buffer. After hybridisation, filters were washed in 2~ SSC containing 0.1% (w/v) SDS for 30 min with three buffer changes, then incubated with RNase A (20 kg/ml) in 2x SSC for 30 min, washed in 0.2x SSC containing 0.1% (w/v) SDS for 30 min with three buffer changes, and finally washed in 0.2~ SSC, 0.1% (w/v) SDS at 65°C for 45 min with three buffer changes. All washes were at room temperature unless otherwise stated. The regions of hybridisation were then visualized by autoradiography using Kodak X-Omat S film. Southern blotting Serum samples (5 ~1) were diluted in 2~ SSC to a final volume of 100 ~1, then digested with proteinase K (400 kg/ml) at 37°C for 3 h, and purified by sequential extraction with phenol/chloroform (1: 1)) chloroform, and diethyl ether. After ethanol precipitation each purified specimen was electrophoresed on a 1% (w/v) neutral agarose gel, and blotted onto a GeneScreen Plus membrane using a procedure modified from Southern (1975), as described in the GeneScreen Plus manual. Filters were probed with a single-stranded RNA probe (specific activity 2 x 10s cpm/pg). Hybridisation and wash conditions were as described for dot hybridisation assays.
Results Synthesis of single-stranded RNA hybridisation probes We have recloned the 700 bp PstI parvovirus DNA fragment from pVTM1 into the PstI site of the polylinker of the vector pGEM 1. This allows RNA to be tran-
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scribed from either + or - viral DNA strand, using the SP6 or T7 promoter (Fig. la). In this instance to generate RNA transcripts, the plasmid (pGEM lpv) was linearized using the restriction enzyme Hind111 (for transcription with T7 RNA polymerase) or BamHI (for transcription with SP6 RNA polymerase), to ensure that a virus specific probe was synthesized (Fig. la). As the virus packages both positive and negative strands in approximately equal amounts, either promoter can be used to generate a single-stranded RNA hybridisation probe. The T7 RNA polymerase was the enzyme of choice, as its transcription efficiency and stability was found to be higher than that of the SP6 polymerase. Agarose gel electrophoresis of restricted plasmid, before and after transcription with T7 RNA polymerase demonstrates the presence of a discrete RNA transcript (Fig. la).
1
2
3
Fig. l(a) Preparation of labelled RNA. Plasmid pGEM-pV was linearized with the restriction enzyme HindIII. Probes prepared by transcription from the T7 RNA promoter were electrophoresed on a 1% agarose gel (lane 2). The sample in lane 1 was treated with DNase. Lane 3 contained plasmid DNA.
Fig. l(b) Sensitivity of the dot hybridisation assay. 5 ~1 of infected serum and serial dilutions thereof, were spotted onto nitrocellulose. Using a ‘ZP-labelled RNA probe (spec. act. 2 x lo* cpmipg of DNA) (A), and a biotinylated RNA probe (200 @ml of hybridisation buffer) (B), virus was detected at serum dilutions of lo-‘, equivalent to approx. 3 pg of DNA, in this specimen.
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Comparison of biotinylated and 32P-labelled RNA probes for the detection of parvovirus sequences
The sensitivities of detection of parvovirus DNA in a reference positive serum (serum AI), were compared using “2P-labelled and biotinylated RNA probes. The reference positive serum has been found to contain 600 ng viral DNA/ml, as described by Anderson et al. (1985). Samples (5 ~1) were applied directly onto the filter, denatured in situ and digested with proteinase K. We found that it was essential that the filters be treated with proteinase K before hybridisation in order to eliminate non-specific interaction of serum with the streptavidin-alkaline phosphate detection system. The results (Fig. lb), demonstrate that whether “P or biotin was used as the detection system, virus could be detected at serum dilutions of lo-“, equivalent to approx. 3 pg of DNA, in this specimen. However, the faint signal at a serum dilution of lo-“, clearly visible on the autoradiograph, did not reproduce when photographed. This result was obtained after (a) a 16-h autoradiographic exposure for 32P, or (b) a 2-h incubation with developing dyes for biotin-streptavidin-alkaline phosphatase. However, a IO-fold increase in sensitivity could be obtained for “2P-labelled probe by the use of longer (72 h) autoradiographic exposure times (data not shown). Rapid, routine analysis of parvovirus sequences in serum
The results described above validate the use and relative sensitivity of biotinylated RNA probes, but involves a procedure which covers 2 days from start to finish. We have now carefully calibrated each step and find that hybridisation times can be reduced from overnight (16 h) to 2 h without loss of sensitivity. Shorter hybridisation time permits us to develop a methodology by which parvovirus DNA can be detected within 8 h of receipt of the serum sample. As a demonstration of the methodology 20 serum samples were tested blind by dot-blotting using {a) biotinylated RNA probe at a concentration of 200 ngiml of hybridisation buffer, and (b) a s2P-labelled RNA probe (specific activity 2 X 10’ dpmipg) in the knowledge that 8 serum samples were from patients with haemolytic anaemia, or hereditary spherocytosis, who had suffered an aplastic crisis, and that the remainder were negative sera from healthy pregnant females who attended an antenatal clinic. The results (Fig. 2) show that parvovirus DNA was detected in all eight specimens using the s2P-labelied RNA probes, but that one specimen which contained viral DNA detectable using a “2P-labelled RNA probe, was not detected in the non-radioactive system. To verify that the observed hybridisation was to viral DNA, in particular in the sample where there was a discrepancy in data, DNA was isolated from two virus positive sera (samples 5 and 15). from the serum in which there was some disparity between the tests (sample 16), and from three control virus-negative sera (samples 7, 12 and 13). Samples were subjected to agarose gel electrophoresis and blotted on to GeneScreen plus and then probed with a “2P-labelled RNA transcript. As shown in Fig. 3, sequences of approx 5.5 kb and 2.2 kb in size, respectively, hybridised to the ‘*P-labelled probe, in the tracks corresponding to the virus-positive samples. The 5.5 kb band probably corresponds to the reannealed viral genome,
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Fig. 2. Screening of 20 sera for parvovirus DNA; comparison of radioactive and non-radioactive probes. Duplicate filters were prepared on which 5 pi of neat serum and a IO-fold dilution of each sample were spotted. One filter was probed with (A) a “‘P-labelled RNA probe (spec. act. 2x 108 cpm/~g) and the other was probed with (B) a biotinylated RNA probe at a concentration of 200 nglml hybridisation buffer.
Fig. 3. An autoradiograph of a Southern blot of viral DNA. Viral DNA was isolated from 5 pl each of serum samples 5, 15 and 16, which were positive on a 1% neutral agarose gel, blotted onto GeneScreen Plus and probed with a 32P-labelled RNA probe (spec. act. 2x 10” cpmipg) (lanes 1, 5 and 6). 5 pl of each of serum samples 7, 12 and 13 were treated in a similar manner as negative controls (lanes 2, 3 and 4). The bars indicate the positions of the A Hind111 size marker fragments 23 130,9419,6557,4371, 2322 and 2028. respectively.
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migrating as a duplex, whereas the smaller band is likely to be the single-stranded native virus. Similar banding patterns of duplex and single-stranded virus have previously been described by Cotmore and Tattersall (1984). No viral DNA was present in the three control serum samples analysed whilst the serum which gave conflicting results, produced a low molecular weight smear suggesting that the viral DNA was degraded (Fig. 3).
Discussion Previous studies have shown that DNA hybridisation can be used in the identification of parvovirus in human serum (Anderson et al., 1985; Clewley, 1984). As the virus cannot be grown in tissue culture and other methods of diagnosis have a requirement for larger quantities of serum, DNA hybridization is replacing other methods of diagnosis. We have described some modifications to the dot hybridisation assay which enable us to reduce the time scale of the assay from a 2-3 day This time scale could conprocess to one day (8 h), without loss of sensitivity. ceivably be reduced still further by the use of an even shorter hybridisation time, and the use of nylon membranes to which nucleic acids can be rapidly crosslinked using ultraviolet light (Church and Gilbert, 1984) as opposed to the baking step used for nitrocellulose. Moreover, within this time scale, the sensitivity of biotinylated RNA probes is comparable to “2P-labelled RNA probes (Fig. lb). Results obtained from the screening of 20 clinical specimens using both dot hybridisation assays were in agreement, with one exception, in which the viral DNA subsequently proved to be degraded. However, the sensitivity of ‘2P-labelled probes could be increased with longer autoradiographic exposures, up to 72 h, detecting viral DNA serum dilutions of 10s4 (0.3 pg). Viral DNA could not be detected at this dilution using biotinylated RNA probes. The use of RNA probes eliminates the need for the time-consuming separation of contaminating plasmid sequences from the probe. Furthermore, biotinylated RNA probes can be generated in large quantities, with ease. We favour the use of single-stranded RNA probes since RNA transcripts can readily be generated in large amounts from strong RNA polymerase promoters. For example, a single unlabelled RNA transcription reaction has yielded up to 20 f_r,gof RNA, from 1 kg of plasmid DNA. Moreover, the plasmid template DNA may then be selectively eliminated using deoxyribonuclease. RNA/DNA hybrids are also more stable than DNA/DNA hybrids. Replacement of radiolabelled probes with biotinylated probes eliminates many of the disadvantages associated with dot hybridisation assays. Biotinylated probes can be stored at -20°C for up to 1 year, without effect on probe size, ability to hybridise, or sensitivity of detection. Thus there is a potential for bulk synthesis of probe and subsequent distribution to other diagnostic laboratories throughout the world, thus standardizing probes used in different laboratories. Testing could then be performed with a minimum of facilities, since the diagnosis of parvovirus infections is particularly important in areas which have a high
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incidence of sickle cell anaemia where infection is likely to result in aplastic crisis. In such situations, a simple kit which included the biotinylated probe would be of value. With the streptavidin-alkaline phosphatase detection system the sensitivity of detection is dependent on the reagent used. Other workers (Chan et al., 1985) have found streptavidin purchased from Amersham International to be more sensitive than that purchased from other manufacturers. We also favour the use of streptavidin from Amersham, as maximum sensitivity was achieved using this reagent in preliminary experiments, as compared to streptavidin, or a streptavidin-alkaline phosphatase conjugate, purchased from other sources (results not shown). The non-specific development of a background colour on the filter sometimes occurs when biotinylated probes are used. The use of freshly prepared buffer and dye solutions and the careful handling of the filter usually reduce background. Thus, although biotinylated probes have many major advantages for routine use considerable care must be exercised with these systems. However, careful choice of reagents should result in improved sensitivity, and so enable us to detect 100% of virus-positive specimens. Clearly, hybridisation assays such as this, using non-radiolabelled RNA probes, will have a role to play in routine diagnostic medical microbiology. DNA hybridisation assays using radiolabelled hybridisation probes have been described for the Epstein-Barr virus, the herpes simplex virus, the hepatitis B virus (for review see Caskey, 1987; Highfield and Dougan, 1985), although some systems require DNA extraction from clinical samples before hybridisation. Most other systems rely on 32P with long autoradiographic exposure times (12 h to 1 week). Our results demonstrate that, using non-radiolabelled RNA probes, assays may be performed over 8 h. As this diagnostic test is easily amenable to automation it may, in the future, be possible to apply it to the routine screening of large numbers of serum samples. Moreover, the technique is applicable for the detection of any foreign nucleic acid sequence (RNA or DNA) in serum, provided that the appropriate hybridisation probe is available.
Acknowledgements We thank the Middlesex Hospital Medical School Special Trustees for supporting this work. Dr. M.J. Anderson for helpful advice and discussion and for making available the sera and plasmid PVTMl, and Mrs. U. Ayliffe for technical help with the latter. References Allison, J., Hall, L., MacIntyre, I. and Craig, R.K. (1981) The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein. Biochem. J. 199, 725-731. Anderson, M.J. and Pattison, J.R. (1984) The human parvovirus: brief review. Arch. Virol. 82, 137-148. Anderson, M. J., Jones S.E., Fisher-Hoch, S.P., Lewis, E., Hall, SM., Bartlett, C.L.R., Cohen, B.J.,
288 Mortimer, P.P. and Pereira, M.S. (1983) The human parvovirus, the cause of erythema infectiosum (fifth disease)? Lancet i, 1378. Anderson, M.J., Jones, S.E. and Minson, A.C. (1985) Diagnosis of human parvovirus infection by dot blot hybridization using cloned viral DNA. J. Med. Virol. 15, 163-172. Caskey, C.T. (1987) Disease diagnosis by recombinant DNA methods. Science 236, 1223-1229. Chan, V.T.W., Fleming, K.A. and McGee, J.O.D. (1985) Detection of subpicogram quantities of specific DNA sequences on blot hybridization with biotinylated probes. Nucleic Acids Res. 13, 8083-8091. Church, G.M. and Gilbert. W. (1984) Genomic sequencing. Biochemistry 81, 1991-1995. Clewley, J.P. (1984) Biochemical characterization of a human parvovirus. J. Gen. Virol. 65. 241-245. Cohen, B.J., Mortimer, P.P. and Pereira, M.S. (1983) Diagnostic assays with monoclonal antibodies for the human serum parvovirus-like virus (SPLV). J. Hyg. (Camb.) 91, 113-130. Cotmore, S.F. and Tattersall, P (1984) Characterization and molecular cloning of a human parvovirus genome. Science 226, 1161-1165. Highfield, P.E. and Dougan, G. (1986) DNA probes for microbial diagnosis. Med. Lab. Sci. 42.352-360. Kelleher, J.F., Luton, N.L.C., Mortimer, P.P. and Kamimura. T. (1983) The human serum ‘parvovirus’: a specific cause of aplastic crisis in hereditary spherocytosis. J. Paediatr. 102, 72@722. Knott, P.D., Welply, G.A.C. and Anderson, M.J. (1984) Serologically proved intrauterine infection with parvovirus. Br. Med. J. 289, 1660. Krumlauf, R.. Holland, P.N., McVey, J. and Hogan. B.L. (1987) Developmental and spatial patterns of expression of the mouse homeobox gene Hox 2.1. Development 99, 603-617. Langer, P.R., Waldrop, A.A. and Ward, D.C. (1981) Enzymatic synthesis of biotin-labelled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633-6637. Leary, J.J., Brigati, D.J. and Ward, D.C. (1983) Rapid and sensitive colourimetric method for visualizing biotin on nitrocellulose: Bioblots. Proc. Natl. Acad. Sci. USA 80, 4045-4049. Mason, W.S., Aldrich. C., Summers, J. and Taylor, J.M. (1982) Asymmetric replication of duck hepatitis B virus DNA in liver cells: free minus strand DNA. Proc. Natl. Acad. Sci. USA 79, 39974001. Melton, D.A., Kreig, P.A., Rebergliat. M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridisation probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12. 2036-2056. Mortimer. P.P., Cohen. B.J.. Buckley, M.M., Cradock-Watson, J.E., Ridehalgh, M.K.S., Burkhardt, F. and Schilt. U. (1985) Human parvovirus and the fetus. Lancet i. 1012. Serjeant. G.R., Mason, K., Topley, J.M., Serjeant. B.E., Pattison, J.R., Jones, S.E. and Mohamed, R. (1981) Outbreak of aplastic crisis in sickle cell anaemia associated with parvovirus-like agent. Lancet i. 595-597 Shneerson, J.M., Mortimer, P.P. and Vandervelde. E.M. (1980) Febrile illness due to a parvovirus. Br. Med. J. 280, 1580. Southern, E. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. Summers, J., Jones, S.E. and Anderson, M.J. (1983) Characterization of the agent of erythrocyte aplasia permits its classification as a human parvovirus. J. Gen. Virol. 64. 2567-2532.