Journal of Virological Methods, 30 (1990) 15-24 Elsevier

VIRMET

15

01060

Development of the polymerase chain reaction for the detection of bluetongue virus in tissue samples A.M. Wade-Evans,

P.P.C. Mertens and C.J. Bostock

AFRC, institute for Animal Health, Pit-bright Laboratory, (Accepted

6 June

Wok@,

U.K.

1990)

Summary Total genomic dsRNA, extracted from purified core particles of bluetongue virus serotype 1 from South Africa (BTVl SA), was used as template to optimise a polymerase chain reaction (PCR) for the detection of bluetongue virus RNA. Pairs of oligonucleotides complemental to the 3’ termini of eight of the ten genome segments were tested. Those representing the 5’ termini of genome segment 7 gave the best amplification results producing a single DNA band with the same mobility during agarose gel electrophoresis as genome segment 7. It was confirmed by cloning and sequence analysis, that this PCR-amplified DNA contained both terminal regions of genome segment 7 and therefore represented full length cDNA. Using these segment 7 oligonucleotides it was not only possible to detect routinely as few as 6 molecules of segment 7 dsRNA per sample, but also to detect purified dsRNAs from isolates of other BTV serotypes (1 Australia (AUS), 2, 3, 4, 10, 16 and 20). However, with the exception of Tilligery virus, isolates from other Orhivirus serogroups tested all gave negative results (African horse sickness, epizootic haemorrhagic disease, Palyam, Warrego and Eubenangee). The PCR was also used to analyse red blood cells (RBC) and buffy coat samples from cattle infected with BTV4. Positive results were obtained from samples taken 7 days post-infection (p.i.) (containing 1.6 x lo” TCIDSO of virus/ml of whole blood) and from the RBC sample only, taken 14 days p.i. (16 TCID=&ml).,However, at 28 days p.i. (< 1.6 TCID&ml) BTV RNA was not detected using the PCR in either sample. Bluetongue

virus; PCR; Diagnosis

Correspondenc’e Road, Woking,

to: A.M. Wade-Evans, AFRC, Surrey, UU24 OND, U.K.

016&8510/90/$03.500

Institute

1990 Elsevier Science Publishers

for Animal

Health,

B.V. (Biomedical

Pirbright

Division)

Laboratory,

Ash

16

Introduction Bluetongue virus (BTV) is the prototype member of the O~hivirus genus, within the family Reoviridae. The BTV serogroup contains 24 serotypes, defined by their reaction in serum neutralisation tests (Gorman et al., 1983). The virus particles contain a ten-segmented, double-stranded RNA genome within a doubleshelled protein capsid. The inner capsid layer is composed of two major and three minor components (VP3, VP7 and VP1 , VP4. VP6 respectively). The outer capsid is composed of two additional major proteins, VP2 and VP5 and in some isolates has been shown to contain protein NS2, as a minor component (Met-tens et al., 1987a). At least three non-structural proteins have also been identified (NSl, NS2 and NS3), although their role in the replication of the virus is as yet unclear. BTV infects cattle, goats, wild ruminants and sheep, although the clinical symptoms in the latter are the most severe. Diagnostic tests which are currently used for the detection of bluetongue virus involve the isolation and growth of virus isolates in eggs or mice, followed by passage in tissue culture. The virus is subsequently characterised by serological means, involving its reaction with reference antisera, for example, agar gel immunodiffusion tests (AGID), or serum neutralisation tests (Gorman et al., 1983). These procedures are time consuming and may fail to detect low levels of infectious virus or strains of BTV which fail to replicate in eggs, mice or tissue culture. The use of the ELISA for the detection of antibodies to BTV in previously infected animals is faster (Anderson, 1984) but in the absence of a highly sensitive and specific test for BTV antigen(s) these serological techniques do not allow direct detection of the virus itself in tissue samples (for example blood or semen). Viruses belonging to the bluetongue (BTV), epizootic haemorrhagic disease (EHD), Eubenangee and Palyam Orhivirus serogroups can exhibit cross-reactions in some serological tests (Borden et al., 1971; Moore and Lee 1972; Della-Porta et al., 1979) and it has been suggested that they cluster to form a subgenus (Gorman, 1979; Gorman et al., 1983; Gorman and Taylor 1985; Della-Porta et al., 1982). However, the use of a BTV-specific monoclonal antibody, against BTV protein VP7 in a competition ELISA, effectively overcomes this problem (Anderson, 1984). We have assessed the use of the polymerase chain reaction (PCR) for the direct detection of BTV RNA sequences using purified genomic dsRNA and total nucleic acid extracts from infected blood samples. The absolute sensitivity of the PCR for detection of BTVlSA genome segment 7 (coding for the core structural protein and BTV group antigen, VP7) together with its ability to distinguish between isolates from several BTV serotypes, was analysed. The potential value of this procedure both as a diagnostic test and for the synthesis of BTV cDNA is discussed.

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Materials and Methods Purification

of viral dsRNA

The method used for the growth of BTV in BHK cells and subsequent virus purification is described by Met-tens et al. (1987a). The isolates of BTVlSA, BTVlAUS, 3, 4, 10 and 20 are as described previously (Mertens et al., 1987b). Purification of RNA and source of isolates of BTV2, 4 and 16, together with EHDVl, EHDV2, Palyam, Warrego and Tilligery viruses are described by Mohammed (1988). Extraction of dsRNA from purified virus particles is described by Mertens et al. (1984). Method of cDNA synthesis The pairs of oligonucleotides used as primers for cDNA synthesis were complementary to the 3’ termini of both strands of each genome segment, the sequences of which were determined by Met-tens et al. (1985). The terminal sequences of segment 7 were obtained from a full length cDNA clone of genome segment 7 from BTVlSA, obtained using a modification of the method described by Gubler and Hoffman (1983) (Wade-Evans and Mertens, manuscript in preparation). The method used for cDNA synthesis is based on that described by Doherty (1989). 200 ng of dsRNA (containing all ten genome segments), in 2.5 ~1, was denatured with an equal volume of 0.02 M methyl mercuric hydroxide prior to being used as a template for cDNA synthesis. After reduction of the methyl mercuric hydroxide by addition of 1 ~1 0.7 M b-mercaptoethanol, 2 ~1 of RNAsin (Promega 40 units/pi) was added. 4 ~1 of the RNA mix (100 ng) was diluted to 20 ~1 in 50 mM Tris, pH 8.8, 75 mM KCl, 1 mM DTT, 1 mM dNTPs, 3 mM MgC12 containing 5 pmol each of a pair of primers (approximately 9.25 PM - kinased prior to use). 200 units of mouse moloney leukaemia virus reverse transcriptase (MMLV-RT-BRL) were added to the reaction mix which was then incubated at 37°C for 30 min. Polymerase

chain reaction (PCR)

The cDNA reaction mix was diluted to 100 ~1 containing a final concentration of 10 mM Tris, pH 8.8, 1.5 mM MgC12, 50 mM KCl, 200 PM dNTPs, 5 pmol of each primer (approximately 0.05 ,QM) and 0.01% gelatin. The mix was heated to 95OC for 2 min, then 2 units of Taq polymerase (Cambio) were added prior to incubation on an intelligent heating block (Hybaid) for 40 cycles of 45OC for 1 min, 70°C for 2 min, 95OC for 1 min, followed by 1 cycle of 45OC for 1 min and 70°C for 10 min. 20 ,ul samples were analysed on 0.8% agarose gels, run at 200 V for 45 min and stained with ethidium bromide (1 fig/ml). A further 2 units of enzyme were added if more PCR cycles were necessary.

Extraction from agarose,

cDNA cloning and sequencing

The methods used have previously been described by Wade-Evans et al. (1988). Calculation

of number of molecules

of dsRNA used as template

The concentration of purified BTV dsRNA was calculated from measurement of optical density at a wavelength of 260 nm (assuming that a solution with an optical density of 1 will contain 66 &ml). The total molecular weight of the BTV genome has been calculated to be 13 x 10” Da (Roy 1989). Therefore 2.1 x lo-l7 g of BTV dsRNA is equivalent to a single copy of the whole genome (one copy of each of the ten segments). Preparation

of blood samples for use in PCR

Blood was collected from the cattle in heparin-treated tubes. The red blood cells (RBC) and buffy coat were separated from the plasma by centrifugation at 2000 rpm for 10 min. Each fraction was washed 3 times in phosphate buffered saline (PBS). The buffy coat was resuspended in hypertonic saline to remove any contaminating RBCs. Both RBC and buffy coat were sonicated in a small volume of PBS prior to treatment with proteinase K (50 @ml) and SDS (0.1%) for 3 h at 37OC. Both fractions were then phenol extracted followed by ethanol precipitation and stored at -2OOC. Immediately prior to the PCR, samples stored under ethanol were warmed to room temperature, spun for 10 min at 2500 rpm and the pellet resuspended in 100 ~1 dH20. 2 ~1 samples were then denatured as described earlier prior to cDNA synthesis followed by the PCR. Virus isolation from cattle inoculated

with B7’V4

Virus isolations from heparinised blood were made using BHK monolayers as indicator cells (Jeggo et al., 1983). The viraemias are expressed as TCIDso/ml of the blood sample.

Results Eight pairs of oligonucleotides, complementary to the 3’ termini of genome segments 1, 2, 3, 5, 6, 7, 8 and 10 of BTVlSA respectively (Table l), were tested as primers in the PCR, using the conditions described in Materials and Methods. As can be seen in Fig. 1 (track 7) the oligonucleotides representing segment 7 sequences gave the best results. Using these primers the PCR amplified a DNA band that co-migrated in 0.8% agarose gels with the dsRNA segment 7, indicating that this material contained full length cDNA copies (1156 nucleotides, Wade-Evans and Mertens, manuscript in preparation). Other pairs of primers, for segments 2, 5 and 10, also produced amplified bands co-migrating with their corresponding

19 TABLE 1 Oligonucleotideprimers used in the FCR 1B 5’ G’ITAAAATGC’M-A A’ITCGTAAAATTG 5’ 1T 2B 5’ G’ITAAAATIUTAGCGCG GTCTAGGCGCGTGATAATCTGAATG 5’ 2T 3B 5’ G’ITAAATTTCCGTA TTGTGTGAATG 5’ 3T 5B 5’ GTTAAAAAGTGCCCCCTTAGCG ‘ITCGTGAATGTGAATG 5’ 5T 6B 5’ GTTAAAAAAGTKTCT TGATCTTGAAAAGTTGAATG 5’ 6T 7B 5’ GTTAAAAATCTATAGAG AGAGAATCTMTGTGAATG 5’ 7T 8B 5’ GTTAAAAAATCCTAGAGT CCCCCCTAAAATGTGAATG 5’ 8T 1OB 5’ G’ITAAAAAGTGTCGCTGCCATGCTATCC TCCCATACGCCGCGATGTGTGAATG 5’ 1OT

genome segments (Fig. 1). However, they also gave numerous other bands, making them less suitable for use under these particujar PCR conditions. When the 70°C elongation step in the PCR cycle was increased from 2 to 5 minutes the quantity of full length material for genome segments 2 and 5 was increased and the other amplified bands decreased in relative intensity (data not shown). Use of the primers for segments 1, 3, 6 and 8 did not give any detectable amplification. cDNAs extracted from bands co-migrating with segment 7 or 10 (tracks 7 and 9 of Fig. 1, respectively) were cloned into pZP19, a derivative of pUC19 (Pan and Bostock 1988) and the resulting clones were analysed to ensure their integrity. 200 bases were sequenced at each terminus from twelve independently derived clones (six of each segment). 400 nucleotides of sequence represent approximately half of segment 10 (822 nucleotides - Wade-Evans and Mertens, manuscript in preparation) and a third of segment 7 (1156 nucleotides - WadeEvans and Mertens, manuscript in preparation). No base changes were observed between any of the cDNAs cloned via the PCR method and the clones produced in a more conventional manner as described by Wade-Evans et al. (1988). Genome segment 7 of BTVlSA encodes the serogroup-specific antigen, VP7 (Gumm and Newman, 1982; Mertens et al., 1984). The results obtained with the PCR suggest that primers complementary to the 3’ termini of segment 7 might be suitable for the development of a BTV serogroup-specific diagnostic test using the PCR technique. The segment 7 primers were used in the PCR with dsRNA from a range of other Orbivirus isolates (Fig. 2). Although an amplified band of cDNA is not clearly visible for BTV3 (track 3), on repeating the PCR with another isolate of BTV3 RNA, freshly prepared from infected BHK cell lysates,

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Fig. 1 Use of oligonucleotides complementary to each genome segment of BTVlSA in the PCR: The samples were analysed by agarose gel elec~ophoresis as in Materials and Methods. The tracks labelled M represent lambda Hind111 digests used as lnigration markers. Tracks I and IO are samples of purified BTVI SA dsRNA (500 ng). The remaining tracks are PCR samples using 40 reaction cycles and 100 ng BTVl SA dsRNA as template (all ten segments) in each case. Each of the reactions contained different primers complementary to the termini of both strands of segment 1 (track 2), segment 2 (track 3), segment 3 (track 4) segment 5 (track 5). segment 6 (track 6) segment 7 (track 7), segment 8 (track 8) and segment IO (track 9).

positive results were obtained with amplified bands equal in intensity to those obtained with BTVlSA (data not shown). From the results it is clear that these primers can be used to amplify, and therefore to detect, RNA of the isolates tested from BTV serotypes 1AUS, 2, 3, 4, 10, 16 and 20. However, with the exception of Tilligery virus (a serotype from the Eubenangee serogroup), these primers did not appear to result in the amplification of genome segment 7 of isolates from other orbivirus serogroups, including EHDV, Palyam and Wart-ego (Fig. 2) and AHS and certain serotypes of Eubenangee (data not shown), and could not therefore be used for their amplification and detection by the PCR. The minimum amount of BTVl SA dsRNA that could be detected under optimised PCR conditions was determined in order to evaluate the sensitivity of the technique. Fig. 3 shows the quantities of cDNA produced by the PCR after sixty reaction cycles using dsRNA at dilutions calculated to be equivalent to between 48 and 1 molecule of genome segment 7 per reaction. A positive result was consistently obtained using 6 (Fig. 3, track 4) or more molecules as template and on several occasions as few as 2 molecules were detectable.

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Fig. 2. PCR on RNA isolates from different Orbivhus serogroups including several distinct BTV serotypes: PCR samples were analysed by agarose gel electropho~is as described in Materials and Methods. Primers complementary to BTVt SA segment 7 were used in all tracks for PCRs of 40 cycles with purified dsRNA from 1) BTVlAUS, 2) BTVZ, 3) BTV3, 4) BTV4, 5) BTVIO, 6) BTVlG, 7) BTV20, 8) EHDl, 9) EHD2, 10) Palyam, 11) Palyam, 12) Warrego, and 13) Tilligery, as template.

The PCR was also used to test blood samples taken from BTV4 infected cattle (Fig. 4). Positive results were obtained with both RBC and buffy coat fractions taken 7 days p.i. (viraemia of 1.6 x lo”), with RBC only at 14 days p.i. (16 TCID~~/ml) and in neither fraction at 28 days p.i. (cl.6 TCID5o/ml).

Discussion The conditions described for the PCR reproducibly allow detection of as few as 6 molecules of dsRNA, after sixty reaction cycles. It seems unlikely that in the majority of experimental procedures that this level of detection will be necessary. Forty ~plification cycles were therefore used routinely as ‘standard’ PCR procedure. This also reduces the risk of false positive results due to very low level contamination of samples with cloned material from the laboratory. The segment 7 primers are currently being tested in PCRs for the amplificatian and detection of RNA from isolates of the remaining BTV serotypes. It seems likely that the failure of the PCR to amplify segment 7 of one isolate of BTV3 was caused by repeated freeze-thawing of the sample. It may therefore be important to store the dsRNA samples in aliquots under ethanol. From the results obtained

22

Fig. 3. Determination of the limit of detection of BTV dsRNA by the PCR: The PCR samples, using 60 cycles and varying quantities of BTVlSA dsRNA template, were analysed by agarose gel electrophoresis. The marker lanes (labelled Mf contain DNA from I~~a-~~~dIII digests. PCR reactions were run containing template dsRNA calculated to be equivalent to If 48 molecules, 2) 24 molecules, 3) 12 molecules, 4) 6 molecules, 5) 3 molecules, 6) 2 molecules, 7) 1 molecule, 8) no dsRNA.

to date it seems likely that the PCR, using segment 7 oligonucleotide primers, will be able to detect RNA from most, if not all, of the 24 BTV serotypes. The positive PCR results obtained with dsRNA of Tilligery virus were confirmed using freshly prepared template from total nucleic acid extracts from BHK cell lysates infected with reference stocks of the virus. The DNA from this PCR-amplified material is currently being sequenced in order to determine the degree of relationship observed between the BTV and Eubenangee serogroups (Della-Porta et al., 1979, Mohammed, 1988), as indicated by the very similar, or identical, RNA genome profiles for BTVlSA and Tilligery virus when analysed by 10% PAGE or 1% agarose gel electrophoresis, respectively. The PCR failed to detect RNA from isolates of the other Orhivirlcs serogroups tested, including EHDV, AHS, Warrego, Palyam and an isolate of Eubenangee virus from the Eubenangee serogroup. It is therefore largely BTV serogroupspecific under the reaction conditions described. Further studies with other virus isolates, possibly using different reaction conditions, or primers from other BTV genome segments may help to increase the specificity of the PCR for BTV diagnostic purposes. The PCR described in this paper can produce full length cDNA copies from very small amounts of dsRNA template directly from infected blood samples.

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Fig. 4. Analysis of PCRs on blood samples from cattle infected with BTV4 by agarose gel electrophoresis: Lane M contains a Hind111 digest of lambda DNA. Tracks 1, 2 and 3 contain samples of PCR using total nucleic acid extracted RBC as template, taken If 7 days post-infection (p.i.), 2) 14 days p.i. and 3) 28 days p.i. Tracks 4, 5 and 6 contain samples of PCR using total nucleic acid extracted from the buffy coat fraction taken 4) 7 days p-i., 5) 14 days p.i. and 6) 28 days pi.

This technique, therefore, seems highly suitable for the synthesis of cDNA for future cloning, sequencing and expression studies.

Acknowledgements We thank Mr R. Chamberlain for advice and assistance in setting up the PCR, Dr J. McCauley for the synthesis of the oligonucleotides, Miss A. Corteyn for virus titrations, Dr M. Jeggo for providing the infected bovine blood samples, Mr J.N. Burroughs for purified preparations of BTV particles and Dr M.E.H. Mohammed for RNA samples.

References Anderson, J. (1984) Use of monoclonal antibodies in a blocking ELISA to detect group specific antibodies to bluetongue virus. J. Immunol. Methods 74, 139-149. Anderson, J. (1986) Investigations on bluetongue virus using monoclonal antibodies. Ph.D. Thesis. Borden, E.C., Shape, R.E. and Murphy, F.A. (1971) Physicochemical and morphological relationship of some arthro~~~me viruses to bluetongue virus - a new taxonomic group: physicochemical

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and serological studies. J. Gen. Virol 13, 261-271. Della-Porta. A.J., McPhee, D.A and Snowdon, W.A. (1979) The serological relationships of Orbiviruses. In: T.D. St. George and E.L. French (Eds), Arbovirus Research in Australia. Proceeding 2nd Arbovirus Symposium, July pp. 64-71. Della-Porta, A.J., McPhee, D.A., Parsonson, I.M. and Snowdon, W.A. (1982) Classification of the Orbiviruses. Confusion in the use of terms bluetongue virus, bluetongue-like viruses. bluetonguerelated viruses and overall nomenclature. Aus. Vet. J. 58. 164-165. Doherty. P.J., Huesca-Contreras, M., Dosch, H.M. and Pan, S. (1989) Rapid amplification of complementary DNA from small amounts of unfractionated RNA. Anal. Biochem. 177, 7-10. Gorman, B.M. (1979) Variation in Orbiviruses. J. Gen. Virol. 44, I-15. German, B.M., Taylor, J. and Walker, P.J. (1983) ‘Orbiviruses’. In: W.K. Joklik (Ed.), The Reoviridae. Plenum Press, New York. German, B.M. and Taylor. J. (1985) Orbiviruses. Virology. 907-925. Gubler. U. and Hoffman, B.J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. Gumm, I.D. and Newman, J.F.E. (1982) The preparation of purified Bluetongue virus group antigen for use as a diagnostic reagent. Arch. Virol. 72, 83-93. Jeggo, M.H., Gumm, I.D. and Taylor, W.P. (1983) Clinical and serological response of sheep to serial challenge with different BTV types. Res. Vet. Sci. 34, 205-211. Mertens, P.P.C., Brown, F. and Sangar, D.V. (1984) Assignment of the genome segments of BTV type 1 to the proteins which they encode. Virology 135, 207-217. Mertens, P.P.C. and Sangar, D.V. (1985) Analysis of the terminal sequences of the genome segments of four orbiviruses. Virology 140, 5567. Mertens. P.P.C., Burroughs, J.N. and Anderson, J. (1987a) Purification and properties of virus particles, infectious subviral particles and cores of bluetongue virus serotypes 1 and 4. Virology 157, 375-386. Mertens, P.P.C., Pedley, S.. Cowley, J. and Burroughs, J.N. (1987b) A comparison of six different bluetongue virus isolates by cross-hybridisation of the dsRNA genome segments. Virology 161, 438447. Mohammed, M.E.H. (1988) Epidemiological studies on some Sudanese arboviruses. Ph. D. Thesis, University of Reading. Moore, D.L. and Lee, V.H. (1972) Antigenic relationship between the virus of epizootic haemorrhagic disease of deer and bluetongue virus. Arch. Ges. Virusforsch. 37. 282-284. Pan, Z.Q. and Bostock. C.J. (1988) A cloning vector allowing excision of inserts with original termini irrespective of their sequence. Nucleic Acids Res. 16. 8183. Roy, P. (1989) Bluetongue virus genetics and genome structure. Virus Res: 13, 179-206. Wade-Evans, A.M., Pan, Z.Q. and Mertens. P.P.C. (1988) Sequence analysis and in vitro expression of a cDNA clone of genome segment 5 from BTV, serotype 1 from South Africa. Virus Res. 1 I, 227-240.

Development of the polymerase chain reaction for the detection of bluetongue virus in tissue samples.

Total genomic dsRNA, extracted from purified core particles of bluetongue virus serotype 1 from South Africa (BTV1SA), was used as template to optimis...
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