Characterization of nonradioactive hybridization probes for detecting infectious bursal disease virus

Journal of Virological Methods, 38 (1992) 81-92 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00

81

VIRMET 01333

Characterization of nonradioactive hybridization probes for detecting infectious bursal disease virus Long Huw Lee Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan, Republic of China (Accepted 9 January

1992)

Summary Reverse transcription followed by the polymerase chain reaction was used to amplify a fragment of infectious bursal disease virus (IBDV) strain P3009 genome. The amplified DNA fragment was annealed into the plasmid pUC18 and used to transform Escherichiu coli strain JM109. A clone that contained IBDV-specific nucleotide sequences was selected and designated pC23. The DNA fragment within pC23 was 320 base pairs in length and designated C23. Radiolabeled probes prepared from C23 hybridized to genome segment A of strain P3009 by a northern-blot hybridization assay. Biotin-labeled probes prepared from C23 and pC23 either by using nick translation (designated C23/ NT and pC23/NT, respectively) or by direct introduction of biotin molecules into C23 and pC32 (designated C23/BH and pC23/BH, respectively) were used in the dot blot hybridization assay for detecting IBDV strains. All four biotinylated probes detected three serotype 1 viruses and one serotype 2 IBDV. However, they did not cross-react with nucleic acids extracted from mockinfected cells or from seven unrelated avian viruses. Probe pC23/BH detected as little as 0.04 ng of IBDV RNA, while the other three probes were less sensitive and detected approximately 1 ng of IBDV RNA. In addition, the probe pC23/ BH detected IBDV RNA in bursa tissues from commercial broiler raising farms following the dot blot hybridization. Infections bursal disease virus (IBDV); Nonradiolabeled RNA detection

hybridization

probe; IBDV

Correspondence to: L.H. Lee, Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China.

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Introduction Infectious bursal disease virus (IBDV) is classified as a birnavirus and is the etiological agent of an immunosuppressive disease in young chicken (Becht, 1981; Kibenge et al., 1988). At least two serotypes of the virus are currently recognized based on an in vitro neutralization assay (McNulty et al., 1979; Jackwood et al., 1982). The same assay system has also been used to define at least six antigenic subtypes of serotype 1 viruses (Jackwood and Saif, 1987). The known isolates of IBDV pathogenic to chickens are all of serotype 1 (Cummings et al., 1986). IBDV genome contains two segments of doublestranded (ds) RNA (Muller et al., 1979) with approximate lengths of 3,400 (segment A) and 2,900 (segment B) base pairs (bp) (Azad et al., 1985). The use of radiolabeled cDNA probes as a diagnostic tool for IBDV infections has been studied (Jackwood et al., 1989; Jackwood, 1990). The probes hybridize to both serotype viruses, however, they do not hybridize to nucleic acids from several unrelated avian viruses (Jackwood et al., 1989). Jackwood et al. (1990) also used the nonradioactive cDNA probes, such as those labeled with biotin following the nick translation, to detect IBDV strains. However, the sensitivity of their probe has not been shown. To introduce biotin molecules into nucleic acids, Reisfeld et al. (1987) used sodium bisulfite to catalyze the interaction of biotin hydrozide (Sigma, St. Louis, MO, USA) with unpaired cytosine residues of denatured DNA molecules. The limit for detecting the biotinylated probes prepared in this manner was c 1 pg. To examine the sensitivity and specificity of biotin-labeled DNA probes for detecting IBDV, a DNA fragment amplified by using reverse transcription-polymerase chain reaction has been cloned into the plasmid pUC18. The DNA fragment and the recombinant plasmid were labeled with biotin following different preparation procedures and used to detect IBDV strains.

Materials and Methods Virus strains Four IBDV isolates representing serotype 1 (P3009, FlO, and LU) and serotype 2 (MO) were used. The LU and MO viruses were obtained from Dr. P.D. Lukert, Department of Medical Microbiology, University of Georgia, Athens, Georgia, USA. P3009 and FlO’ are the local isolates from chickens in Taiwan and have been identified as serotype 1 viruses based on the virus neutralization assay with antiserum to LU as described previously (Lee et al., 1988). Seven avian viruses were used for the determination of specificity of probes prepared in this study. The viruses tested are the commercially available vaccine strains. They were sampled for the evaluation of the efficacy and safety tests by the Taiwan Provincial Research Institute for Animal Health, Taiwan, ROC and included infectious bronchitis virus (Intervet International B.V.,

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Holland), infectious laryngotracheitis virus and fowl poxvirus (Nippon Institute for Biological Science, Tokyo, Japan), LaSota and Bl Newcastle disease viruses and turkey herpesvirus (Marek’s disease vaccine) (Salsbury Inc., Charles City, Iowa, USA) and an avian reovirus strain 1133 (Vineland Laboratories, Vineland, NJ, USA). Virus propagation

and purification

The procedures for virus propagation and purification were made essentially as described by Muller et al. (1979). Briefly, viruses were propagated in chicken embryo libroblasts (CEF). After extensive cytopathic effects were observed, the culture was treated by freezing and thawing three times followed by low speed centrifugation. The supernatant obtained was concentrated loo-fold using polyethylene glycol 6,000 and then extracted with fluorocarbon (Merck, Darmstadt, Germany). The supernatants containing virions were isopycnically centrifuged at 128,000 x g for 18 h at 4°C on a CsCl-density gradient. The viruses banded at a buoyant density of 1.32 g/ml were collected and pelleted through a 35% sucrose gradient. The pellet was finally resuspended in proteinase K buffer (0.01 M Tris, 0.1 M NaCl, 0.001 M EDTA, 0.5% sodium dodecyl sulfate (SDS), pH 8.0). RNA extraction

and pur$ication

Virion in a proteinase K buffer was digested with proteinase K (1 mg/ml) (Boehringer Mannheim, Germany) at 37°C for 1 h. The viral RNA was extracted with phenol and chloroform (1: 1) and recovered by precipitation with ethanol. The dsRNA was then purified by differential salt precipitation (DiazRuiz and Kaper, 1978). The concentration of viral RNA was calculated by determination of the optical density at 260 nm. Preparation

of nucleic acid from

avian viruses

Seven unrelated avian viruses listed above and the IBDV strain P3009 were used to determine the specificity of the probes. The nucleic acids extracted directly from virions contained in vaccine vials without further passage in cell culture. Each vaccine vial included a minimum of 1,000 doses of infectious viruses. The lyophilized viruses were resuspended in proteinase K buffer and digested with proteinase K (2 mg/ml) at 37°C for 2 h. Following incubation, the nucleic acids were extracted with phenol and chloroform. An equal volume of formamide was then added. Preparation

of IBDV

RNA from

bursa specimens

Bursa tissues were collected from chickens on six poultry farms with suspected cases of infectious bursa disease. Tissue samples were homogenized

84

Fig. 1. IBDV genome segment A and PCR fragments. The bar represents the genome segment A of IBDV strain Cu-1. The respective regions of the genome encoding viral proteins are indicated by boxes. The C23 displays the region amplified with PCR. Numbers indicate the corresponding fragment size in base pairs (bp).

in proteinase K buffer, frozen and thawed three times, and then digested with proteinase K (2 mg/ml) at 37°C for 2 h. The nucleic acids were extracted and treated as described for unrelated avian viruses. Reverse transcription-polymerase chain reaction

A set of primers designed for reverse transcription (RT)-polymerase chain reaction (PCR) were chosen according to the cDNA sequences of IBDV genomic segment A of strain Cu-1 reported by Spies et al. (1989). The sequences of primer pairs are as follows: Pl, SGAGATCAGACAAACGATCGCA3’ (identical to nucleotides 72 to 92, numbered according to Spies et al., 1989), and P2, SGCAGTAGTTGTAACTGGCCGG3 (complementary to nucleotides 372 to 392) (Fig. 1). Approximately 4 pg of viral RNA were denatured by heating followed by addition of methylmercuric hydroxide as described previously (Azad et al., 1985). Denatured viral RNA was then used as template for the first strand cDNA synthesis using a commercial kit with cloned Moloney murine leukemia virus (M-MLV) reverse transcriptase (cDNA synthesis system, Bethesda Research Lab., Gaithersburg, MD, USA) according to the manufacturer’s instructions. The single-stranded cDNA was extracted with a standard phenol and chloroform extraction, 2 M ammonium acetate and ethanol precipitation and resuspended in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.6). PCR was conducted in a total volume of 100 ~1 containing 67 mM Tris-HCl, pH 8.8, 16.6 mM (NH&S04, 6.7 mM MgCl*, 10 mM 2-mercaptoethanol, 6.7 PM EDTA, 170 pg/ml bovine serum albumin, 1.5 mM of each dNTP, 1 PM of each primer, 10 ~1 of sample and 2 units Taq polymerase (Boehringer, Mannheim, Germany). After addition of 300 ~1 mineral oil to prevent evaporation, the samples were subjected to 30 cycles (denaturation 30 s at 94°C; annealing 1 min at 55°C; extension 1 min at 74°C) in a programmable cyclic reactor (Perkin-Elmer Corp., Norwalk, CT, USA).

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Preparation of the DNA clone

The amplified DNA was extracted and precipitated as described above. The DNA fragment was C tailed with terminal transferase (Boehringer), annealed to G-tailed PstI-cut pUCl8 (Boehringer), and cloned in E. coli JM 109 cells (Maniatis et al., 1982). Clones containing DNA fragment were identified by using a colony hybridization procedure (Grunstein and Wallis, 1979). A clone was selected and designated as pC23. Insert within pC23 was named C23. The specificity of pC23 to the viral genome was determined by using northern-blot hybridization. Northern-blot hybridization

Purified dsRNA from IBDV strain P3009 was denatured and separated on 1% agarose gel containing 2.2 M formaldehyde as previously described (Maniatis et al., 1982). Separated RNA segments were transferred to a Zeta probe blotting membrane (Bio-Rad Lab., Richmond, CA, USA) as described by Maniatis et al. (1982) and the membrane was baked at 65°C for 1 h before hybridization. Preparation of radiolabeled probes

For colony hybridization and northern-blot hybridization, random cDNA probes were prepared directly from the purified viral dsRNA genome by using random primer (Boehringer) to initiate cDNA synthesis using the same cDNA synthesis system (BRL) as described for reverse transcription and 5 $i of [a-32P]dCTP (NEN Research Products, Boston, MA, USA). In addition, radiolabeled DNA probes for northern-blot hybridization were also prepared from the insert C23. Recombinant plasmids pC23 were extracted as described previously (Birnboim and Doly, 1979). Insert C23 was then excised by the digestion of pC23 with PstI and separated on 2% agarose gels in TBE buffer (90 mM Tris, 90 mM boric acid, 1 mM EDTA, pH 8.3). Insert C23 was eluted from the gels using the Geneclean II kit (Bio 101, La Jolla, CA, USA) before nick translation reactions. Nick translations were conducted essentially as described by Rigby et al. (1977). All radioactively labeled probes were purified from unreacted isotopes by precipitation three times from 2 M ammonium acetate and ethanol. Preparation of biotin-labeled probes

For dot blot hybridization, four kinds of biotinylated probes were prepared. Nick translation procedures were conducted to insert C23 and recombinant plasmid pC23 as described for radiolabeled probes, except that biotin-7-dATP (BRL) was used to label the probes. Probes prepared in this manner were designated C23/NT and pC23/NT, respectively. In order to label the insert C23

86

and the recombinant plasmid pC23 directly, sodium bisulfite was used to catalyze the interaction of biotin hydrazide with them as described by Reisfeld et al. (1987). The transamination reactions between hydrazide and cytosine residue in DNA were carried out for 36 h and were determined by an increase in UV absorption at 260 nm. Probes prepared in this manner were designated C23/BH and pC23/BH, respectively. All biotin-labeled probes were purified by centrifugation through Sephadex G50 spin columns (Maniatis et al., 1982). Dot blot hybridization

IBDV dsRNA extracted from cell culture or bursa specimens and viral nucleic acid from vaccine viruses were heated at 100°C for 3 min before being placed on Zeta probe membrane. The samples were applied with strong suction using a 96-well filtration manifold (Bio-Rad Laboratories) and processed as described by Jackwood et al. (1990). Zeta probe membranes containing viral nucleic acids were air-dried, packed at 80°C for 1 h, and then prehybridized in a hybridization solution containing 5 x SSC (1 x SSC: 0.15 M NaCl, 15 mM sodium citrate buffer, pH 7.0) 5 x Denhardt’s solution (0.1% Ficoll (w/v), 0.1% polyvinylpyrrolidone (w/v), 0.1% bovine serum albumin (w/v)), 50 mM sodium phosphate (pH 6.5) 0.1% SDS, 250 pg/ml salmon sperm DNA, and 50% formamide (v/v). Following incubation at 42°C for at least 4 h, the buffer was removed and fresh hybridization solution with biotinylated probes (0.5 pg/ ml) was added. The reaction was carried out at 42°C for 18 h. After washing twice at room temperature for 30 min each in 2 x SSC containing 0.1% SDS (w/v) and two times for 30 min each in 0.1 x SSC containing 0.1% SDS (w/v), the signal on the membrane was detected using a nonradioactive nucleic acid detection system according to the instruction manual (Bluegene, BRL).

Results RNA purification

and RT-PCR

The IBDV dsRNA extracted from CsCl-density gradient purified virions could be further purified by differential LiCl precipitation. The result from a representative strain P3009 is indicated in Fig. 2. The viral RNA was subjected to 0.8% agarose gels and appeared to be highly pure. The lengths of both segments were approximately 3400 bp and 2900 bp. The RNA from IBDV strain P3009 was reverse-transcribed to cDNA and amplified as described in materials and methods. DNA with a length of 320 bp was amplified in accordance with the reaction cycles (Fig. 3. cf. Fig. 1). The amplified DNA was as long as that expected from the cDNA sequences of the designated region. No amplified product could be observed when nucleic acids from mock-infected CEF cells were used as templates.

87

!3130bp #ii 4361 3400 2900

587 bp434 bp267 bp-

310

bp

184 bp124 bp-

564

Fig. 2. Agarose gel electrophoresis of IBDV genomic RNA following purification with differential LiCl precipitation. The gel was stained with ethidium bromide and nucleic acid bands were visualized. Lambda bacteriophage DNA digested with Hind111 was included for size references (lane A). Viral genome segments (strain P3009) with the mol wt. of 3,400 base pairs and 2,900 base pairs are indicated (lane B). Fig. 3. Amplification of the DNA fragment from IBDV strain P3009. Lane I, DNA of plasmid pBR322 digested with HueIII; lane 2, amplification products; lane C, negative control, amplification starting from the preparation of mock-infected CEF cells.

Characterization

of the pC23 clone

With colony hybridizations, IBDV-specific clones were identified (data not shown). Clone pC23 was selected as a probe for dot blot hybridization. Northern-blot hybridization results indicated that insert C23 hybridized to viral genome segment A (Fig. 4, lane 2). The random cDNA probes prepared directly from the P3009 dsRNA genome were used as a control in northern-blot hybridization. This probe hybridized to both segments of the genome (Fig. 4, lane 1). The length of insert C23 was determined and was similar to that of amplified DNA (data not shown). Detection of IBDV strains using biotinylated probes in dot blot hybridization The viruses tested were P3009, LU, FlO, and MO. Four biotionylated probes (C23/NT, pC23/NT, C23/BH, and pC23/BH)

kinds of prepared

88

Fig. 4. Northern-blot hybridization. IBDV genomic RNA (strain P3009) and nucleic acid from mockinfected CEF cells were denatured, separated on a 0.8% agarose-formaldehyde gel, and transferred onto Zeta probe membrane. Lane 1, the membrane was incubated with a probe prepared from the P3009 genome; lanes 2 and C, the membranes were incubated with a probe prepared from the insert C23. Viral genome segments A and B are indicated.

- 25.00 ng -

:

I

-

5.00 ng -

-

1 .OO ng --

-

0.20 ng -

-

0.04

-

0.008ng

ng -

Fig. 5. Dot blot hybridization of IBDV genomes. Three serotype 1 viruses (P3009, LU, and FlO) and one serotype 2 virus (MO) were tested. The nick translated probe prepared from C23 (A) and probe prepared by the interaction of biotin hydraaide with pC23 (B) were used to detect various concentrations of dsRNA from IBDV strains. Following 36 h of autoradiography, the lowest concentrations (approximately 1 ng in A, 0.04 ng in B) of viral RNA were detected. Nucleic acid from CEF cells was used as a negative control.

89

TABLE I The lowest quantities of IBDV RNA detected by biotinylated following different procedures Probe

C23 /NT PC23/NT C23 /BH pC23/BH

probes prepared

from C23 or PC23

Virus strain MO

FlO

LU

P3009

5.00” 1.00 1.oo 0.04

5.00 5.00 1.oo 0.04

I .oo

1.oo 1.oo 1.oo 0.04

aNumbers represent the lowest quantities

1.00 1.oo 0.04

in ng detected.

using different procedures were used to detect the viral RNA. A serial live-fold dilutions of each viral RNA with TE buffer were made. Quantities ranging from 25 ng to 0.008 ng of viral RNA were placed on Zeta probe membranes. Similar quantities of total nucleic acid extracted from mock-infected CEF cells were included as a negative control. The representative results detected by probes C23/NT and pC23/BH are shown in Fig. 5. The intensity of the signal was determined by visual inspection. A result that could easily be distinguished from the CEF background signal was considered positive. Both probes reacted with all virus samples which included 3 serotype 1 viruses and 1 serotype 2 virus, while the level limits of viral RNA detection were significantly different. Dot blot hybridization using probe C23/NT led to the detection of approximately 1 ng target RNA (Fig. 5A). On the other hand, probe pC23/ BH detected viral RNA as little as 0.04 ng (Fig. 5B). In addition, both probes did not react with nucleic acid prepared from mock-infected CEF cells. Using 4 biotinylated probes for detecting target viral RNA prepared from different IBDV strains, different level limits of detection were recognized. The results are summarized in Table 1. All virus samples were detected in various level limits using these four probes. Generally, similar limits (approximately 1 ng) of viral RNA detection were obtained by probes C23/NT, C23/BH, and pC23/NT. The most sensitive results were obtained by using probe pC23/BH (0.04 ng). The specificity of these 4 probes for the detection of IBDV strains was determined by using nucleic acids prepared from seven unrelated avian viruses which contained both RNA and DNA viruses. Genome RNA of IBDV strain P3009 was used as a positive control. The probe did not hybridize with the nucleic acid from any of the unrelated viruses tested; but it did hybridize with the IBDV genome RNA from the strain P3009 (data not shown). Detection of IBDV RNA from bursal specimens To determine the value of the probe pC23/BH and dot hybridization procedure for detecting IBDV in clinical bursa tissues, six samples A to F from different commercial broiler raising farms were collected. Each sample contained a pool of 3 to 5 bursas which were homogenized in proteinase K

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BURSA A

B

C

SAMPLE 0

E

F

C

Fig. 6. Dot blot hybridization using bursa tissue from commercially raising broilers in which IBDV infections were suspected. Two separate nucleic acid extractions and hybridizations (1 and 2) were made with the bursa tissues. Six bursa samples (A to F) from different farms were tested. Bursa from mockinfected chickens was used as a negative control (C). Each sample dot contained 200 ~1 of the original bursal homogenate.

buffer (1 ml per bursa). A volume of 0.5 ml homogenized bursa tissue was digested with proteinase K as described previously. Each sample spotted on the membrane contained a 200~~1 volume of the original bursa homogenate. Dot blot hybridization using probe pC23/BH showed that all bursa samples tested contained IBDV, however, hybridization of probe pC23/BH to bursa samples from a mock-infected control bird was not observed (Fig. 6).

Discussion The use of cloned cDNA probes labeled with biotin (Jackwood et al., 1990) or with radioisotope (Jackwood et al., 1989) for the detection of IBDV has been studied. Jackwood (1990) also used the probes prepared from IBDV genomic RNA to detect IBDV strains. However, because it may be difficult and time consuming to prepare sufficient quantities of purified genomic IBDV RNA for the production of cDNA probes, the use of probes prepared in this manner may be limited as a practical diagnostic tool. In addition, although the availability of molecular clones of the IBDV genome may eliminate isolation and purification of the dsRNA for the production of the probes, the use and detection of radiolabeled probes is not practical for most diagnostic laboratories (Jackwood, 1990). It was concluded that the use of nonradioactive IBDV-specific probe such as those labeled with biotin, prepared from cloned viral cDNA may be a reliable and fast tool for the diagnosis of IBDV (Jackwood et al., 1990). The DNA molecules can be labeled with biotinylated nucleotide using nick translation with the commercial available kit. Chemically, biotin can be introduced directly into DNA molecules (Reisfeld et al., 1987). In 3 labeling experiments, the reactions were carried out for 36 h. Following reaction the absorbances were determined and their optical density values were consistent and were approximately 0.33 (data not shown). Results of the recent study showed that probes (C23/BH and pC23/BH) have been prepared successfully

91

by directly labelling insert C23 or the recombinant plasmid pC23 with biotin hydrazide and the labeling procedures are stable and reproducible. All four kinds of the biotinylated probes prepared in this study detect viral dsRNA in dot blot hybridization assays. The detection of IBDV RNA using probe C23/ NT has similar results to that detected with probe C23/BH. However, probe pC23/BH exhibits significant differences of sensitivity for detecting IBDV RNA. These differences could be due to the efficiency of biotin incorporation, but not the G:C ratio that is higher than that of A:U in IBDV genome (Becht, 198 1). In addition, probe pC23/BH contains plasmid DNA which could also be labeled with biotin. Therefore, the relatively high quantities of biotin molecules which were incorporated into probe pC23/BH and gave a more intense signal may contribute to this difference. The viruses tested include three strains (P3009, LU, and FlO) of serotype 1 IBDV and one serotype 2 virus (MO). Each of 4 probes prepared to virus strain P3009 hybridizes not only to all three serotype 1 viruses, but also to virus MO. Furthermore, bursa tissues collected from six different commercial broiler rearing farms were tested with the probe pC23/BH. IBDV RNA in all six samples were detected. These results indicate the potential for detecting several different strains of IBDV using nucleic acid probes. Additionally, the probe pC23/BH appears to be specific for IBDV RNA. because it did not hybridize with nucleic acid extracted from seven unrelated avian pathogens or from mock-infected CEF cells in dot blot hybridization. The results demonstrate that dsRNA extracted from IBDV strain P3009 can be reverse-transcribed to cDNA followed by further amplification with Tuq polymerase. The amplified DNA fragment was as long as that expected from the cDNA sequences of the designated region and was not observed when mock-infected nucleic acids were used (Fig. 3C). Following cloning in bacterial cells, insert C23 was confirmed to be located on genome segment A (Figs. 1 and 4) based on the northern-blot hybridization. Together, these observations indicated that the amplified DNA did not originate from a contaminated unknown DNA but was a product of the designated region of IBDV. Baylis et al. (1990) analyzed the nucleotide sequences of three strains of serotype 1 IBDV and compared with those of an Australian strain 002-73 (Hudson et al., 1986). The results showed that the nucleotide sequences located in the region which was amplified in this study are highly conserved. Therefore, the probe pC23/ BH prepared by directly labeling with biotin seems to be practical for the diagnosis of IBDV for most diagnostic laboratories.

Acknowledgement Financial support for this research was provided by the National Council (NSC-79-0409-B-005-06), Republic of China.

Science

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