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Development and characterization of nucleic acid probes to infectious bursal disease viruses
Veterinary Microbiology, 24 (1990) 253-260 Elsevier Science Publishers B.V., Amsterdam
253
Development and characterization of nucleic acid probes to infectious bursal disease viruses Daral J. Jackwood* Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, U.S.A.
ABSTRACT Jackwood, D.J., 1990. Development and characterization of nucleic acid probes to infectious bursal disease viruses. Vet. Microbiol., 24: 253-260. Radiolabeled cDNA probes were prepared using both segments of double-stranded genomic infectious bursal disease virus (IBDV) RNA as template. The probes were synthesized and labeled with 32p using random primers and reverse transcriptase. Probes were prepared to the genomic RNA extracted from a pathogenic serotype 1 virus (ST-C) and from an attenuated serotype 1 vaccine virus (D-78 ) which is commercially available. These probes were determined to range in size from approximately 200 to 1500 nucleotides in length using a 6% denaturing polyacrylamide gel. The probes were used in a dot hybridization assay and detected approximately 10 ng of IBDV RNA. In addition, they detected genomic RNA from five different subtypes of IBDV serotype 1 and from two serotype 2 viruses. The probes appeared to be specific for viral RNA since hybridization to cell culture nucleic acid was not detected.
INTRODUCTION
Infectious bursal disease virus (IBDV) is a member of the birnavirus family and causes an immunosuppressive disease-in young chickens (Becht, 1981; Kibenge et al., 1988 ). Two serotypes, designated 1 and 2, of IBDV have been described (Jackwood et al., 1982; McNulty et al., 1979). These two serotypes can be distinguished using an in vitro virus neutralization assay. This assay has also been used to define at least six antigenic subtypes of the serotype 1 viruses (Jackwood and Saif, 1987 ). All the known pathogenic isolates of IBDV belong to serotype 1. The IBDV genome consists of two segments of double-stranded (ds) RNA. Both genome segments of an Australian IBDV isolate 002-73 have been cloned and sequenced (Azad et al., 1985; Hudson et al., 1986; Morgan et al., 1988). *Salaries and research support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript number 330-88.
0378-1135/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
2 54
D.J. JACKWOOD
The length of the larger genome segment (A) is approximately 3400 base pairs (bp) and the smaller genome segment (B) is approximately 2900 bp. Diagnostic assays for infectious bursal disease include the enzyme-linked immunosorbent assay (ELISA), the virus neutralization assay, the agar-gel precipitin test and the immunofluorescence (IF) assay. The IF assay, ELISA, and agar-gel precipitin test can not distinguish among IBDV strains. Although the virus neutralization assay can distinguish between serotypes and among serotype 1 subtypes, this assay is laborious and time consuming. A fast and reliable diagnostic assay which can distinguish among antigenically different IBDV isolates is needed. Diagnostic assays which employ nucleic acid probes have the potential to differentiate closely related viruses (Tenover, 1988). To examine the possibility of detecting IBDV strains using DNA probes, radiolabeled cDNA molecules were prepared directly from the viral genome and used to detect IBDV isolates. MATERIALS A N D M E T H O D S
Viruses
cDNA probes were prepared against the genomes of two serotype 1 IBDV isolates; the ST-C virus which is highly pathogenic in chicks (Rosenberger et al., 1975 ) and the D-78 vaccine virus (Intervet America Inc., Millsboro, DE ). Other viruses which were used in these studies include the OH and MO serotype 2 viruses isolated from turkeys (Jackwood et al., 1982 ), the NC serotype 1 virus isolated from turkeys (Jackwood et al., 1982), and several serotype 1 vaccine viruses; SAL (Bursine: Salsbury Laboratories Inc., Charles City, IA ), BVM (Bursa-Vac-M: Sterwin Laboratories, Inc., Millsboro, DE), BV (BursaVac: Sterwin Laboratories, Inc.), and 2512 (IBD-BLEN: Ceva Laboratories Inc., Overland Park, KS).
Purification of virus IBDV strains were purified from cell culture or bursal homogenates. Virus was collected on CsC1 cushions (density= 1.54 g/ml) and then purified by isopycnic ultracentrifugation in CsC1 as previously described (Jackwood et al., 1982 ). Virus particles from gradient fractions at a density of 1.29 to 1.32 g/ml were used to obtain viral RNA.
Extraction of viral RNA Genomic viral RNA was collected from virus particles using proteinase K digestion. Viral particles from CsC1 gradients were pelleted at 132 000 × g for 3 h at 4°C. Viral pellets were resuspended in TNE ( 10 mM Tris [pH 8.0], 100 mM NaC1, 1 mM EDTA) containing 0.5% SDS. Alternatively, vaccine viruses in Experiment 2 were used directly from the commercial supplier without further passage in cell culture. The lyophilized viruses were resus-
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NUCLEIC ACID PROBES TO IBDV
pended in TNE containing 0.5% SDS. Proteinase K (Sigma Chemical Co., St. Louis, MO ) was added to a final concentration of 2.0 mg/ml and all samples were incubated at 37 °C for 1 h. Due to the possible presence of cellular DNA, the samples were treated with RNase-free DNase I (Boehringer Mannheim Biochemicals, Indianapolis, IN) at a concentration of 100 U / m g of total nucleic acid and incubated at 37°C for 1 h.
Synthesis of probes Radiolabeled cDNA probes were prepared as described in Fig. 1, using both segments of double-stranded genomic IBDV RNA as the template. Approximately 1.0 #g of dsRNA extracted from purified viral particles was used to prepare probes. The viral genome segments were denatured in 90% DMSO for 1 h at 65°C and then at 100°C for 3 min. Denatured RNA was added to a reaction mixture containing 0.01 M Tris-base (pH 8.3), 60 mM KC1, 10 mM MgC12, 10 mM dithiothreitol, 1.5 mM dGTP, 1.5 mM dATP, 1.5 mM dTTP, random calf thymus DNA primers (Pharmacia Inc., Piscataway, NJ ) (separately denatured at 100 ° C for 3 min ), 32p-dCTP ( 25/zCi, specific activity= 600 C i / m M ) , and 25 U of avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer Mannheim Biochemicals, Indianapolis, IN). Incubation was conducted at 42 °C for 2 h. The RNA template was digested by IBDV
ds RNA] 90% Heat
DMSO
RNADenatured]
xU
dATP, dGTP, dTTP, 3zP-dCTP Random p r i m e r s AMV- RT
RNA/eDNA Hybrids
RNase H IBDV cDNA Probe Fig. 1. Synthesis of cDNA from the IBDV dsRNA genome. The dsRNA is denatured in a 90% DMSO buffer using heat. The denatured genornic RNA is then used as template to prepare c D N A / R N A hybrids. The cDNA is synthesized using deoxynucleotide triphosphates (dATP, dGTP, dTTP, and 3zp-labeled dCTP), random primers and reverse transcriptase (AMV-RT). The enzyme RNase H is then used to remove the RNA template leaving the radiolabeled cDNA probe.
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adding 0.85 U RNase H (Boehringer Mannheim Biochemicals, Indianapolis, IN) and incubation was continued for 1 h at 37 °C. Following incubation the probe reaction mixture was phenol-chloroform extracted and purified by centrifugation through Sephadex G-50 columns (Maniatis et al., 1982 ).
Hybridizations A dot hybridization procedure was used in these studies. Viral RNAs were denatured at 100 ° C for 5 min and then quick-chilled on ice. Denatured RNA samples in a volume of 5.0 #1 or less were placed directly onto nylon membranes (Biodyne, Pall Ultrafine Filtration Corp., Glen Cove, NY) and allowed to dry for 30 min. The filters were than baked at 80°C for 2 h. The membranes were prehydridized at 42°C for 4 h in a solution containing 50 mM sodium phosphate (pH 6.5 ), 5 × Denhardt's solution (0.001 g/ml each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin), 0.75 M sodium chloride, 0.075 M sodium citrate, 250/tg/ml salmon sperm DNA, and 50% formamide. Hybridization was conducted in the same buffer used for prehybridization. Approximately 1.0N 10 6 counts per min (cpm) of 3zp-labeled probe was added to each filter and incubation was continued at 42 °C for 12 h. Following hybridization the filters were washed at room temperature in 2 × SSC (0.15 M sodium chloride, 0.015 M sodium citrate ) containing 0.1% (w/v) SDS and then in 0.1 ×SSC containing 0.1% (w/v) SDS. The results were observed using autoradiography. RESULTS
Synthesis of IBD V probes The sizes of DNA probes prepared using AMV reverse transcriptase were determined by electrophoresis on a denaturing 6% polyacrylamide-urea gel (Fig. 2). When a 1 : 1 weight ratio of random primers (1.0pg) to IBDV RNA ( 1.0/~g) was used, the probe size ranged from approximately 200 nucleotides (nt) to 1500 nt. At higher ratios of random primers (50 pg) to IBDV RNA ( 1.0/tg), the average size of the probes was smaller. The cpm per #g of viral RNA template observed in probes which were produced using a 1 : 1 ratio of random primers ranged from 0.9× 10 6 cpm//tg RNA to 2.2× 10 6 cpm//tg RNA.
Sensitivity of lBD V probes Radiolabeled probes (1.0N 10 6 cpm) prepared to the D-78 vaccine virus were hybridized to 1, 10, 100, and 1000 ng of homologous (D-78) and heterologous (ST-C) viral RNA (Fig. 3 ). The lowest quantity of viral RNA detected was 10 rig. No difference in the sensitivity of detection was observed for homologous or heterologous viral RNA.
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NUCLEIC ACID PROBES TO IBDV
Kb
A
B
Kb !
1.500~
--I,310 -Q920
-~070
-Q580
0.zoo
J
¸
Fig. 2. Radiolabeled cDNA probes prepared from both segments of the ST-C genomic RNA were denatured at 100 °C for 3 rain and separated on a denaturing 6% polyacrylamide-urea gel similar to those used for nucleotide sequencing. Lane A, ST-C probe was prepared using 1.0/tg viral RNA and 1.0/tg random primers. Lane B, ST-C probe was prepared using 1.0/~g viral RNA and 50 Ftgrandom primers. Lane C, lambda bacteriophage DNA which was digested with HindlII and EcoRI and then radiolabeled using 32p-ATP and polynucleotidekinase. The sizes of the lambda restriction fragments and IBDV probes are listed in kilobases (kb).
Specificity of lB D V probes The specificity o f the c D N A probes for both IBDV genomic R N A segments was initially d e m o n s t r a t e d using a northern blot hybridization assay (Jackwood et al., 1989).
Experiment 1. The ST-C viral R N A segments obtained from CsC1 gradient purified viral particles were used to synthesize probes. Radiolabeled probe ( 1.1 × 106 cpm ) was a d d e d to a m e m b r a n e containing viral R N A extracted from CsC1 gradient purified ST-C, SAL, NC, OH, and MO viruses. Total nu-
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Fig. 3. Viral RNAs extracted from the D-78 and ST-C viruses were adjusted to 1000, 100, 10 and 1 ng and placed on the hybridization membrane. Radiolabeled cDNA probe (l.0X l 0 6 cpm) prepared to the D-78 viral RNA was used. CEF
Fig. 4. Hybridization Experiment l. Radiolabeled cDNA probes to both RNA segments of STC were hybridized to genomic RNA from homologous and heterologous IBDV isolates. ST-C #1 contained RNA extracted from virus in CsCI gradient fractions with a density of 1.29 to 1.30 g/ml. ST-C #2 contained RNA extracted from virus in CsC1gradient fractions with a density of 1.32 g/ml. Total nucleic acid (DNA and RNA) extracted from CEF cells was included as a negative control. All test samples and the CEF control contained > 50 ng of nucleic acid. cleic acid (DNA and R N A ) extracted from chicken-embryo-fibroblast (CEF) cells was included as a negative control. The ST-C cDNA probe hybridized to all the viral R N A samples tested but not to the CEF control (Fig. 4 ).
Experiment 2. The D-78 viral R N A segments obtained from CsC1 gradient purified viral particles were used to synthesize cDNA probes. Radiolabeled probe (0.9 X 106 c p m ) was hybridized to viral RNAs from the BVM, BV, D78, 2512, and ST-C viruses. Virus isolates BVM, BV, D-78, and 2512 were resuspended directly from vaccine vials in TNE buffer containing 0.5% SDS. Viral RNA was extracted from these samples using proteinase K without prior purification of the viral particles through CsC1 gradients. Each 5 ~tl sample contained 25/~g of nucleic acid which presumably contained both viral nucleic acid and nucleic acid from CEF cells which where used to propagate the vaccine viruses. The genomic R N A sample from the ST-C virus was prepared as described in Experiment 1 and contained approximately 50 ng o f nucleic
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CEF
Fig. 5. Hybridization Experiment 2. Radiolabeled cDNA probes to both RNA segments of D78 were hybridized to viral RNAs from the virulent ST-C virus and from the vaccine viruses BVM, BV, D-78, and 2512. Total nucleic acid (DNA and RNA) extracted from CEF cells was included as a negative control. The vaccine viral samples and CEF control contained 25/tg of nucleic acid and the ST-C sample contained approximately 50 ng of nucleic acid.
acid. Total nucleic acid from CEF cells was included as a control. Samples were placed onto a nylon membrane for hybridization. The D-78 probe hybridized to all the viral RNA samples (Fig. 5). No hybridization was observed to the CEF cellular nucleic acid. DISCUSSION
The vaccine viruses used in these studies represent five different antigenic subtypes of serotype 1 IBDV (Jackwood and Saif, 1987). Probes prepared to the vaccine virus D-78 or the virulent ST-C virus hybridized not only to these five viral subtypes, but also to two serotype 2 viruses (OH and MO). These results indicate the potential for detecting several different strains of IBDV using nucleic acid probes. Since the D N A - R N A hybrid is more stable than D N A - D N A hybrids, and since the stringency of hybridization and wash conditions used in these studies were maintained relatively low, it may be possible to increase the specificity of these probes for different IBDV strains by increasing the stringency of the hybridization and wash conditions. The ability of these probes to hybridize to the IBDV isolates under conditions of higher stringency was not determined. Thomas (1980) detected 1.0 pg of RNA using a dot hybridization assay. The lowest quantity of sample RNA which was detected using radiolabeled cDNA probes to the D-78 RNA was 10 ng. This is not surprising because of the relatively low quantity of radioactivity which was incorporated into the cDNA probes. These studies demonstrate that cDNA probes can be produced from genomic IBDV RNA and used to detect viral dsRNA in a dot hybridization assay. The probes appeared to be specific for IBDV RNA and did not hybridize to cellular nucleic acid. The use of probes prepared directly from IBDV genomic RNA, however, may be limited as a practical diagnostic tool. It is difficult and time consuming to obtain sufficient quantities of purified genomic IBDV RNA for the production o f c D N A probes. However, the availability of molecular clones of the IBDV genome would eliminate isolation and purification of the dsRNA for the production of probes. The use and detection of
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r a d i o l a b e l e d p r o b e s is n o t p r a c t i c a l f o r m o s t d i a g n o s t i c l a b o r a t o r i e s . N o n r a d i o a c t i v e p r o b e s h o w e v e r , s u c h as t h o s e l a b e l e d w i t h b i o t i n , m a y e l i m i n a t e this s h o r t c o m i n g . T h u s , a reliable a n d fast d i a g n o s t i c a s s a y for I B D V m a y be p o s s i b l e u s i n g n o n r a d i o a c t i v e p r o b e s p r e p a r e d f r o m c l o n e d v i r a l genes. ACKNOWLEDGEMENTS T h e t e c h n i c a l s e r v i c e s o f J e r r y M e i t z l e r are g r a t e f u l l y a c k n o w l e d g e d .
REFERENCES Azad, A.A., Barrett, S.A. and Fahey, K.J., 1985. The characterization and molecular cloning of the double-stranded RNA genome of an Australian strain of infectious bursal disease virus. Virology, 143: 35-44. Becht, H., 1981. Infectious bursal disease virus. Curr. Top. Microbiol. Immunol., 90:107-121. Hudson, P.J., McKern, N.M., Power, B.E. and Azad, A.A., 1986. Genomic structure of the large RNA segment of infectious bursal disease virus. Nucleic Acids Res., 14:5001-5012. Jackwood, D.H. and Saif, Y.M., 1987. Antigenic diversity of infectious bursal disease viruses. Avian Dis., 31: 766-770. Jackwood, D.J., Saif, Y.M. and Hughes, J.H., 1982. Characteristics and serologic studies of two serotypes of infectious bursal disease virus in turkeys. Avian Dis., 26:871-882. Jackwood, D.J., Kibenge, F.S.B. and Mercado, C.C., 1989. Detection of infectious bursal disease viruses by using cloned cDNA probes. J. Clin. Microbiol., 27: 2437-2443. Kibenge, F.S.B., Dhillon, A.S. and Russell, R.G., 1988. Biochemistry and immunology of infectious bursal disease virus. J. Gen. Virol., 69:1757-1775. Maniatis, T., Fritsch, E.F. and Sambrook, J., 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 466-467. Morgan, M.M., Macreadie, I.G., Harley, V.R., Hudson, P.J. and Azad, A,A., 1988. Sequence of the small double-stranded RNA genomic segment of infectious bursal disease virus and its deduced 90-kDa product. Virology, 163: 240-242. McNulty, M.S., Allan, G.M. and McFerran, J.B., 1979. Isolation of infectious bursal disease virus from turkeys. Avian Pathol., 8:205-212. Rosenberger, J.K., Klopp, S., Eckroade, R.J. and Krauss, W.C., 1975. The role of the infectious bursal agent and several avian adenoviruses in hemorrhagic-aplastic-anemia syndrome and gangrenous dermatitis. Avian Dis., 19:717-729. Tenover, F.C., 1988. Diagnostic dexoyribonucleic acid probes for infectious diseases. Clinical Microbiol. Rev., 1: 82-101. Thomas, P.S., 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci., 77: 5210-5205.
253
Development and characterization of nucleic acid probes to infectious bursal disease viruses Daral J. Jackwood* Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, U.S.A.
ABSTRACT Jackwood, D.J., 1990. Development and characterization of nucleic acid probes to infectious bursal disease viruses. Vet. Microbiol., 24: 253-260. Radiolabeled cDNA probes were prepared using both segments of double-stranded genomic infectious bursal disease virus (IBDV) RNA as template. The probes were synthesized and labeled with 32p using random primers and reverse transcriptase. Probes were prepared to the genomic RNA extracted from a pathogenic serotype 1 virus (ST-C) and from an attenuated serotype 1 vaccine virus (D-78 ) which is commercially available. These probes were determined to range in size from approximately 200 to 1500 nucleotides in length using a 6% denaturing polyacrylamide gel. The probes were used in a dot hybridization assay and detected approximately 10 ng of IBDV RNA. In addition, they detected genomic RNA from five different subtypes of IBDV serotype 1 and from two serotype 2 viruses. The probes appeared to be specific for viral RNA since hybridization to cell culture nucleic acid was not detected.
INTRODUCTION
Infectious bursal disease virus (IBDV) is a member of the birnavirus family and causes an immunosuppressive disease-in young chickens (Becht, 1981; Kibenge et al., 1988 ). Two serotypes, designated 1 and 2, of IBDV have been described (Jackwood et al., 1982; McNulty et al., 1979). These two serotypes can be distinguished using an in vitro virus neutralization assay. This assay has also been used to define at least six antigenic subtypes of the serotype 1 viruses (Jackwood and Saif, 1987 ). All the known pathogenic isolates of IBDV belong to serotype 1. The IBDV genome consists of two segments of double-stranded (ds) RNA. Both genome segments of an Australian IBDV isolate 002-73 have been cloned and sequenced (Azad et al., 1985; Hudson et al., 1986; Morgan et al., 1988). *Salaries and research support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript number 330-88.
0378-1135/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
2 54
D.J. JACKWOOD
The length of the larger genome segment (A) is approximately 3400 base pairs (bp) and the smaller genome segment (B) is approximately 2900 bp. Diagnostic assays for infectious bursal disease include the enzyme-linked immunosorbent assay (ELISA), the virus neutralization assay, the agar-gel precipitin test and the immunofluorescence (IF) assay. The IF assay, ELISA, and agar-gel precipitin test can not distinguish among IBDV strains. Although the virus neutralization assay can distinguish between serotypes and among serotype 1 subtypes, this assay is laborious and time consuming. A fast and reliable diagnostic assay which can distinguish among antigenically different IBDV isolates is needed. Diagnostic assays which employ nucleic acid probes have the potential to differentiate closely related viruses (Tenover, 1988). To examine the possibility of detecting IBDV strains using DNA probes, radiolabeled cDNA molecules were prepared directly from the viral genome and used to detect IBDV isolates. MATERIALS A N D M E T H O D S
Viruses
cDNA probes were prepared against the genomes of two serotype 1 IBDV isolates; the ST-C virus which is highly pathogenic in chicks (Rosenberger et al., 1975 ) and the D-78 vaccine virus (Intervet America Inc., Millsboro, DE ). Other viruses which were used in these studies include the OH and MO serotype 2 viruses isolated from turkeys (Jackwood et al., 1982 ), the NC serotype 1 virus isolated from turkeys (Jackwood et al., 1982), and several serotype 1 vaccine viruses; SAL (Bursine: Salsbury Laboratories Inc., Charles City, IA ), BVM (Bursa-Vac-M: Sterwin Laboratories, Inc., Millsboro, DE), BV (BursaVac: Sterwin Laboratories, Inc.), and 2512 (IBD-BLEN: Ceva Laboratories Inc., Overland Park, KS).
Purification of virus IBDV strains were purified from cell culture or bursal homogenates. Virus was collected on CsC1 cushions (density= 1.54 g/ml) and then purified by isopycnic ultracentrifugation in CsC1 as previously described (Jackwood et al., 1982 ). Virus particles from gradient fractions at a density of 1.29 to 1.32 g/ml were used to obtain viral RNA.
Extraction of viral RNA Genomic viral RNA was collected from virus particles using proteinase K digestion. Viral particles from CsC1 gradients were pelleted at 132 000 × g for 3 h at 4°C. Viral pellets were resuspended in TNE ( 10 mM Tris [pH 8.0], 100 mM NaC1, 1 mM EDTA) containing 0.5% SDS. Alternatively, vaccine viruses in Experiment 2 were used directly from the commercial supplier without further passage in cell culture. The lyophilized viruses were resus-
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pended in TNE containing 0.5% SDS. Proteinase K (Sigma Chemical Co., St. Louis, MO ) was added to a final concentration of 2.0 mg/ml and all samples were incubated at 37 °C for 1 h. Due to the possible presence of cellular DNA, the samples were treated with RNase-free DNase I (Boehringer Mannheim Biochemicals, Indianapolis, IN) at a concentration of 100 U / m g of total nucleic acid and incubated at 37°C for 1 h.
Synthesis of probes Radiolabeled cDNA probes were prepared as described in Fig. 1, using both segments of double-stranded genomic IBDV RNA as the template. Approximately 1.0 #g of dsRNA extracted from purified viral particles was used to prepare probes. The viral genome segments were denatured in 90% DMSO for 1 h at 65°C and then at 100°C for 3 min. Denatured RNA was added to a reaction mixture containing 0.01 M Tris-base (pH 8.3), 60 mM KC1, 10 mM MgC12, 10 mM dithiothreitol, 1.5 mM dGTP, 1.5 mM dATP, 1.5 mM dTTP, random calf thymus DNA primers (Pharmacia Inc., Piscataway, NJ ) (separately denatured at 100 ° C for 3 min ), 32p-dCTP ( 25/zCi, specific activity= 600 C i / m M ) , and 25 U of avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer Mannheim Biochemicals, Indianapolis, IN). Incubation was conducted at 42 °C for 2 h. The RNA template was digested by IBDV
ds RNA] 90% Heat
DMSO
RNADenatured]
xU
dATP, dGTP, dTTP, 3zP-dCTP Random p r i m e r s AMV- RT
RNA/eDNA Hybrids
RNase H IBDV cDNA Probe Fig. 1. Synthesis of cDNA from the IBDV dsRNA genome. The dsRNA is denatured in a 90% DMSO buffer using heat. The denatured genornic RNA is then used as template to prepare c D N A / R N A hybrids. The cDNA is synthesized using deoxynucleotide triphosphates (dATP, dGTP, dTTP, and 3zp-labeled dCTP), random primers and reverse transcriptase (AMV-RT). The enzyme RNase H is then used to remove the RNA template leaving the radiolabeled cDNA probe.
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adding 0.85 U RNase H (Boehringer Mannheim Biochemicals, Indianapolis, IN) and incubation was continued for 1 h at 37 °C. Following incubation the probe reaction mixture was phenol-chloroform extracted and purified by centrifugation through Sephadex G-50 columns (Maniatis et al., 1982 ).
Hybridizations A dot hybridization procedure was used in these studies. Viral RNAs were denatured at 100 ° C for 5 min and then quick-chilled on ice. Denatured RNA samples in a volume of 5.0 #1 or less were placed directly onto nylon membranes (Biodyne, Pall Ultrafine Filtration Corp., Glen Cove, NY) and allowed to dry for 30 min. The filters were than baked at 80°C for 2 h. The membranes were prehydridized at 42°C for 4 h in a solution containing 50 mM sodium phosphate (pH 6.5 ), 5 × Denhardt's solution (0.001 g/ml each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin), 0.75 M sodium chloride, 0.075 M sodium citrate, 250/tg/ml salmon sperm DNA, and 50% formamide. Hybridization was conducted in the same buffer used for prehybridization. Approximately 1.0N 10 6 counts per min (cpm) of 3zp-labeled probe was added to each filter and incubation was continued at 42 °C for 12 h. Following hybridization the filters were washed at room temperature in 2 × SSC (0.15 M sodium chloride, 0.015 M sodium citrate ) containing 0.1% (w/v) SDS and then in 0.1 ×SSC containing 0.1% (w/v) SDS. The results were observed using autoradiography. RESULTS
Synthesis of IBD V probes The sizes of DNA probes prepared using AMV reverse transcriptase were determined by electrophoresis on a denaturing 6% polyacrylamide-urea gel (Fig. 2). When a 1 : 1 weight ratio of random primers (1.0pg) to IBDV RNA ( 1.0/~g) was used, the probe size ranged from approximately 200 nucleotides (nt) to 1500 nt. At higher ratios of random primers (50 pg) to IBDV RNA ( 1.0/tg), the average size of the probes was smaller. The cpm per #g of viral RNA template observed in probes which were produced using a 1 : 1 ratio of random primers ranged from 0.9× 10 6 cpm//tg RNA to 2.2× 10 6 cpm//tg RNA.
Sensitivity of lBD V probes Radiolabeled probes (1.0N 10 6 cpm) prepared to the D-78 vaccine virus were hybridized to 1, 10, 100, and 1000 ng of homologous (D-78) and heterologous (ST-C) viral RNA (Fig. 3 ). The lowest quantity of viral RNA detected was 10 rig. No difference in the sensitivity of detection was observed for homologous or heterologous viral RNA.
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NUCLEIC ACID PROBES TO IBDV
Kb
A
B
Kb !
1.500~
--I,310 -Q920
-~070
-Q580
0.zoo
J
¸
Fig. 2. Radiolabeled cDNA probes prepared from both segments of the ST-C genomic RNA were denatured at 100 °C for 3 rain and separated on a denaturing 6% polyacrylamide-urea gel similar to those used for nucleotide sequencing. Lane A, ST-C probe was prepared using 1.0/tg viral RNA and 1.0/tg random primers. Lane B, ST-C probe was prepared using 1.0/~g viral RNA and 50 Ftgrandom primers. Lane C, lambda bacteriophage DNA which was digested with HindlII and EcoRI and then radiolabeled using 32p-ATP and polynucleotidekinase. The sizes of the lambda restriction fragments and IBDV probes are listed in kilobases (kb).
Specificity of lB D V probes The specificity o f the c D N A probes for both IBDV genomic R N A segments was initially d e m o n s t r a t e d using a northern blot hybridization assay (Jackwood et al., 1989).
Experiment 1. The ST-C viral R N A segments obtained from CsC1 gradient purified viral particles were used to synthesize probes. Radiolabeled probe ( 1.1 × 106 cpm ) was a d d e d to a m e m b r a n e containing viral R N A extracted from CsC1 gradient purified ST-C, SAL, NC, OH, and MO viruses. Total nu-
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Fig. 3. Viral RNAs extracted from the D-78 and ST-C viruses were adjusted to 1000, 100, 10 and 1 ng and placed on the hybridization membrane. Radiolabeled cDNA probe (l.0X l 0 6 cpm) prepared to the D-78 viral RNA was used. CEF
Fig. 4. Hybridization Experiment l. Radiolabeled cDNA probes to both RNA segments of STC were hybridized to genomic RNA from homologous and heterologous IBDV isolates. ST-C #1 contained RNA extracted from virus in CsCI gradient fractions with a density of 1.29 to 1.30 g/ml. ST-C #2 contained RNA extracted from virus in CsC1gradient fractions with a density of 1.32 g/ml. Total nucleic acid (DNA and RNA) extracted from CEF cells was included as a negative control. All test samples and the CEF control contained > 50 ng of nucleic acid. cleic acid (DNA and R N A ) extracted from chicken-embryo-fibroblast (CEF) cells was included as a negative control. The ST-C cDNA probe hybridized to all the viral R N A samples tested but not to the CEF control (Fig. 4 ).
Experiment 2. The D-78 viral R N A segments obtained from CsC1 gradient purified viral particles were used to synthesize cDNA probes. Radiolabeled probe (0.9 X 106 c p m ) was hybridized to viral RNAs from the BVM, BV, D78, 2512, and ST-C viruses. Virus isolates BVM, BV, D-78, and 2512 were resuspended directly from vaccine vials in TNE buffer containing 0.5% SDS. Viral RNA was extracted from these samples using proteinase K without prior purification of the viral particles through CsC1 gradients. Each 5 ~tl sample contained 25/~g of nucleic acid which presumably contained both viral nucleic acid and nucleic acid from CEF cells which where used to propagate the vaccine viruses. The genomic R N A sample from the ST-C virus was prepared as described in Experiment 1 and contained approximately 50 ng o f nucleic
259
NUCLEIC ACID PROBES TO IBDV
CEF
Fig. 5. Hybridization Experiment 2. Radiolabeled cDNA probes to both RNA segments of D78 were hybridized to viral RNAs from the virulent ST-C virus and from the vaccine viruses BVM, BV, D-78, and 2512. Total nucleic acid (DNA and RNA) extracted from CEF cells was included as a negative control. The vaccine viral samples and CEF control contained 25/tg of nucleic acid and the ST-C sample contained approximately 50 ng of nucleic acid.
acid. Total nucleic acid from CEF cells was included as a control. Samples were placed onto a nylon membrane for hybridization. The D-78 probe hybridized to all the viral RNA samples (Fig. 5). No hybridization was observed to the CEF cellular nucleic acid. DISCUSSION
The vaccine viruses used in these studies represent five different antigenic subtypes of serotype 1 IBDV (Jackwood and Saif, 1987). Probes prepared to the vaccine virus D-78 or the virulent ST-C virus hybridized not only to these five viral subtypes, but also to two serotype 2 viruses (OH and MO). These results indicate the potential for detecting several different strains of IBDV using nucleic acid probes. Since the D N A - R N A hybrid is more stable than D N A - D N A hybrids, and since the stringency of hybridization and wash conditions used in these studies were maintained relatively low, it may be possible to increase the specificity of these probes for different IBDV strains by increasing the stringency of the hybridization and wash conditions. The ability of these probes to hybridize to the IBDV isolates under conditions of higher stringency was not determined. Thomas (1980) detected 1.0 pg of RNA using a dot hybridization assay. The lowest quantity of sample RNA which was detected using radiolabeled cDNA probes to the D-78 RNA was 10 ng. This is not surprising because of the relatively low quantity of radioactivity which was incorporated into the cDNA probes. These studies demonstrate that cDNA probes can be produced from genomic IBDV RNA and used to detect viral dsRNA in a dot hybridization assay. The probes appeared to be specific for IBDV RNA and did not hybridize to cellular nucleic acid. The use of probes prepared directly from IBDV genomic RNA, however, may be limited as a practical diagnostic tool. It is difficult and time consuming to obtain sufficient quantities of purified genomic IBDV RNA for the production o f c D N A probes. However, the availability of molecular clones of the IBDV genome would eliminate isolation and purification of the dsRNA for the production of probes. The use and detection of
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r a d i o l a b e l e d p r o b e s is n o t p r a c t i c a l f o r m o s t d i a g n o s t i c l a b o r a t o r i e s . N o n r a d i o a c t i v e p r o b e s h o w e v e r , s u c h as t h o s e l a b e l e d w i t h b i o t i n , m a y e l i m i n a t e this s h o r t c o m i n g . T h u s , a reliable a n d fast d i a g n o s t i c a s s a y for I B D V m a y be p o s s i b l e u s i n g n o n r a d i o a c t i v e p r o b e s p r e p a r e d f r o m c l o n e d v i r a l genes. ACKNOWLEDGEMENTS T h e t e c h n i c a l s e r v i c e s o f J e r r y M e i t z l e r are g r a t e f u l l y a c k n o w l e d g e d .
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