Detection of DNA viruses by radioactive and non ... - Springer Link

Archives of Virology

Arch Virol (1987) 92:233-242

© by Springer-Vcrlag 1987

Detection of DNA Viruses by Radioactive and Non Radioactive DNA Probes: Application to African Swine Fever Virus By E. TABAI¢£ S Departamento de Microbiologla, Facultad de Medicina, Universidad Aut6noma de Madrid, Madrid, Spain With 7 Figures Accepted June 13, 1986

Summary A molecular hybridization technique using radioactive and non radioactive DNA probes, has been used to detect ASFV DNA immobilized on nitrocellulose paper. It is based on the use of plasmid pRPEL-2 as a hybridization probe. This plasmid containe the H-ClaI DNA fragment (size 5.6 Kbp) from the Spain-70 strain of ASFV. The sensitivity of detection using radioactive 32P-probes (specific activity about 2 x l0 s cpm per jig) was about 20 pg of viral DNA. The ~2P-pRPEL-2 DNA probe can detect about 100 infected MS cells and failed to hybridize to DNA from HSV-2, MS cells or salmon sperm. The sensitivity with non radioactive probes was about 4 ng of viral DNA for a sulfonated DNA probe and 400 pg for a biotinylated DNA probe. The effiency of DNA fixation to the filter, the effect of EDTA and of ultrasonic treatment of the sample were also investigated.

Introduction Molecular hybridization of viral DNA to complementary sequences of int~cted cells immobilized on nitrocellulose paper, provides a specific method for virus detection (4, 10). Probes for nucleic acid hybridization are usually labeled with radioisotopes. However, non radioactive probes are more suitable for routine use in the diagnosis of virus infection. Recent developments offer alternative methods using non radioactive probes such as DNA labelled with biotin (8) by nick translation and chemically modified DNA that is recognized by specific antibodies (12). Hybridization can be detected b y using an enzyme label and either a eolorimetrie, fluorescent or a

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e h e m i l u m i n e s e e n t e n d p o i n t (9). N o n r a d i o a c t i v e D N A p r o b e s h a v e b e e n u s e d i n t h e d e t e c t i o n of D N A v i r u s e s (3) a n d c o u l d b e v e r y u s e f u l i n t h e d e t e c t i o n o f A f r i c a n S w i n e F e v e r ( A S F ) i n l o c a t i o n s w h e r e it, is not, p o s s i b l e to use radioisotopes. T h e p u r p o s e of t h i s w o r k w a s t o e x t e n d p r e v i o u s s t u d i e s o n t h e d e t e c t i o n of A S F V b y D N A - D N A h y b r i d i z a t i o n u s i n g the r e c o m b i n a n t p l a s m i d p R P E L - 2 (2) a n d n o n r a d i o a c t i v e p r o b e s . T h e s e n s i t i v i t y of t h i s m e t h o d w a s c o m p a r e d w i t h t h a t of 32P-DNA p r o b e s .

Materials and Methods Cells and Viras The Spain-70 strain of ASFV was used after fourty seven passages in the MS monkey kidney cell line (5). It is rei~rred to as E 70 MS 47 in the text. HSV-2 (strain lovelace) was passaged in ~¢~ro cells.

Preparation of Viral and CeUular DNA8 The MS cell DNA was obtained by treatment with SDS and proteinase K. Cells pelleted by centrifugation were resuspended in 0.15 MNaC1, 10 m ~ Tris (pH 8), I mM ED[I~ and lysed by the addition of SDS at a final concentration of 0.4 per cent w/v. They were then digested with 200 l~g/ml proteinase K for 30 minutes at 45° C and then at 37° C overnight. The cell extract was deproteinized by sequential extractions with phenol (7) (70 ml m-cresol, 55 ml water and 0.5 g 8-hydroxyquinoline were mixed with 500 g phenol) and chloroform-isoamylalcohol (24 : 1). Following the precipitation of nucleic acids with 2.5 vol of ethanol, tile sample was dissolved in 10 mM Tris, lmM EDTA (pH 7.5) (TE) and treated with 200 t~g/ml of ]~NAse for 30 minutes at 37° C. After deproteinization by a further sequential extraction this time with phenol, chloroform-isoamylalcohol and then ether, the DNA was precipitated with 2.5 volumes of ethanol in the presence of 0.3 ~ sodium acetate. Finally, the MS DNA was dissolved in TE. ASFV DNA was isolated from the cytoplasmic fraction of infected cells (11). MS cells infected with ASFV were pelleted by centrifugation for 2 minutes in an Eppendorf centrifuge, washed with 0.34 M sucrose, 5 mM Tris ptt 8, before being resuspcnded in 67 mM sucrose, 5 m ~ Tris pH 8 and incubated at 0° C for 10 minutes. Cells were lysed by adding NP 40 to a final concentration of 1 per cent (w/v) followed by incubation at 0° C for 10 minutes in the presence of 200 ~g/ml of RNAse. The nuclei were collected by centrifugation for 2 minutes in an Eppendorf centrifuge through a cushion of 0.34 M sucrose in 5 mM Tris pH 8. The supernatant was removed, made up to 50 M ~ ME, 50 mM Tris, pH 8, 2 mM MgC12 and 100 ~g/ml DNAse and incubated at 37°C for 30 minutes. This treatment digested remains of cellular DNA while the DNA inside sub viral particles was protected. The sample was then made to 10 m ~ EDTA, 1 per cent SDS and 500 Itg/ml proteinase K before a further incubation for 30 minutes at 45 ° C. After deproteinization first with phenol (7) : chloroform : isoamylalcohol (25 : 24 : 1), and then with chlorotbrm : isoamylatcohol (24 : 1) the sample was precipitated with 2.5 volumes of ethanol. The precipitated DNA was washed with 50 mM NaC1 in 70 per cent ethanol and finally dissolved in TE. HSV-2 DNA was isolated and purified by NaI density gradient centrifugation of infected Vcro cell extracts (13). The determinations of DNA were made fluorometrically (6) and by absorption at 260 nm.

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Viral DNA Probes pRPEI~2 and MS cell DNAs were used as probes. The 32p. and biotin labelled probes were prepared by nick translation following the instructions supplied with the Bethesda Research Laboratories Kit. The sulfonated probe was obtained according to the method supplied with the Chemiprobe Kit (Organics Ltd. Israel).

Hybridization and Visualization of Probes DNA was extracted from saline buffer containing viral DNA, infected or non infected MS cells essentially as described previously (2). The above samples (0.1 ml) were treated with 0.15 mt of a mixture containing 2 per cent SDS and 0.2 • NaOH for 5 minutes at room temperature. After neutralization, by the addition of 0.3 ml of 0.1 N HC1 in 1 M Tris (pH 7), the samples were deproteinized with 0.4 ml of a mixture of phenol (7), chloroform and isoamylalcohol (25 : 24 : 1) equilibrated in 0.5 M Tris (pH 7.5) and centrifngated in the Eppendorf centrifuge for 2 minutes. The aqueous phase was applied to nitrocellulose paper (ScuLEIC~ER and SC~ffLL) pre-soaked with 4 × SSC in a Biodot vacuum manifold (Bio Rad) (filter 7 mm2/sample) and filtered. The filters were rinsed in 2 x SSC, dried and baked at 80 ° C for 2 hours. Before hybridization, the filters were incubated at 55 ° C for 1 hour in prehybridization buffer (30 per cent formamide, 6 × SSC, 0.1 per cen~LSDS and 2 mg/ml each of bovine serum albumin, ficolI and polyvinylpyrrolidone). For hybridization, the filters were incubated at 55 ° C for 20 hours in sealed plastic bags together with the labeled probe in prehybridization buffer containing 25 ~g/ml of calf thymus DNA. After hybridization the filters were rinsed three times in 2 × SSC at room temperature and once in prehybridization buffer at 55 ° C for 15 minutes. The above washing procedure was then repeated before the filters were finally washed in 2 x SSC and dried. The hybrids were

Fig. 1. Viral and cellular DNAs. Agarose gel electrophoresis ofl I~gofASFV DNA digested with ClaI (A) and 1 t~g of ttSV-2 DNA digested with Bgl II (B). 2 I~g each MS cell DNA (C), salmon sperm DNA (D), sonicated salmon sperm DNA (E) and the ClaI viral DNA fragment ([t) are also shown

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visuMised by autoradiography or by development with avidin-biotinylated alkaline phosphatase and monoclonal antibody-alkaline phosphatase for non radioactive probes, according to the manufacturer's instructions.

Results

Dot Hybridizations D o t hyhridizations w e r e c a r r i e d out to d e ~ r m i n e the limits of sensitivity a n d o p t i m u m conditions for d e t e c t i o n of v i r a l DNA, u s i n g e i t h e r radiolabeled or non r a d i o l a b e l e d D2~\~ p r o b e s . T h e D N A s u s e d in the p r e s e n t w o r k w e r e f r o m A S F V , HSV-2, MS cells a n d s a l m o n s p e r m (Fig. 1). The A S F V D N A w a s i s o l a t e d f r o m MS infected cells following a r a p i d m e t h o d involving E p p e n d o r f centrifugation. T h e level of c o n t a m i n a t i o n b y cellular D N A w a s a b o u t t w e l v e p e r cent, as e s t i m a t e d b y h y b r i d i z a t i o n to 32P-MS D N A (Fig. 2, Ac, Be). S o n i e a t e d or n o n s o n i c a t e d s a m p l e s p r o d u c e d t h e s a m e results (Fig. 2, Aa, Ab, Ba, Bb). Similarly, the p r e s e n c e of 5 mM E D T A in the original s a m p l e did n o t a l t e r the results (data n o t shown). H o w e v e r ,

Fig. 2. Spot hybridization of viral and cellular DNAs. Serial I: 10 dilutions of MS cells (d), beginning with 1.1 x 10~cells per spot (d 1), MS infected eells (a) and sonieated MS infected cells (b) and MS infected cells without deproteinization (c) were processed and applied to nitrocellulose filter as described in Materials and Methods. The same procedure was followed with scrim 1 : 10 dilution of: ASFV DNA (e) beginning with 200 ng (e 1) and MS cell DNA (f) beginning with 214 ng (fl). These spots were hybridized with 2 × 106 cpm of 32P-MS DNA (specific activity 6.1 × 10a cpm/t~g) (A) and 2 × 108 epm of 3-~P-pRPEL-2 (specific activity 6.7 x 106 epm/vg) (B). The autoradiographs developed after 2 days (A) and 7 days (B) of exposure are shown. (C) Several preparations ofvirM DNAs (g 1 to 5), of l~g MS (g6), 0.811g of HSV-2 (g7) and 1.5txg of sMmon sperm (gS) DNAs were hybridizated with 32P-pRPED2 under the same conditions as in B

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the D N A did n o t bind to the filters if the s a m p l e w a s n o t d e p r o t e i n i z e d (Fig. 2, Ae, Bc). T h e efficiency of fLxation of D N A to the nitrocellulose filter, studied with a2p-labelled s a l m o n s p e r m DNA, w a s a b o u t s e v e n t y p e r c e n t for a m o u n t s b e l o w 1.2 ~g. This v a l u e d e c r e a s e d with i n c r e a s i n g a m o u n t s of D N A (Fig. 3). T h e p r o b e u s e d for A S F V D N A d e t e c t i o n w a s t h e p l a s m i d p R P E L - 2 (5) which did n o t p r o d u c e d e t e c t a b l e h y b r i d i z a t i o n to D N A f r o m MS cells (Fig. 2, g 6), H S V - 2 (Fig. 2, g 7) or s a l m o n s p e r m (Fig. 2, g 8).

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Sensitivity The sensitivity of ASFV DNA detection by blot hybridization was determined after the application of serial 10 fold dilutions of purified and denatured genome viral DNA to nitrocellulose membranes. Using the pRPEL-2 probe (containing the 5.6 Kbp ClaI-H fragment of ASFV DNA), labelled with 32p by nick translation, as little as 20 pg of viral DNA was detectable after 4 days of exposure (Fig. 4, F 5). The amount ofs2P-DNA on the filter was about 0.3 pg. The size of ASFV DNA (E 70 MS 45) was approximately 156 Kbp (E. MART~N and E. TABAR£S, manuscript in preparation). Therefore, 20 pg of -v~al DNA contained 0~72 pg of Cla I H fragment. This represents an efficiency of hybridization of about 42 per cent. The sensitivity was about 4 ng of viral DNA with the sulfonated DNA probe (Fig. 4, H 3) and 400 pg with the biotin-labelled probe (Fig. G 4). Furthermore, the background with the biotin-avidin probe was lower than that with the sulfonated probe. The sensitivity with biotin was similar to that observed for the 32P-DNA probe that had a specific activity of 6.7 × 10~ cpm/~g (Fig. 2, Be).

Fig. 4. Sensitivity and specifier of the hybridization reaction. Serial 1:10 dilutions of ASFV DNA beginning with 200 ng, were hybridized with 3 ~g (1 txg/ml of hybridization buffer) of sultbnated pt~PEL-2 DNA (A) or 20 × 10~ epm of 32P-pRPEI~2 (specific activity 1.92 × 10s epm/~g). The autoradiographs were developed after 2 hours (1t), 4 hours (C), 16 hours (D), 2 days (L) and 4 days ~). Serial 1 : 10 dilutions ofASFV DNA, beginning with 400 ng, were hybridized with 500 ng (! txg/ml of hybridization buffer) of sulphonated (H) or biotinylated pRPEL-2 DNA (G). The specificity was tested with 16 ~g of MS cell DNA (G 7, H 7) and 16 ~g of sMmon sperm DNA (G 8, H 8)

Detection of ASFV by DNA Hybridization

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Fig. 5. Hybridization with 3~P-pRPEL-2 DNA, Radioactivity of the spots of ASFV DNA hybridized with 3~P-pRPEI;2 DNA {specificactivity 6.4 × l06 cpm/~tg) (Fig. 2, Be) (©) ~nd hybridized with 3~P-pRPEL-2 DNA {specificactivity 1.92 × l0 s cpm/~g) (O). ~I~ecpm were determined 20 days after hybridization and are plotted ~gainst log nanograms DNA

Quantification of Spot Hybridization Direct quantification, achieved by scintillation counting of individual spots (Fig. 5), indicated t h a t the sensitivity of detection of ASFV DNA using ~2P-DNA probes with a specific activity of 1.93 × 10s cpm/~g was about ten times higher than t h a t found when probes containing 6.7 × 106 cpm per ~g were used.

Detection of Viral DNA in Infected MS Cells The assay for the detection of the number of infected cells was performed on MS cells infected with E 70 MS 46 virus. The limit of sensitivity was about 1.2 × 1Oa infected cells using a a2p-pRPEL-2 probe with a specific activity of 6.7 × 106 cpm per ~g (Fig. 6). This value could be increased to about l0 s

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infected cells using 3~P-pRPEL-2 p r o b e s with a specific activity of 1.93 × l0 s cpm p e r ~g. T h e c o n c e n t r a t i o n of viral DNA in the MS infected cells was a b o u t 312 ng viral DNA p e r million cells, which r e p r e s e n t e d a b o u t 1800 copies p e r cell.

Discussion P r e v i o u s studies s h o w e d t h a t the p R P E L - 2 plasmid could be u s e d to d e t e c t A S F V in field samples (2). This plasmid contains essentially the H-ClaI DNA f r a g m e n t from the Spain-E 70 strain of A S F V growing in pig

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leukocytes. The H-ClaI DNA fragment has a high genetic stability (2) and is localized between 0.495 and 0.53 map units (Fig. 7) (E. MARTIN and E. TABAR£S, manuscript in preparation). Of particular interest to the present study, were the optimal conditions for detection of ASFV by molecular hybridization with 32p., sulfonated or biotin-labeled probes. While as little as 20 pg of viral DNA, or 102 infected MS cells, were detectable using the 32p. pRPEI~2 probe, and 400 pg could be detected with the biotin-pRPEL-2 probe. This sensitivity is similar to that reported in other systems (1) and could be increased by using more of the viral genome in the DNA probe. This could be achieved with several recombinant plasmids in the same DNA probe. The fact t h a t t h e pI~PEL-2 DNA probe fails to hybridize to DNA from pig leukocytes (2), HSV-2, MS cells and salmon sperm indicates a high specifity for this method. The use of non radioactive DNA probes offers several advantages over the use of radioactive probes. Because of their chemical stability non radioactive probes can be used over long periods of time [at least 1-2 years (8)] while the radioactive probes present a hazard to the operator and have a very short half life. However the 32P-DNA probe is about 20 times more sensitive. Nevertheless, the use of non radioactive probes will be useful for detection of ASFV in laboratories where it is not possible to use radioisotopes.

Acknowledgments

I am grateful to J. Alvarez and M. C. Aguado for excellent technical assistance, to Dr. E. Domingo and A. J. Hargreaves for the help with the manuscript and M. A. Moratilla for typing it. This investigation has been. aided by a grant from Comision Asesora para el Desarrollo de la Investlgaei6n Cientifca y T6emca (Spain). o

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E. TABARI~S: Detection of ASFV by DNA Hybridization References

1. BRA~-DS~IAJ, MILLER G (1980) Nucleic acid spot hybridization: Rapid quantitative screening of lymphoid cell lines for Epstein-Barr viral DNA. Proc Natl Acad Sci USA 77:6851-6855 2. CABALLERORG, TARAR~S E (1986) Application ofpRPEL-2 plasmid to detectAfrican Swine Fever virus by DNA-DNA hybridization. Arch Virol 87:119-125 3. DORI~AN MA, BLAIR CD, COLLINS JK, BEAT¥ BJ (1985) Detection of Bovine Herpes virus 1 DNA immobilized on nitrocellulose by hybridization with biotinylated DNA probes. J Clin Microbiot 2 2 : 9 9 0 - 9 9 5 4. HYYPIA T, STALttAUDSKE P, VAINIONPAA 1:~, PETTERSSON V (1984) Detection of enteroviruses by spot hybridization. J Clin Microbiol 1 9 : 4 3 6 - 4 3 8 5. KANDA J, MELNICK JL (1959) In vitro differentiation of virulent and attenuated polioviruses by their growth characteristics on MS cells. J Exp Med 1 0 9 : 9 - 2 4 6. KAPUSCINSKY J, SKOOZYLASB (1977) Simple and rapid fluorimetric method for DNA microassay. Anal Biochem 8 3 : 2 5 2 - 2 5 7 7. Krl~BY KS (1965) Isolation and charaCterization of ribosomal ribonucleie acid. Biochem J 9 6 : 2 6 6 - 2 6 9 8. LEARY JJ, BRmATI DJ, WARD DC (1983) l~apid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or l~NA immobilized on nitrocellulose: Bio-blots. Proe Natl Aead Sei USA 8 0 : 4 0 4 5 - 4 0 4 9 9. MATTKEWS JA, BATKI A, [-IYND8 C, KRICKA LJ (1985) Enhanced chemiluminescent method for the detection of DNA dot hybridization assays. Anal Biochem 151: 2 0 5 209 10. ST~LItANDSKE P, PETTERSSON U (1982) Identification of DNA viruses by membrane filter hybridization. J Clin Microbiot t 5 : 7 4 4 - 7 4 7 11. TABARt~S E, FERN~-NDEZ M, SALVADOR TEMPRANO E, CARNERO ME, S/~NCHEZ BOTIZA C (1981) A reliable enzyme linked immunosorbent assay for African Swine Fever virus using the major structural protein as antigenic reagent. Arch Virol 70: 297-300 12. TcHE~¢ P, F u c R s RPP, SAGE E, LEna M (1984) Chemically modified nucleic acids as immunodetectable probes in hybridization experiments. Proc Natl Aead Sei USA 81: 3466-3470 13. WALBOOMERSJMM, TEn SCHEGaET J (1976) A new method for the isolation of herpes simplex virus Vype 2 DNA. Virology 7 9 : 2 5 6 - 2 5 8 Author's address: Dr. E. TARAR:g]S, Departamento de Microbiologia, Facultad de Medicina, Universidad Aut6noma de Madrid. Arzobispo Morcillo 4, E-28029 Madrid, Spain. l~eceived March 3, 1986