J Vet Diagn Invest 4:8-12 (1992)

Detection of bluetongue virus using a cDNA probe derived from genome segment 4 of bluetongue virus serotype 2 Jarasvech Chinsangaram, Salah Hammami, Bennie I. Osburn Abstract. The double-stranded (ds) RNA genome segment 4 of bluetongue virus (BTV) serotype 2 was cloned and used as a serogroup-specific complementary (c) DNA probe for BTV diagnosis. A cDNA representing a 60% copy of genome segment 4 of BTV-2 prototype was produced. The specificity of the cDNA probe was determined by hybridizing this probe to a northern blot of dsRNA (separated by polyacrylamide gel electrophoresis) of plaque-purified BTV-2 prototype. This cDNA probe was then used to hybridize to the RNA samples. Because the probe hybridized to all BTV samples but not to epizootic hemorrhagic disease virus samples, it appears to be a group-specific probe that could be used in BTV diagnosis.

Bluetongue virus (BTV), a double-stranded (ds) RNA virus, is the prototype virus in the genus Orbivirus, which is 1 of 6 genera in the family Reoviridae.5,12 There are 24 serotypes of BTV identified worldwide,7,10 among which BTV-2 has been either isolated or serologically detected in animals from most parts of the world, including Africa, the Middle East, India, Southeast Asia, North America, and South America. l5 The genome of BTV is composed of 10 segments of dsRNA, each of which encodes for at least 1 protein.11 The genome segments 2 and 5 encoding for the outer capsid proteins VP2 and VP5, which are responsible for the neutralization characteristic of the virus, have the highest sequence variability.9 Presently, laboratory methods for diagnosis are embryonated chicken egg (ECE) inoculation, serum neutralization (SN) test, agar gel immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA), and fluorescent antibody (FA) test. However, a cross-reaction between BTV and epizootic hemorrhagic disease virus (EHDV, of deer and other ruminants) has been reported using the serologic method, and the ECE inoculation method is time consuming.19 Consequently, the nucleic acid hybridization technique has been employed as an alternative method to increase the specificity, rapidity, and sensitivity of BTV diagnosis.17,19,22 The purpose of this study was to clone complementary (c) DNA of genome segment 4 of BTV-2, to use this radiolabeled cDNA as a probe for BTV diagnosis using a hybridization technique, and to compare the northern blot and slot blot hybridization techniques in the detection of BTV.

Materials and methods Virus. BTV-2, BTV-10, BTV-11, BTV-13, BTV-17, EHDV-1, and EHDV-2 were prototype virusesa used in this study. BTV field isolates were obtained during an epidemiologic study from 1979 to 1981 in 4 western states of the United States. Heparinized blood samples from sheep, cattle, goats, and elk were processed as described13 and inoculated into 10-11-day-old ECE. The ECE-adapted virus isolates were subsequently adapted to cell culture in either African green monkey kidney (VERO) cells or mouse fibroblast (L929) cells grown in Eagle’s medium. The virus-infected cells were incubated at 37 C until 100% cytopathic effect (CPE) or approximately 108 plaque-forming units/ml were observed, and the infective material (cell debris plus medium) was sonicated and stored at 4 C as previously described.18 The following numbers of field isolates were studied: 20 BTV10, 29 BTV-11, 20 BTV-13, 26 BTV-17, 5 EHDV.b,c Three milliliters of each virus isolate was added to a monolayer of baby hamster kidney (BHK-21) cells in a 75cm2 flask at an approximate multiplicity of infection of 10. The infected cells were incubated at 37 C in 5% CO, until 100% CPE was observed (about 72 hr). The cells were pelleted and transferred to a microcentrifuge tube for the extraction of the dsRNA. Virus purification. BTV-2 was purified by plaque picking 3 times. The serotype of the virus was confirmed by a plaque inhibition (disc) method as described.20 The serotype of the virus was also examined by comparing its electropherotype to that of all US prototypes (BTV-2, -10, -11, -13, and - 17). RNA extraction and purification. Monolayers of VERO cells were infected with plaque-picked BTV-2. The dsRNA was extracted by phenol as described2 and modified.18 The extracted dsRNA was purified and concentrated with a purification columnd following the manufacturer’s recommendation. The amount of RNA was measured using a spectrophotometer at 260 nm wavelength. The extracted dsRNA was separated into 10 segments on a 1% agarose gel. Genome segment 4 was excised, electroeluted in a dialysis bag containing TBE buffer (0.089 M Tris, pH 8.0; 0.089 M boric

From the Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616. Received for publication April 18, 1991. 8

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Detection of BTV using a cDNA probe

acid; 0.02 M ethylenediaminetetraacetic acid [EDTA], and ethanol precipitated. Polyadenylation of RNA. The dsRNA genome segment 4 of BTV-2 was polyadenylated at the 3' ends using Escherichia e 1 coli poly A polymerase as described. The reaction was stopped by the addition of 10% sodium dodecyl sulfate (SDS) and 250 mM EDTA. The polyadenylated RNA was recovered by Tris-EDTA-phenol : chloroform extraction and ethanol precipitation. cDNA synthesis and bacterial transformation. Purified polyadenylated RNA was used as a template for the cDNA synthesis using a cDNA synthesis system as described by the manufacturer.” The cDNA was inserted in the SmaI site of a dephosphorylated pUC-13 plasmid.e The cDNA inserted vector was used to transform the competent E. coli JM105 cells as previously described.16 The white colonies were checked for possible inserts by a rapid miniscreen procedure.3 Preparation of the cDNA probe. The plasmid DNA was extracted from JM105 cells, purified with a spin column,f and cleaved with SalI and SstI. The insert was electroeluted, 32 P-labeled using a random primer labeling system,g and used as a probe for the hybridization assay. The specific activity of 3 x 106 counts per minute/ml of this probe was used in every hybridization reaction. Northern blot hybridization. A 13% gel was used for polyacrylamide gel electrophoresis (PAGE) of 500 ng of each sample. Samples were then electroblotted onto the Zeta-probe membranes,h which were then air-dried and baked in a vacuum oven for 2 hr at 80 C and incubated in prehybridization buffer4 at 42 C for 3 hr in a continuous shaking condition. The prehybridization buffer was then replaced with cDNA probes and hybridization buffer.4 The hybridization reaction was allowed to proceed for 16 hr at 42 C in a continuous shaking condition. The membranes were then washed 3 times at room temperature for 5 min with 0.2 x standard saline citrate (SSC) and 0.1% SDS, and then washed 2 times at 42 C for 15 min with 0.1% SSC and 0.1% SDS. The membranes were then exposed to X-ray films for 2 hr. Slot blot hybridization. The extracted RNA of BTV and EHDV from the virus-infected cell culture was resuspended in diethylpyrocarbonate-H2O and denatured with 0.3 x SSC and 7.4% formaldehyde at 65 C for 20 min. Five hundrednanogram RNA samples were blotted onto a nitrocellulose membrane of 0.45-µm-diameter pore size using a vacuum filtration apparatus.’ The nitrocellulose papers were baked in a vacuum oven for 2 hr at 80 C and used in the hybridization reaction as described above. To compare the sensitivity between northern blot and slot blot techniques, BTV-2 RNA was 2-fold serially diluted and subjected to both northern blot and slot blot hybridizations. Membranes were exposed to the X-ray film for 3, 24, and 72 hr.


Figure 1. Northern blot hybridization of US BTV prototypes. BTV-2, BHK-21 cell, BTV-10, BTV-11, BTV-13, BTV-17, EHDV1, and EHDV-2 were fractionated on 13% polyacrylamide gels, northern blotted onto a Zeta-probe membrane, and hybridized with 32 P-cDNA. The cDNA hybridized to genome segment 4 of every BTV prototype but not the BHK-21 cell, EHDV-1, or EHDV-2.

PAGE-separated genome segments of plaque-purified BTV-2 prototype (Fig. 1). The RNA samples obtained from the field isolates of BTV and EHDV were assembled by serotype, and 500 ng of each sample were electrophoresed by PAGE. The gels showed the inconsistency of the electrophoretic pattern of each RNA sample even within the same serotype. The electropherotypes of EHDV samples were indistinguishable from those of BTV samples. The cDNA probe was hybridized to US BTV prototypes (BTV-2, -10, -11, -13, and -17) and EHDV prototypes (EHDV-l and -2). The strongest hybridization signal was obtained from BTV-2 prototype (Fig. 1). The other US BTV prototypes yielded similar signal intensities, whereas neither EHDV prototype provided a hybridization signal. All signals were detected on the X-ray film at the position of genome segment 4 of each prototype. There was no hybridization signal between this probe and the extract of uninfected BHK-21 cells. Ninety-five BTV field isolates hybridized to the probe equally well (11 isolates of each serotype shown in Fig. 2), in spite of the differences in serotypes, host species, time, and locations of isolations. However, this probe Results failed to hybridize to all 5 EHDV field isolates (data The cDNA insert had internal restriction sites for not shown). In the slot blot hybridization, the RNA samples were EcoRI and BamHI; therefore, SalI and SstI were used to cut the cDNA insert out of the plasmid. The recov- arranged according to the serotype and the host species. ered cDNA had an estimated size of 1,200 base pairs The probe hybridized to every BTV prototype and (bp). The 32P-labeled probe hybridized specifically to BTV field isolate but failed to hybridize to EHDV genome segment 4 of BTV-2 in a northern blot of prototypes and EHDV field isolates (Fig. 3). However,

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Chinsangaram, Hammami, Osbum

Figure 2. Northern blot hybridization of BTV field isolates. All samples were fractionated on 13% polyacrylamide gels, northern blotted onto a Zeta-probe membrane, and hybridized with 32P-cDNA. A. BTV-10. B. BTV-11. C. BTV-13. D. BTV-17. Lane 2 is BTV-2 prototype and lane BHK is BHK-21 cell (negative control).

the intensities of the signals were inconsistent and different from those of the same samples in northern blot hybridization even though identical samples and probe were used in both methods. Under the same stringency, the northern blot hybridization provided better sensitivity than did the slot blot hybridization. The sensitivity of the norther blot hybridization was at 0.78 ng or 3.6 x 1O7 virus particles (13 x 103 kD of total genome per virus particle)6 (Fig. 4), whereas the sensitivity of the slot blot hybridization was at 6.25 ng or 2.9 x 108 virus particles (Fig. 5). Discussion The electropherotype of BTV field isolates was inconsistent even within the same serotype and was indistinguishable from that of EHDV field isolates. Similar results have previously been reported,8,18 indicating that electropherotyping alone is not sufficient for BTV diagnosis. Genome segment 4 encodes for the viral protein VP4, which is a minor component of the inner core of BTV. This genome segment is highly conserved within BTV serogroups, making its cDNA suitable as a serogroup-specific probe for BTV diagnosis. Based on the length of genome segment 4 of BTV10 that was previously reported,15 this cDNA probe represents 60% of the length of genome segment 4 of BTV-2 (1,200/2,011 bp). The cDNA probe was tested for specificity and hybridized to genome segment 4 on a northern blot of plaque-purified BTV-2. The result indicated that this cDNA probe was indeed the desired cDNA. One hundred field isolates and 7 prototypes of BTV and EHDV were used to test the efficiency of this cDNA

Figure 3. Slot blot hybridization. The field isolates of BTV-10, BTV-11, BTV-13, BTV-17, and EHDV were sorted according to serotypes and host species, blotted onto a nitrocellulose membrane, and hybridized with 32P-cDNA. Left. 1A, BTV-2. 1B, BHK-21. Row 2-4, 5A, row 7-9, 10A; BTV-10 field isolates. Row 12-14, 15A, 15B, row 17-19, row 21-23; BTV-11 field isolates. Right. 1A, BTV2. 1B, BHK-21. Row 2-5, 6A, 6B, 8A, 8B, 9A, 9B; BTV-13 field isolates. Row 11, 12, 13A, 13B, row 15-18, row 20, 21; BTV-17 field isolates. Row 23, 24A, 24B; EHDV field isolates.

as a diagnostic tool. The probe was serogroup specific for BTV diagnosis with no cross-reaction to EHDV. By using 2 different hybridization techniques, the same result was obtained; this cDNA probe hybridized to all BTV samples but not to EHDV samples. In a previous study, a cDNA cone of genome segment 4 of BTV-10 was hybridized to 20 serotypes of BTV (BTV-1-20), EHDV-1, and EHDV-2.14 The hybridization signal was detected from all 20 serotypes of BTV. A weak hybridization signal was also detected from EHDV-2, indicating a cross-hybridization. There are 3 possible explanations for the cross-hybridization with EHDV noted in the previous study and the lack of cross-hybridization observed in our study. First, cDNA in the previous study might have contained a higher proportion of conserved sequence/variable sequence between the BTV and EHDV genomes as compared with the cDNA used in our study. The probability that the previous cDNA would anneal to genome segment 4 of EHDV (a virus closely related to BTV) would then be higher. Second, the difference in the

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Detection of BTV using a cDNA probe


Figure 5. Slot blot hybridization-sensitivity test. BTV-2 prototype was 2-fold serially diluted starting at 200 ng/µl. These samples were dot blotted onto a nitrocellulose membrane and hybridized with 32P-cDNA. 1A = 200 ng/µl; 1B = 100 ng/µl; 1C = 50 ng/µl; 2A = 25 ng/µl; 2B = 12.5 ng/µl; 2C = 6.25 ng/µl. Figure 4. Northern blot hybridization-sensitivity test. BTV-2 prototype was 2-fold serially diluted starting at 200 ng/µl. These samples were fractionated on 13% polyacrylamide gels, northern blotted onto a Zeta-probe membrane, and hybridized with 32P-cDNA.

hybridization requires several more steps and takes more time than does the slot blot technique. Hybridization tests can effectively reduce the time required to identify BTV in samples. Conventional methods of serotyping are more costly and require l3 days longer to interpret than does the hybridization assay. Furthermore, hybridization assays are specific, and as such they are more reliable than fluorescent antibody techniques in the hands of inexperienced technicians.

hybridization buffer systems, including the ion concentrations and ion components used in these 2 experiments, may be responsible for the different outcomes. Third, genome segment 4 of BTV-10 could have higher sequence homology with the corresponding segment of EHDV-2 than does genome segment 4 of BTV-2. Based on this assumption, the cDNA of genome segment 4 of BTV-2 would not hybridize to Acknowledgements that of EHDV-2. On the other hand, the cDNA of genome segment 4 of BTV-10 containing partially We thank Dr. J. Pearson USDA/APHIS, NVSL, Ames, homologous sequence to that of EHDV-2 would hy- IA, and Dr. A. Galina, Washington Animal Disease Diagbridize to EHDV-2. nostic Laboratory, Pullman, WA, for providing us with EHDV The slot blot hybridization is very useful for diag- field isolates. nostic purposes because it requires less time than does Sources and manufacturers the northern blot hybridization (1 day vs. 2 days). However, an inconsistency in the signal intensity has a. Arthropod-Borne Animal Disease Research Laboratory, Denver, CO. been reported21 and was also observed in this study. Because of this inconsistency, quantitative analysis us- b. National Veterinary Services Laboratory, USDA/APHIS, Ames, IA. ing slot blot hybridization would be difficult. The reac. Washington Animal Disease Diagnostic Laboratory, Pullman, son for the inconsistency has not been explained. WA. The sensitivity of the northern blot and slot blot d. DuPont Co., NEN Research Products, Boston, MA. hybridizations was compared using this cDNA probe. e. Pharmacia Co., Piscataway, NJ. f. 5 Prime — 3 Prime, Inc., West Chester, PA. The northern blot hybridization was 8 times more seng. Amersham Co., Arlington Heights, IL. sitive than the slot blot hybridization under the same h. Bio-Rad Laboratories, Richmond, CA. stringency condition (0.78/6.25 ng of extracted BTV i. Schleicher & Schuell, Woburn, MA. dsRNA). The northern blot hybridization has many advanReferences tages over the slot blot hybridization besides the greater 1. Cashdollar LW, Esparza L, Hudson GR, et al.: 1982, Cloning sensitivity. The northern blot hybridization of PAGEthe double-stranded RNA genes of reovirus: sequence of the separated RNA provides the information about the cloned S2 gene. Proc Nat1 Acad Sci USA 79:7644-7648. relatedness between 2 samples and specifies the ge- 2. Clarke IN, McCrae MA: 1981, A rapid and sensitive method for analysing the genome profiles of field isolations of rotavirus. nome segment that is hybridized. Moreover, false posJ Virol Methods 2:203-209. itives due to nonspecific binding can also be ruled out 3. Close TJ, Rodriguez RL: 1982, Construction and characterif the hybridization signal is detected on the membrane ization of the chloramphenicol-resistant gene cartridge: a new in a position other than that of the fixed corresponding approach to the transcriptional mapping of extrachromosomal RNA genome segment. Nevertheless, the northern blot elements. Gene 20:305-316.

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4. de Mattos CC, de Mattos CA, Osbum BI: 1989, Recombinant DNA probe for serotype-specific identification of bluetongue virus 17. Am J Vet Res 50:536-541. 5. Els H J, Verwoerd DW: 1969, Morphology of bluetongue virus. Virology 38:213-219. 6. Fukusho A, Yu Y, Yamaguchi Y, Roy P: 1989, Completion of the sequence of bluetongue virus serotype 10 by the characterization of structural protein VP6 and a nonstructural protein NS2. J Gen Virol 70:1677-1689. 7. Gorman BM, Taylor J, Walker PJ: 1983, The Reoviraedae. In: Orbiviruses, ed. Joklik WK, pp. 287-357. Plenum, New York, NY. 8. Gorman BM, Taylor J, Walker PJ, et al.: 1981, Comparison of bluetongue type 20 with certain viruses of the bluetongue and Eubenangee serological groups of orbiviruses. J Gen Virol 57: 251-261. 9. Huismans H, Cloete M, Le Roux A: 1987, The genetic relatedness of a number of individual cognate genes of viruses in the bluetongue and closely related serogroups. Virology 16 1: 421-428. 10. Knudson DL, Shope RE: 1985, Overview of the orbiviruses. In: Bluetongue and related orbiviruses, ed. Barber TL, Jochim MM, pp. 255-266. Alan R. Liss, New York, NY. 11. Mertens PPC, Brown F, Sanger DV: 1984, Assignment of the genome segments of bluetongue virus type 1 to the proteins they encode. Virology 135:207-217. 12. Murphy FA, Borden EC, Shope RE, Harrison A: 1971, Physicochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus- a new taxonomic group. Electron microscope studies. J Gen Virol 13:273-280. 13. Osbum BI, McGowan B, Heron B, et al.: 1981, Epizootiologic

study of bluetongue: virologic and serologic results. Am J Vet Res 42:884-887. 14. Ritter DG, Roy P: 1988, Genetic relationships of bluetongue virus serotypes isolated from different parts of the world. Virus Res 11:33-44. 15. Roy P: 1989, Bluetongue virus genetics and genome structure. Virus Res 12: 179-206. 16. Sambrook J, Fritsch EF, Maniatis T: 1989, Molecular cloning: a laboratory manual, 2nd ed., pp. 1.82-1.84. Cold Spring Harbor Laboratory Press, New York, NY. 17. Schoepp RJ, Blair CD, Roy P, Beaty BJ: 1991, Detection of bluetongue virus RNA by in situ hybridization: comparison with virus isolation and antigen detection. J Vet Diagn Invest 3:22-28. 18. Squire KRE, Osbum BI, Chuang RY, Doi RH: 1983, A survey of electropherotype relationships of bluetongue virus isolates from the western United States. J Gen Virol 64:2103-2115. 19. Squire KRE, Stott JL, Dangler CA, Osbum BI: 1987, Application of molecular techniques to the diagnosis of bluetongue virus infection. Prog Vet Microbiol Immun 3:235-250. 20. Stott JL, Barber TL, Osbum BI: 1978, Serotyping bluetongue virus: a comparison of plaque inhibition (disc) and plaque neutralization methods. Proc Annu Meet Am Assoc Vet Lab Diagn 21:399-410. 21. Unger RE, Chuang RY, Chuang LF, et al.: 1988, Comparison of dot-blot and northern blot hybridizations in the determination of genetic relatedness of United States bluetongue virus serotypes. J Virol Methods 22:273-282. 22. Wechsler SJ, Austin KJ, Wilson WC: 1990, Limits of detection of bluetongue virus with different assay systems. J Vet Diagn Invest 2:103-106.

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Detection of bluetongue virus using a cDNA probe derived from genome segment 4 of bluetongue virus serotype 2.

The double-stranded (ds) RNA genome segment 4 of bluetongue virus (BTV) serotype 2 was cloned and used as a serogroup-specific complementary (c) DNA p...
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