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Real-time RT-PCR differentiation and quantitation of infectious bursal disease virus strains using dual-labeled fluorescent probes
Journal of Virological Methods 127 (2005) 87–95
Real-time RT-PCR differentiation and quantitation of infectious bursal disease virus strains using dual-labeled fluorescent probes Michelle A. Peters, Tsang Long Lin, Ching Ching Wu ∗ Department of Veterinary Pathobiology, Purdue University, 406 S. University St., West Lafayette, IN 47907-2065, USA Received 27 May 2004; received in revised form 28 February 2005; accepted 1 March 2005 Available online 21 April 2005
Abstract A real-time RT-PCR assay was developed utilizing dual-labeled fluorescent probes binding to VP4 sequence that are specific to the classical (Cl), variant (V) and very virulent (vv) strains of infectious bursal disease virus (IBDV). The assay was highly sensitive and could detect as little as 3 × 102 to 3 × 103 copies of viral template. Viral genomic copy number could be accurately assayed over a broad range of 7–8 logs of viral genome. The variant sequence-specific probe was found to be highly specific in detecting isolates classified as variant A, D, E, G and GLS-5, and did not react with classical strains. A total of 130 field and experimental variant strain isolates were tested using this assay. The classical sequence-specific probe also demonstrated high sensitivity and specificity, and positively detected a total of 87 STC isolates, both field and experimental isolates, while differentiating between isolates that were variant and classical strains. The very virulent sequence-specific probe detected positively the Holland vvIBDV isolate and did not react with classical or variant strains. Rapid identification of viral strain is a primary concern to poultry flock health programs to ensure administered vaccines will protect against current strains of virus circulating in the flock. The ability to quantify virus concurrently is also of assistance in identifying the progression of disease outbreaks within the flock. © 2005 Elsevier B.V. All rights reserved. Keywords: Infectious bursal disease virus; Classical; Variant; VP4; Real-time RT-PCR; Dual-labeled fluorescent probes
1. Introduction Infectious bursal disease virus (IBDV) infection of young chickens results in lymphocytolysis of the Bursa of Fabricius and immunosuppression, leading to an increased incidence of vaccination failure and secondary infections within the flock (Allan et al., 1972; Cosgrove, 1962). IBDV is a nonenveloped virus with a bi-segmented, double-stranded, RNA genome, classified within the genus Avibirnavirus and within the Birnaviridae family (Kibenge et al., 1988; Murphy et al., 1999; van Regenmortel et al., 2000). Genome segment A is 3.3 kb in size and encodes two partially overlapping open reading frames (ORF) (Bayliss et al., 1990; Hudson et al., 1986). The first ORF encodes the 17 kDa, non-structural, VP5 gene, and the second encodes a 110 kDa polyprotein VP243 ∗
Corresponding author. Tel.: +1 765 494 7459; fax: +1 765 494 9181. E-mail address: [email protected] (C.C. Wu).
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that is cleaved by the auto-protease VP4 into constituent proteins pVP2 (48 kDa), structural protein VP3 (32 kDa) and VP4 (24 kDa) (Birghan et al., 2000; Muller and Becht, 1982). The precursor pVP2 protein is then further processed to the mature structural protein VP2 (38 kDa) (Muller and Becht, 1982). Genome segment B is 2.8 kb in size and encodes a single 90 kDa protein VP1, the putative viral RNA-dependent RNA polymerase (Muller and Nitschke, 1987; Spies et al., 1987). VP1 protein is also found within the viral particle complexed to both genome segments A and B. Of concern in the control of IBDV infection is the steady progression of viral evolution over recent decades and the emergence of both new antigenic variants (V) and strains with enhanced virulence. Before 1985, all classical (Cl) strains of IBDV were indistinguishable antigenically by virus neutralization (VN) assays (Jackwood and Saif, 1987; Jackwood et al., 1985). Classical strains varied in virulence, mortality ranged from 1 to 30%, and protection was achieved by the
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administration of inactivated vaccines to breeder birds. Vaccination is the primary means of control of infectious bursal disease in the poultry industry (Lukert and Saif, 1991). In 1986, variant viruses were first described in the USA as newly emergent viruses due to a major antigenic shift within serotype 1 (Snyder et al., 1988a; Snyder et al., 1992). Antigenic variants could not be cross-neutralized by antiserum raised against classical strains (except at very high titers) (personal communication) and could evade immunity to current classical vaccines within a flock (Ismail and Saif, 1991). Very virulent (vv) strains (vvIBDV) emerged in Europe in 1986, and were identified subsequently in Africa and Asia, but not in the USA (Abdel-Alim and Saif, 2001; Cao et al., 1998; Jackwood and Sommer, 2002; Liu et al., 2001; Rautenschlein et al., 2003; Sun et al., 2003; Tsukamoto et al., 1999). Very virulent IBDV is capable of breaking through high-levels of maternal-antibody protection and producing 70% flock mortality. The very virulent strains are similar antigenically to the classical strains. Effective control of IBD is contingent on the availability of rapid and efficient diagnostic procedures for identifying and differentiating IBDV strains within a flock. Currently, there is no single-step assay available that will rapidly identify and differentiate IBDV strains. Nucleic acid-based technologies for the diagnosis of IBDV flock status have targeted largely the VP2 gene sequence (Bayliss et al., 1990; Lee et al., 1992; Lin et al., 1994; Vakharia et al., 1992), or the combined VP2 and VP3 regions (Lee et al., 1992; Tham et al., 1995), with the exception of recent studies targeting the VP1 gene (Islam et al., 2001; Tiwari et al., 2003), and one study targeting the VP4 gene (Wu and Lin, 1992). A quantitative real-time RT-PCR assay targeting VP2 was developed by Moody et al. (2000) that would detect all IBDV strains. Virus neutralization assays or virulence studies in SPF chickens are direct methods to differentiate variation in antigenicity and virulence. Jackwood and Saif (1987) classified six subtypes of IBDV using VN and cross-neutralization assays. Vakharia et al. (1994) demonstrated that antigenic variation was due to amino acid changes in VP2, and Heine et al. (1991) showed that changes in four residues were sufficient for escape of the variant Del E strain. VP2 gene sequence between residues 206 and 350 has been identified as a hypervariable region and contains major serum-neutralization and conformational epitopes. Within the hypervariable region, major hydrophilic peaks have been identified with maximal variation between residues 212–224 and 314–324, and minor hydrophilic peaks between residues 248–252 and 279–290 (Dormitorio et al., 1997; Smiley and Jackwood, 2001). Marked sequence heterogenicity exists within the epitopes responsible for antigenicity and immunogenicity. Sequence flanking the hypervariable region is highly conserved across all IBDV isolates and is a target region for specific amplification of serotypes 1 and 2 IBD virus. A series of predicted restriction enzyme sites were identified within the hypervariable region of VP2, that could be employed for the
differentiation of antigenic variant, classical and very virulent strains (Jackwood and Jackwood, 1994; Jackwood and Sommer, 1997, 1999; Kataria et al., 1999; Liu et al., 1994; Zierenberg et al., 2000). Using either the RT-PCR-RE or RTPCR-RFLP techniques, strains have been classified into six molecular groups: groups 1 and 2 contained variant viruses, groups 3 and 4 contained classical viruses, group 5 was characterized by the Lukert strain, contains both classical, variant and hybrid strains and group 6 contains the Intervet vaccine strain RS593, 50% of field strains originating outside the USA and also very virulent strains (Jackwood and Sommer, 1999). Virulence determinants and the markers for virulent phenotypes are currently unknown, and are not associated with antigenic subtypes, as very virulent strains are similar antigenically to less virulent classical strains. The objective of the present study was to develop a real-time RT-PCR assay utilizing fluorescent probes binding to strain-specific sequence from either classical, variant or very virulent strains. Sequence from the VP4 gene was targeted due to its sequence conservation within strains and therefore its capacity to differentiate strain lineage. Such an assay would allow the rapid, single-step classification of isolates during the cycling reaction and would negate the need for separation and visualization of PCR products by electrophoresis in an agarose gel, and the practice of restriction digestion to differentiate strains. The utility, high sensitivity and specificity of real-time RT-PCR make it an ideal assay for management of poultry health programs.
2. Materials and methods 2.1. Sequence analysis and design of primer/probe sets IBDV genomic sequences were aligned using ClustalX via the interface (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/) (Thompson et al., 1997). PrimerSelect (DNAStar Inc., Madison, WI) software was used to select primer-probe sets for real-time RT-PCR assays of the IBDV genome. Oligonucleotides were selected to amplify a region of between 120 and 200 bp that was conserved across all aligned IBDV sequences. Amplicons were selected further to contain 20–30 bp probe sequences that were conserved within but varied between classical, variant or very virulent strains of IBDV. Fig. 1 illustrates the alignment of primer/probe pairs to VP4 sequences (bases 2026–2161) present in the GenBank database. The forward primer 5 -GTCGAGTGGATATTGGCCCC 3 and reverse primer 5 -GGCTCCTGCGTTATTCTTGC-3 (Fischer Scientific, Itasca, IL, USA) were used to amplify an IBDV segment A amplicon between bases 2026 and 2161 (numbering based on the IBDV variant E sequence, #AF133904) within the VP4 gene. A dual-labeled, fluorescent probe JOE – 5 -CAATGCTTGTGGCGAGATTGAGAAAGTAAG-3 – BHQ1 was designed with sequence specific to aligned classical IBDV strains (probe 1), and
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Fig. 1. Sequence alignments of variant, classical and very virulent IBDV segment A sequences from residues 2026 to 2180 bp using classical strain STC as the reference. Virus sequences are clustered into variant, classical and very virulent strains. Variant E IBDV sequence is used as the consensus sequence for variant strains, STC IBDV sequence is used as the consensus sequence for classical strains and OKYM sequence is used as the consensus sequence for very virulent strains. Universal flanking primer sequences are shown as boxed text at the top of the alignment, and probe sequences conserved with the isolate subtypes are shown as boxed text above each cluster of isolates. Within the region targeted by the probe consensus residues are shown as asterices, residues identical to the consensus sequence are illustrated by capitals and non-homologous residues are italicized. Row 1: IBDV isolate STC (D00499); 2: IBDV isolate IM (AY029166); 3: IBDV isolate Edgar (A33255); 4: IBDV isolate PBG-98 (D00868); 5: IBDV isolate Cu-1 (D00867); 6: IBDV isolate 52/70 (D00869); 7: IBDV isolate GBF-1 (D16828); 8: IBDV isolate Cu-1 (X16107); 9: IBDV isolate Mundt (X84034); 10: IBDV isolate Purdue 2003 (our sequence not submitted to GenBank); 11: IBDV isolate variant E (AF133904); 12: IBDV isolate GLS (M97346); 13: IBDV isolate Vakharia (X54858); 14: IBDV isolate Purdue 2003 (our sequence not submitted to GenBank); 15: IBDV isolate OKYM (D49707); 16: IBDV isolate UK661 (X92760); 17: IBDV isolate HK46 (AF092943); 18: IBDV isolate Tasik94 (AF322444); 19: IBDV isolate D6948 (AF240686); 20: IBDV isolate KS (L42284); 21: IBDV Holland isolate.
a second dual-labeled, fluorescent probe FAM – 5 -CAACGCTTGTGGCGAGATTGAGAAAATAAG-3 – BHQ1 (probe 2) was designed with sequence specific to aligned variant IBDV strains (Biosearch Technologies, Novato, CA, USA). A third probe FAM – 5 -CAACGCCTATGGCGAGATTGAGAACGTGAG-3 – BHQ1 (probe 3) was designed with sequence specific to aligned very virulent IBDV strains. 2.2. Viral RNA samples Bursae were obtained both from chickens inoculated experimentally with IBD viruses within the animal housing facilities and from field samples. The types of isolates, number of isolates and dates of acquisition are outlined in Table 1. The bursal tissue was collected onto dry ice and either stored at −80 ◦ C or processed immediately. Bursal tissue was homogenized in chilled sterile phosphate buffered saline (PBS: 0.136 M NaCl, 0.003 M KCl, 0.01 M Na2 HPO4 , 0.002 M KH2 PO4 ; pH 7.4). The homogenate was clarified by centrifugation at 2000 × g for 5 min, and the cellular debris was re-suspended in PBS wash buffer, homogenized a second time and clarified by centrifugation at 2000 × g for 5 min.
The clarified homogenates were combined and total RNA extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s instructions. Purified RNA was eluted in a final 50 l volume of 0.1% diethyl pyrocarbonate (DEPC) treated water. 2.3. Real-time RT-PCR assay for IBDV Reverse transcription was performed at 42 ◦ C for 50 min in a 25 l reaction mix, containing 5 l RNA preparation, 2.5 M of each IBDV primer, 250 M each of dATP, dCTP, dGTP and dTTP (Promega), 1× Superscript RNaseH− Reverse Transcriptase buffer (Invitrogen), 1 U Superscript RNaseH− Reverse Transcriptase (Invitrogen) and 0.01 M DTT (Invitrogen). The cDNA was then amplified by realtime PCR. A 25 l reaction mixture was prepared containing 250 M each of dATP, dCTP, dGTP and dTTP (Promega, Madison, MA, USA), 3 mM MgCl2 , 2.5 l of 10× Taq DNA polymerase buffer (Promega), 1 U of Taq DNA polymerase (Promega), 2.5 M of each primer, either 3 M JOE-labeled classical IBDV probe, 3 M FAM-labeled variant IBDV probe or 3 M FAM-labeled very virulent IBDV probe, and 4 l of template cDNA. Polymerase 5 -nuclease activity
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Table 1 IBDV isolates assayed by real-time RT-PCR assay to detect variant, classical and very virulent strain-specific VP4 sequences Isolate typea
Field samplesb
Experimental samplesb
Acquisition dates
Classical probe assay positives
Variant probe assay positives
Very virulent probe assay positives
Variant E (V)
10 (Bursae)
80 (Bursae)
1c
90
0
2–21
Variant A (V) Variant D (V) Variant G (V) GLS-5 (V) STC (Cl) D78 (Cl) Holland (vv)
5 (Bursae) 5 (Bursae) 5 (Bursae) 5 (Bursae) 1 (Bursae) – –
5 (Bursae) 5 (Bursae) 5 (Bursae) 5 (Bursae) 80 (Bursae) 6 (Bgm cell line) 1 (Bursa)
9/1997, 1/2000, 6/2002, 9/2002, 8/1995, 6/2000 8/1995, 6/2000 8/1995, 6/2000 6/2000, 6/2002 9/1997, 6/2002, 9/2002 6/2002, 8/2003 11/2004
0 0 0 0 81 6 0
10 10 10 10 0 0 0
0 0 0 0 0 0 1
8–20 6–15 10–12 10–13 3–20 4–5 2–20
a b c
Log range of copy number detected
Name of isolate and broad classification as variant (V), classical (Cl) strain or (vv) very virulent. Isolate obtained by experimental IBDV infection of chickens or from field poultry samples. Sample was negative when the assay was repeated.
during PCR amplification resulted in a reduction in fluorophore quenching by the BHQ1 dye, and this fluorescent signal was measured in the Rotor Gene, real-time, thermal cycling system (Corbett Research, Mortlake, NSW, Australia). The cycle profile was 1 cycle of 60 ◦ C for 3 min, 95 ◦ C for 3 min and 50 cycles of 95 ◦ C for 20 sec, 60 ◦ C for 1 min. Cycle time (Ct ) was measured for each assay. One-step, real-time, RT-PCR assays were also carried out, combining the superscript reverse transcriptase (RT) with Platinum Taq DNA polymerase in a single reaction mix. A 25 l reaction mix was prepared containing 12.5 l of 2× reaction buffer (Invitrogen), 2.5 M of each primer, 0.5 l Superscript RT/Platinum Taq DNA polymerase mix (Invitrogen), 3 M MgSO4 , either 3 M JOE-labeled classical IBDV probe, 3 M FAM-labeled variant IBDV probe or 3 M FAM-labeled very virulent IBDV probe and 4 l of template RNA. The cycle profile was 1 cycle of 45 ◦ C for 20 min, 95 ◦ C for 3 min, and 40 cycles of 95 ◦ C for 20 s, 60 ◦ C for 1 min.
reverse primer 5 -GACGACCGATTTGCACTGC-3 and the probe FAM – 5 -AGGACCGCTACGGACCTCCACCA-3 – BHQ1. The sequences of the target amplicons from the variant E, STC and Holland vvIBD viruses were established by bidirectional, automated sequencing. 2.5. Time course of infection in bursa using the IBDV real-time RT-PCR assay One-day-old SPF chickens were inoculated orally with either: (i) 106 EID50 variant E IBDV in 200 l DMEM; (ii) 106 EID50 IBDV strain STC in 200 l DMEM; or with (iii) 200 l DMEM. For each treatment group, five chickens were euthanased at 12, 24 and 48 h after infection and the bursae were removed and placed into chilled PBS. Bursal tissue RNA was extracted for RT-PCR as described above.
3. Results 2.4. IBDV real-time RT-PCR standard curves 3.1. Sequence analysis IBDV large segment ORF was amplified from preparations of bursal RNA infected with either variant E, STC or Holland vvIBD virus, and cloned into the pCR3.1 vector (Invitrogen), as described previously (Chang et al., 2001). Tenfold dilution series, starting with 2 g/ml, of either variant E or STC IBDV template cloned into pCR3.1 vector, were used to standardize Ct values from the real-time PCR assays against template copy number. Sample template copy number was calculated from cycle time by linear regression using the respective standard curves. The linear ranges of the real-time, RT-PCR assays were established from eight serial 10-fold dilutions of sample bursal RNA purified from chickens inoculated experimentally with either the variant E or STC viruses. Sample Ct values were normalized against Ct values for amplification of the 28S internal standard. The 28S gene was amplified using forward primer 5 -GGCGAAGCCAGAGGAAACT-3 ,
A target region of 135 bp sequence between bases 2026 and 2161 of VP4 (numbering based on the IBDV variant E sequence, GenBank number AF133904) was identified from the alignment of the VP4 gene of IBDV isolates (Fig. 1). Many of the IBDV GenBank entries are limited to VP2 gene sequence and do not include the VP4 sequence. The alignment was composed of a total of nine classical strain sequences, three variant strain sequences and seven very virulent strain sequences. The probe sequences contain six potentially variant bases. Probes designed for classical strains differed from variant strains by two bases, and both differed from very virulent strains by five bases (the differences between classical and very virulent, and variant and very virulent strain probes were not identical). Two classical strain isolates and one very virulent strain isolate each had one out of the six variant bases mismatched with the consensus sequences. Variation in VP4
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Fig. 2. Standard curves of real-time RT-PCR assays of IBDV using probe 1 (sequence specific to classical strain sequence), probe 2 (sequence specific to variant strain sequence) and probe 3 (sequence specific to very virulent strain sequence). (a) Fluorescence readings for the assay using probe 1 of a dilution series of classical STC IBDV RNA; (b) fluorescence readings for the assay using probe 2 of a dilution series of variant E IBDV RNA; (c) fluorescence readings for the assay using probe 3 of a dilution series of Holland vvIBDV RNA; (d) standard curve for probe 1 assay of a dilution series of the STC IBDV large segment ORF cloned into the pCR3.1 vector; (e) standard curve for probe 2 assay of a dilution series of the variant E IBDV large segment ORF cloned into the pCR3.1 vector; (f) standard curve for probe 3 assay of a dilution series of the Holland very virulent IBDV large segment ORF cloned into the pCR3.1 vector (color version on www.ScienceDirect.com).
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Fig. 2. (Continued ).
sequence is not as great between isolates as for VP2 and VP3 sequences. No target amplicons suitable for real-time RT-PCR were found using alignments of sequences of the IBDV genes VP2, VP3 and VP1. Requirements for such sequences would be between 120 and 200 bp amplicons, with terminal sequences conserved in all strains, suitable targets for flanking primers, and contain intervening sequence conserved within, but variable between strains as a target for strain-specific probes. The probe sequences are also required to have a Tm of 70 ◦ C (10 ◦ C greater than the primer sequence), no runs of nucleotide repeats, low GC content in the 3 -terminus, no Gclamping on the 5 -terminus and suitable GC content. The high degree of variation evident in sequence alignments between isolates of the same strain within the VP2 and VP3 genes made it difficult to identify target amplicons (data not shown). The probe target region was chosen in order to differentiate classical, variant and very virulent strains.
was 0.01, and the cycle threshold (Ct ) range was cycles 5–35. The linear range of the standard curve was between 3 × 102 and 3 × 109 template copies. A linear relationship was established between Ct value and concentration of RNA in bursal tissue from chickens experimentally inoculated with variant E virus. IBDV sequence was not detected using real-time RT-PCR in RNA samples from the bursae of control chickens inoculated with DMEM. IBDV variant E RNA was detected at 12, 24 and 48 h after inoculation in bursal tissue of all chickens and mean titers were highest at 24 h after infection (Fig. 3). Variants A, D and G and GLS-5 RNA isolates of IBDV were detected using the probe based on conserved variant virus sequence (Table 1). The variant IBDV real-time, RT-PCR assay did not amplify STC or D78 classical strain sequences or Holland very virulent strain sequence (Table 1).
3.2. Real-time RT-PCR assay for IBDV variant strains
As for variant strains, a standard curve was derived from the STC.pCR3.1 plasmid standard (Fig. 2). The R2 value for the STC standard curve was 0.905, the Rn significance threshold was 0.01, and the Ct threshold range was cycles 10–40. The linear range of the standard curve was between 3 × 102 and 3 × 108 template copies.
A standard curve of template copy number against Ct value was plotted using the variant E. pCR3.1 plasmid standard (Fig. 2). The coefficient of determination (R2 ) for the variant E standard curve was 0.999, the significance threshold (Rn )
3.3. Real-time RT-PCR assay for IBDV classical strains
Fig. 3. Detection of IBDV sequence using real-time RT-PCR in RNA extracted from the bursae of experimentally infected 1-day-old chickens. Groups of 20 chickens were inoculated with either (a) STC strain IBDV or (b) variant E IBDV, and five chickens were sampled at time points 0, 12, 24 and 48 h after infection. IBDV log genomic copy number in the bursa of each chicken is illustrated.
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Using the classical sequence-specific probe, there was no signal detected from control RNA samples from chickens inoculated with DMEM. IBDV STC RNA was detected in bursal tissue at all time points following inoculation with STC strain virus (Fig. 3). Isolates of the D78 classical strain were also detected in RNA samples using the probe based on conserved classical strain sequence (Table 1). A weak signal was detected for RNA samples containing variant E virus using the classical sequence-specific probe, however, the amplitude of the signal was consistently lower. Therefore, raising the threshold for signal detection allowed differentiation of classical and variant isolates (Table 1). The classical sequence-specific probe did not amplify the Holland very virulent strain sequence. 3.4. Real-time RT-PCR assay for IBDV very virulent strains A standard curve was derived from the Holland vvIBDV.pCR3.1 plasmid standard (Fig. 2). The R2 value for the Holland vvIBDV standard curve was 0.999, the Rn significance threshold was 0.01, and the Ct threshold range was cycles 3–35. The linear range of the standard curve was between 2 × 102 and 2 × 1010 template copies. The vvIBDV-sequence-specific probe detected Holland strain virus in RNA purified from infected bursal samples, but did not amplify the STC or D78 classical strains, nor the variants A, D and G and GLS-5 variant strains. 3.5. 28S internal standard Standard curves were derived for the real-time RT-PCR assays of the 28S internal standard, based on the method described by Moody et al. (2000) (Kaiser et al., 2000). The 28S gene was amplified by real-time RT-PCR from a 10-fold dilution series of sample RNA. The coefficient of determination (R2 value) for the standard curve was 0.997, the Rn significance threshold was 0.05, and the Ct threshold range was cycles 10–40. The 28S standard assay was used to standardize sample differences in cell and RNA content. 3.6. Differentiation of classical, variant and very virulent subgroups of IBDV The real-time RT-PCR assays developed can be used to differentiate between isolates of classical, variant and very virulent strains of IBDV. Classical sequence-specific probe detects all IBDV classical viruses tested, with a weak sub-threshold signal for variant isolates. Variant sequencespecific probe detects and is specific to variant virus isolates. The very virulent sequence-specific probe detects and is specific to very virulent virus isolates tested. There was no significant difference observed when quantifying both the plasmid standards or the RNA samples from IBDV isolates, when using either the single-step, real-time, RT-PCR or the two-step method from cDNA (data not shown).
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4. Discussion In this study, we report the development of an IBDV diagnostic assay using dual-labeled fluorescent probes and realtime RT-PCR to differentiate IBDV isolates into classical and variant strains. The assay is highly sensitive and can detect as little as 3 × 103 (in the case of classical sequence-specific probes), 3 × 102 (for variant sequence-specific probes) or 2 × 102 (for very virulent sequence-specific probes) genomic templates in tissue samples. The variant sequence-specific probe detected all 130 variant isolates tested, including both field and experimental isolates of variants A, D, E and G and GLS-5. The variant sequence-specific probe did not detect the 88 classical isolates tested. The 88 classical isolates, field and experimental isolates, were specifically detected by the classical sequence-specific probe. A probe with very virulent strain-specific sequence was designed for the purpose of extending the application of the assay to the diagnosis of very virulent IBDV where this strain is prevalent. Testing of the very virulent strain-specific probe was limited to a single isolate as vvIBDV is currently not found in the USA and due to the difficulty of importing vvIBDV into the USA. The real-time RT-PCR assay has considerable advantages for application to flock diagnosis of IBDV. The speed and ease of the reaction facilitates its application to the analysis of multiple samples. Because the fluorescence signal is detected in real-time, results are obtained rapidly and without further analysis, such as separation of PCR products by electrophoresis for visualization. The assay has a broad range of between 7 and 8 logs in which viral genomic template can be accurately quantified. Very low amounts of viral genome can be detected, as little as 300 copies. Therefore, changes in viral titers occurring during the progression of infection can be accurately assessed in flock samples. Assessment of titer as well as viral strain is valuable in tracking the status of flock infections and pinpointing the beginning of disease outbreaks. The planning of effective vaccination schedules requires knowledge of the strain of IBDV currently in the flock, as the majority of vaccines in commercial use are strain-specific. The utility of the assay in charting the progression of infection is demonstrated by the time-course study of experimental infection of 1-day-old chickens with either classical or variant strains of IBDV (Fig. 3). Viral titer in the bursa was assayed at 12, 24 and 48 h after oral infection of 1-day-old birds. The real-time RT-PCR assay detected a peak average STC strain titer of 9 × 1017 genomic copies as early as 12 h after infection, and titers declined to an average of 4 × 1016 copies by 48 h. A peak variant E strain titer of 2 × 1019 genomic copies was observed 24 h after infection, and as with STC strain infection, titers declined by 48 h to an average of 1 × 1017 copies. These two patterns of infection were evident for all chickens in the respective treatment groups. The results point to an early acute infection with classical strain viruses, and a slightly longer latent period for variant strain viruses. The higher levels of titer observed for variant E than for STC
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strain suggests a greater capacity of variant viruses for replication in the bursa of newborn chickens. Variant viruses are reported to cause rapid atrophy of the bursa without bursal haemorrhage and inflammation or high mortality (Snyder et al., 1988a). The application to flock surveillance of sensitive assays that can detect not only strain but also viral titer will lead to the accumulation of data that clearly relates viral titer to the incidence of clinical disease and immune suppression for the different strains of IBDV. It is anticipated that these studies be extended to the analysis of mixed IBDV infection, both with different IBDV strains and mixed pathogen infections. The variant sequence-specific and the very virulent sequence-specific probes were highly specific to variant and very virulent isolates, respectively, and no signals were detected for other subgroup templates. The classical sequencespecific probe was also specific to sequences of classical isolates, however, in some assays of variant isolates a lowamplitude, sub-threshold, fluorescence signal was detected. The amplitude of the fluorescence signal is dependent on the affinity of the probe for the template during PCR amplification, which in turn leads to the release of the fluorophore from the vicinity of the quencher by the 3 -exonuclease activity of the polymerase. A weak sub-threshold signal is indicative of the classical probe 1 having a low affinity for the variant isolate template. This does not interfere with the ability of the assay to specifically identify classical isolates, as raising the threshold for detection eliminates signal from amplification of variant isolates. Neither are false negatives likely to occur with increased template concentration. Cycle time required for the fluorescence signal to cross the threshold is a function of template concentration, and signal amplitude is not dependent on concentration. The real-time RT-PCR assay developed for IBDV is unique in that it targets sequence from the VP4 gene. Differential diagnostic assays have been largely directed to the hypervariable region of VP2 due to the abundance of sequence variability within this region, however, this variability exists both within and between strains and the sequence in this region does not serve as a consistent marker for the differentiation of variant, classical and very virulent strains. While heterogeneity between vaccine and laboratory strains is low for this region of VP2, heterogeneity between field strains is marked and can be problematic, as evident by RFLP patterns from field strains in epidemiological surveys which consistently do not fit into the established molecular groupings, and differences between studies in the findings as to the utility of particular restriction endonuclease sites as group specific markers. Jackwood et al. in (Jackwood and Sommer, 1999) found as many as 19 field strains with RFLP patterns that could not be classified into the established groups using this system. The VP4 gene is highly conserved in IBDV most probably due to its essential function in the processing of the VP243 polyprotein. No association has been identified between VP4 sequence variation and isolate virulence, VP4 is
a non-structural protein, and does not contain major protective antigens. Therefore, it is expected that there is minimal selective pressure on the gene due to host factors or immune selection, and mutations present in VP4 are expected to be restricted to conservative mutations that do not disrupt protease function. The VP4 gene is targeted as a marker of strains of classical, very virulent, and variant viruses, and VP4 sequence alignments identified a sequence region that is highly conserved within these strains, but varies between strains. There are several factors that support the use of conserved VP4 sequence as a marker of virus strains. Sequence analysis of isolates of either classical, antigenic variant or very virulent strains indicate that viruses within these strains are more closely related in sequence similarity to each other, than to isolates from different strains. A close phylogenetic relationship within either the classical, variant or virulent strains is further suggested by the emergence of variant viruses at a single time-point in the history of IBDV infection, and also of very virulent strains simultaneously in Europe, Asia and Africa. The availability of VP4 sequence data from a greater number of IBDV isolates is required to further validate its usage as a marker.
Acknowledgement The authors thank Dr. Y.M. Saif, The Ohio State University, for kindly supplying the Holland strain vvIBDV RNA.
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Real-time RT-PCR differentiation and quantitation of infectious bursal disease virus strains using dual-labeled fluorescent probes Michelle A. Peters, Tsang Long Lin, Ching Ching Wu ∗ Department of Veterinary Pathobiology, Purdue University, 406 S. University St., West Lafayette, IN 47907-2065, USA Received 27 May 2004; received in revised form 28 February 2005; accepted 1 March 2005 Available online 21 April 2005
Abstract A real-time RT-PCR assay was developed utilizing dual-labeled fluorescent probes binding to VP4 sequence that are specific to the classical (Cl), variant (V) and very virulent (vv) strains of infectious bursal disease virus (IBDV). The assay was highly sensitive and could detect as little as 3 × 102 to 3 × 103 copies of viral template. Viral genomic copy number could be accurately assayed over a broad range of 7–8 logs of viral genome. The variant sequence-specific probe was found to be highly specific in detecting isolates classified as variant A, D, E, G and GLS-5, and did not react with classical strains. A total of 130 field and experimental variant strain isolates were tested using this assay. The classical sequence-specific probe also demonstrated high sensitivity and specificity, and positively detected a total of 87 STC isolates, both field and experimental isolates, while differentiating between isolates that were variant and classical strains. The very virulent sequence-specific probe detected positively the Holland vvIBDV isolate and did not react with classical or variant strains. Rapid identification of viral strain is a primary concern to poultry flock health programs to ensure administered vaccines will protect against current strains of virus circulating in the flock. The ability to quantify virus concurrently is also of assistance in identifying the progression of disease outbreaks within the flock. © 2005 Elsevier B.V. All rights reserved. Keywords: Infectious bursal disease virus; Classical; Variant; VP4; Real-time RT-PCR; Dual-labeled fluorescent probes
1. Introduction Infectious bursal disease virus (IBDV) infection of young chickens results in lymphocytolysis of the Bursa of Fabricius and immunosuppression, leading to an increased incidence of vaccination failure and secondary infections within the flock (Allan et al., 1972; Cosgrove, 1962). IBDV is a nonenveloped virus with a bi-segmented, double-stranded, RNA genome, classified within the genus Avibirnavirus and within the Birnaviridae family (Kibenge et al., 1988; Murphy et al., 1999; van Regenmortel et al., 2000). Genome segment A is 3.3 kb in size and encodes two partially overlapping open reading frames (ORF) (Bayliss et al., 1990; Hudson et al., 1986). The first ORF encodes the 17 kDa, non-structural, VP5 gene, and the second encodes a 110 kDa polyprotein VP243 ∗
Corresponding author. Tel.: +1 765 494 7459; fax: +1 765 494 9181. E-mail address: [email protected] (C.C. Wu).
0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.03.009
that is cleaved by the auto-protease VP4 into constituent proteins pVP2 (48 kDa), structural protein VP3 (32 kDa) and VP4 (24 kDa) (Birghan et al., 2000; Muller and Becht, 1982). The precursor pVP2 protein is then further processed to the mature structural protein VP2 (38 kDa) (Muller and Becht, 1982). Genome segment B is 2.8 kb in size and encodes a single 90 kDa protein VP1, the putative viral RNA-dependent RNA polymerase (Muller and Nitschke, 1987; Spies et al., 1987). VP1 protein is also found within the viral particle complexed to both genome segments A and B. Of concern in the control of IBDV infection is the steady progression of viral evolution over recent decades and the emergence of both new antigenic variants (V) and strains with enhanced virulence. Before 1985, all classical (Cl) strains of IBDV were indistinguishable antigenically by virus neutralization (VN) assays (Jackwood and Saif, 1987; Jackwood et al., 1985). Classical strains varied in virulence, mortality ranged from 1 to 30%, and protection was achieved by the
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administration of inactivated vaccines to breeder birds. Vaccination is the primary means of control of infectious bursal disease in the poultry industry (Lukert and Saif, 1991). In 1986, variant viruses were first described in the USA as newly emergent viruses due to a major antigenic shift within serotype 1 (Snyder et al., 1988a; Snyder et al., 1992). Antigenic variants could not be cross-neutralized by antiserum raised against classical strains (except at very high titers) (personal communication) and could evade immunity to current classical vaccines within a flock (Ismail and Saif, 1991). Very virulent (vv) strains (vvIBDV) emerged in Europe in 1986, and were identified subsequently in Africa and Asia, but not in the USA (Abdel-Alim and Saif, 2001; Cao et al., 1998; Jackwood and Sommer, 2002; Liu et al., 2001; Rautenschlein et al., 2003; Sun et al., 2003; Tsukamoto et al., 1999). Very virulent IBDV is capable of breaking through high-levels of maternal-antibody protection and producing 70% flock mortality. The very virulent strains are similar antigenically to the classical strains. Effective control of IBD is contingent on the availability of rapid and efficient diagnostic procedures for identifying and differentiating IBDV strains within a flock. Currently, there is no single-step assay available that will rapidly identify and differentiate IBDV strains. Nucleic acid-based technologies for the diagnosis of IBDV flock status have targeted largely the VP2 gene sequence (Bayliss et al., 1990; Lee et al., 1992; Lin et al., 1994; Vakharia et al., 1992), or the combined VP2 and VP3 regions (Lee et al., 1992; Tham et al., 1995), with the exception of recent studies targeting the VP1 gene (Islam et al., 2001; Tiwari et al., 2003), and one study targeting the VP4 gene (Wu and Lin, 1992). A quantitative real-time RT-PCR assay targeting VP2 was developed by Moody et al. (2000) that would detect all IBDV strains. Virus neutralization assays or virulence studies in SPF chickens are direct methods to differentiate variation in antigenicity and virulence. Jackwood and Saif (1987) classified six subtypes of IBDV using VN and cross-neutralization assays. Vakharia et al. (1994) demonstrated that antigenic variation was due to amino acid changes in VP2, and Heine et al. (1991) showed that changes in four residues were sufficient for escape of the variant Del E strain. VP2 gene sequence between residues 206 and 350 has been identified as a hypervariable region and contains major serum-neutralization and conformational epitopes. Within the hypervariable region, major hydrophilic peaks have been identified with maximal variation between residues 212–224 and 314–324, and minor hydrophilic peaks between residues 248–252 and 279–290 (Dormitorio et al., 1997; Smiley and Jackwood, 2001). Marked sequence heterogenicity exists within the epitopes responsible for antigenicity and immunogenicity. Sequence flanking the hypervariable region is highly conserved across all IBDV isolates and is a target region for specific amplification of serotypes 1 and 2 IBD virus. A series of predicted restriction enzyme sites were identified within the hypervariable region of VP2, that could be employed for the
differentiation of antigenic variant, classical and very virulent strains (Jackwood and Jackwood, 1994; Jackwood and Sommer, 1997, 1999; Kataria et al., 1999; Liu et al., 1994; Zierenberg et al., 2000). Using either the RT-PCR-RE or RTPCR-RFLP techniques, strains have been classified into six molecular groups: groups 1 and 2 contained variant viruses, groups 3 and 4 contained classical viruses, group 5 was characterized by the Lukert strain, contains both classical, variant and hybrid strains and group 6 contains the Intervet vaccine strain RS593, 50% of field strains originating outside the USA and also very virulent strains (Jackwood and Sommer, 1999). Virulence determinants and the markers for virulent phenotypes are currently unknown, and are not associated with antigenic subtypes, as very virulent strains are similar antigenically to less virulent classical strains. The objective of the present study was to develop a real-time RT-PCR assay utilizing fluorescent probes binding to strain-specific sequence from either classical, variant or very virulent strains. Sequence from the VP4 gene was targeted due to its sequence conservation within strains and therefore its capacity to differentiate strain lineage. Such an assay would allow the rapid, single-step classification of isolates during the cycling reaction and would negate the need for separation and visualization of PCR products by electrophoresis in an agarose gel, and the practice of restriction digestion to differentiate strains. The utility, high sensitivity and specificity of real-time RT-PCR make it an ideal assay for management of poultry health programs.
2. Materials and methods 2.1. Sequence analysis and design of primer/probe sets IBDV genomic sequences were aligned using ClustalX via the interface (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/) (Thompson et al., 1997). PrimerSelect (DNAStar Inc., Madison, WI) software was used to select primer-probe sets for real-time RT-PCR assays of the IBDV genome. Oligonucleotides were selected to amplify a region of between 120 and 200 bp that was conserved across all aligned IBDV sequences. Amplicons were selected further to contain 20–30 bp probe sequences that were conserved within but varied between classical, variant or very virulent strains of IBDV. Fig. 1 illustrates the alignment of primer/probe pairs to VP4 sequences (bases 2026–2161) present in the GenBank database. The forward primer 5 -GTCGAGTGGATATTGGCCCC 3 and reverse primer 5 -GGCTCCTGCGTTATTCTTGC-3 (Fischer Scientific, Itasca, IL, USA) were used to amplify an IBDV segment A amplicon between bases 2026 and 2161 (numbering based on the IBDV variant E sequence, #AF133904) within the VP4 gene. A dual-labeled, fluorescent probe JOE – 5 -CAATGCTTGTGGCGAGATTGAGAAAGTAAG-3 – BHQ1 was designed with sequence specific to aligned classical IBDV strains (probe 1), and
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Fig. 1. Sequence alignments of variant, classical and very virulent IBDV segment A sequences from residues 2026 to 2180 bp using classical strain STC as the reference. Virus sequences are clustered into variant, classical and very virulent strains. Variant E IBDV sequence is used as the consensus sequence for variant strains, STC IBDV sequence is used as the consensus sequence for classical strains and OKYM sequence is used as the consensus sequence for very virulent strains. Universal flanking primer sequences are shown as boxed text at the top of the alignment, and probe sequences conserved with the isolate subtypes are shown as boxed text above each cluster of isolates. Within the region targeted by the probe consensus residues are shown as asterices, residues identical to the consensus sequence are illustrated by capitals and non-homologous residues are italicized. Row 1: IBDV isolate STC (D00499); 2: IBDV isolate IM (AY029166); 3: IBDV isolate Edgar (A33255); 4: IBDV isolate PBG-98 (D00868); 5: IBDV isolate Cu-1 (D00867); 6: IBDV isolate 52/70 (D00869); 7: IBDV isolate GBF-1 (D16828); 8: IBDV isolate Cu-1 (X16107); 9: IBDV isolate Mundt (X84034); 10: IBDV isolate Purdue 2003 (our sequence not submitted to GenBank); 11: IBDV isolate variant E (AF133904); 12: IBDV isolate GLS (M97346); 13: IBDV isolate Vakharia (X54858); 14: IBDV isolate Purdue 2003 (our sequence not submitted to GenBank); 15: IBDV isolate OKYM (D49707); 16: IBDV isolate UK661 (X92760); 17: IBDV isolate HK46 (AF092943); 18: IBDV isolate Tasik94 (AF322444); 19: IBDV isolate D6948 (AF240686); 20: IBDV isolate KS (L42284); 21: IBDV Holland isolate.
a second dual-labeled, fluorescent probe FAM – 5 -CAACGCTTGTGGCGAGATTGAGAAAATAAG-3 – BHQ1 (probe 2) was designed with sequence specific to aligned variant IBDV strains (Biosearch Technologies, Novato, CA, USA). A third probe FAM – 5 -CAACGCCTATGGCGAGATTGAGAACGTGAG-3 – BHQ1 (probe 3) was designed with sequence specific to aligned very virulent IBDV strains. 2.2. Viral RNA samples Bursae were obtained both from chickens inoculated experimentally with IBD viruses within the animal housing facilities and from field samples. The types of isolates, number of isolates and dates of acquisition are outlined in Table 1. The bursal tissue was collected onto dry ice and either stored at −80 ◦ C or processed immediately. Bursal tissue was homogenized in chilled sterile phosphate buffered saline (PBS: 0.136 M NaCl, 0.003 M KCl, 0.01 M Na2 HPO4 , 0.002 M KH2 PO4 ; pH 7.4). The homogenate was clarified by centrifugation at 2000 × g for 5 min, and the cellular debris was re-suspended in PBS wash buffer, homogenized a second time and clarified by centrifugation at 2000 × g for 5 min.
The clarified homogenates were combined and total RNA extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s instructions. Purified RNA was eluted in a final 50 l volume of 0.1% diethyl pyrocarbonate (DEPC) treated water. 2.3. Real-time RT-PCR assay for IBDV Reverse transcription was performed at 42 ◦ C for 50 min in a 25 l reaction mix, containing 5 l RNA preparation, 2.5 M of each IBDV primer, 250 M each of dATP, dCTP, dGTP and dTTP (Promega), 1× Superscript RNaseH− Reverse Transcriptase buffer (Invitrogen), 1 U Superscript RNaseH− Reverse Transcriptase (Invitrogen) and 0.01 M DTT (Invitrogen). The cDNA was then amplified by realtime PCR. A 25 l reaction mixture was prepared containing 250 M each of dATP, dCTP, dGTP and dTTP (Promega, Madison, MA, USA), 3 mM MgCl2 , 2.5 l of 10× Taq DNA polymerase buffer (Promega), 1 U of Taq DNA polymerase (Promega), 2.5 M of each primer, either 3 M JOE-labeled classical IBDV probe, 3 M FAM-labeled variant IBDV probe or 3 M FAM-labeled very virulent IBDV probe, and 4 l of template cDNA. Polymerase 5 -nuclease activity
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Table 1 IBDV isolates assayed by real-time RT-PCR assay to detect variant, classical and very virulent strain-specific VP4 sequences Isolate typea
Field samplesb
Experimental samplesb
Acquisition dates
Classical probe assay positives
Variant probe assay positives
Very virulent probe assay positives
Variant E (V)
10 (Bursae)
80 (Bursae)
1c
90
0
2–21
Variant A (V) Variant D (V) Variant G (V) GLS-5 (V) STC (Cl) D78 (Cl) Holland (vv)
5 (Bursae) 5 (Bursae) 5 (Bursae) 5 (Bursae) 1 (Bursae) – –
5 (Bursae) 5 (Bursae) 5 (Bursae) 5 (Bursae) 80 (Bursae) 6 (Bgm cell line) 1 (Bursa)
9/1997, 1/2000, 6/2002, 9/2002, 8/1995, 6/2000 8/1995, 6/2000 8/1995, 6/2000 6/2000, 6/2002 9/1997, 6/2002, 9/2002 6/2002, 8/2003 11/2004
0 0 0 0 81 6 0
10 10 10 10 0 0 0
0 0 0 0 0 0 1
8–20 6–15 10–12 10–13 3–20 4–5 2–20
a b c
Log range of copy number detected
Name of isolate and broad classification as variant (V), classical (Cl) strain or (vv) very virulent. Isolate obtained by experimental IBDV infection of chickens or from field poultry samples. Sample was negative when the assay was repeated.
during PCR amplification resulted in a reduction in fluorophore quenching by the BHQ1 dye, and this fluorescent signal was measured in the Rotor Gene, real-time, thermal cycling system (Corbett Research, Mortlake, NSW, Australia). The cycle profile was 1 cycle of 60 ◦ C for 3 min, 95 ◦ C for 3 min and 50 cycles of 95 ◦ C for 20 sec, 60 ◦ C for 1 min. Cycle time (Ct ) was measured for each assay. One-step, real-time, RT-PCR assays were also carried out, combining the superscript reverse transcriptase (RT) with Platinum Taq DNA polymerase in a single reaction mix. A 25 l reaction mix was prepared containing 12.5 l of 2× reaction buffer (Invitrogen), 2.5 M of each primer, 0.5 l Superscript RT/Platinum Taq DNA polymerase mix (Invitrogen), 3 M MgSO4 , either 3 M JOE-labeled classical IBDV probe, 3 M FAM-labeled variant IBDV probe or 3 M FAM-labeled very virulent IBDV probe and 4 l of template RNA. The cycle profile was 1 cycle of 45 ◦ C for 20 min, 95 ◦ C for 3 min, and 40 cycles of 95 ◦ C for 20 s, 60 ◦ C for 1 min.
reverse primer 5 -GACGACCGATTTGCACTGC-3 and the probe FAM – 5 -AGGACCGCTACGGACCTCCACCA-3 – BHQ1. The sequences of the target amplicons from the variant E, STC and Holland vvIBD viruses were established by bidirectional, automated sequencing. 2.5. Time course of infection in bursa using the IBDV real-time RT-PCR assay One-day-old SPF chickens were inoculated orally with either: (i) 106 EID50 variant E IBDV in 200 l DMEM; (ii) 106 EID50 IBDV strain STC in 200 l DMEM; or with (iii) 200 l DMEM. For each treatment group, five chickens were euthanased at 12, 24 and 48 h after infection and the bursae were removed and placed into chilled PBS. Bursal tissue RNA was extracted for RT-PCR as described above.
3. Results 2.4. IBDV real-time RT-PCR standard curves 3.1. Sequence analysis IBDV large segment ORF was amplified from preparations of bursal RNA infected with either variant E, STC or Holland vvIBD virus, and cloned into the pCR3.1 vector (Invitrogen), as described previously (Chang et al., 2001). Tenfold dilution series, starting with 2 g/ml, of either variant E or STC IBDV template cloned into pCR3.1 vector, were used to standardize Ct values from the real-time PCR assays against template copy number. Sample template copy number was calculated from cycle time by linear regression using the respective standard curves. The linear ranges of the real-time, RT-PCR assays were established from eight serial 10-fold dilutions of sample bursal RNA purified from chickens inoculated experimentally with either the variant E or STC viruses. Sample Ct values were normalized against Ct values for amplification of the 28S internal standard. The 28S gene was amplified using forward primer 5 -GGCGAAGCCAGAGGAAACT-3 ,
A target region of 135 bp sequence between bases 2026 and 2161 of VP4 (numbering based on the IBDV variant E sequence, GenBank number AF133904) was identified from the alignment of the VP4 gene of IBDV isolates (Fig. 1). Many of the IBDV GenBank entries are limited to VP2 gene sequence and do not include the VP4 sequence. The alignment was composed of a total of nine classical strain sequences, three variant strain sequences and seven very virulent strain sequences. The probe sequences contain six potentially variant bases. Probes designed for classical strains differed from variant strains by two bases, and both differed from very virulent strains by five bases (the differences between classical and very virulent, and variant and very virulent strain probes were not identical). Two classical strain isolates and one very virulent strain isolate each had one out of the six variant bases mismatched with the consensus sequences. Variation in VP4
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Fig. 2. Standard curves of real-time RT-PCR assays of IBDV using probe 1 (sequence specific to classical strain sequence), probe 2 (sequence specific to variant strain sequence) and probe 3 (sequence specific to very virulent strain sequence). (a) Fluorescence readings for the assay using probe 1 of a dilution series of classical STC IBDV RNA; (b) fluorescence readings for the assay using probe 2 of a dilution series of variant E IBDV RNA; (c) fluorescence readings for the assay using probe 3 of a dilution series of Holland vvIBDV RNA; (d) standard curve for probe 1 assay of a dilution series of the STC IBDV large segment ORF cloned into the pCR3.1 vector; (e) standard curve for probe 2 assay of a dilution series of the variant E IBDV large segment ORF cloned into the pCR3.1 vector; (f) standard curve for probe 3 assay of a dilution series of the Holland very virulent IBDV large segment ORF cloned into the pCR3.1 vector (color version on www.ScienceDirect.com).
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Fig. 2. (Continued ).
sequence is not as great between isolates as for VP2 and VP3 sequences. No target amplicons suitable for real-time RT-PCR were found using alignments of sequences of the IBDV genes VP2, VP3 and VP1. Requirements for such sequences would be between 120 and 200 bp amplicons, with terminal sequences conserved in all strains, suitable targets for flanking primers, and contain intervening sequence conserved within, but variable between strains as a target for strain-specific probes. The probe sequences are also required to have a Tm of 70 ◦ C (10 ◦ C greater than the primer sequence), no runs of nucleotide repeats, low GC content in the 3 -terminus, no Gclamping on the 5 -terminus and suitable GC content. The high degree of variation evident in sequence alignments between isolates of the same strain within the VP2 and VP3 genes made it difficult to identify target amplicons (data not shown). The probe target region was chosen in order to differentiate classical, variant and very virulent strains.
was 0.01, and the cycle threshold (Ct ) range was cycles 5–35. The linear range of the standard curve was between 3 × 102 and 3 × 109 template copies. A linear relationship was established between Ct value and concentration of RNA in bursal tissue from chickens experimentally inoculated with variant E virus. IBDV sequence was not detected using real-time RT-PCR in RNA samples from the bursae of control chickens inoculated with DMEM. IBDV variant E RNA was detected at 12, 24 and 48 h after inoculation in bursal tissue of all chickens and mean titers were highest at 24 h after infection (Fig. 3). Variants A, D and G and GLS-5 RNA isolates of IBDV were detected using the probe based on conserved variant virus sequence (Table 1). The variant IBDV real-time, RT-PCR assay did not amplify STC or D78 classical strain sequences or Holland very virulent strain sequence (Table 1).
3.2. Real-time RT-PCR assay for IBDV variant strains
As for variant strains, a standard curve was derived from the STC.pCR3.1 plasmid standard (Fig. 2). The R2 value for the STC standard curve was 0.905, the Rn significance threshold was 0.01, and the Ct threshold range was cycles 10–40. The linear range of the standard curve was between 3 × 102 and 3 × 108 template copies.
A standard curve of template copy number against Ct value was plotted using the variant E. pCR3.1 plasmid standard (Fig. 2). The coefficient of determination (R2 ) for the variant E standard curve was 0.999, the significance threshold (Rn )
3.3. Real-time RT-PCR assay for IBDV classical strains
Fig. 3. Detection of IBDV sequence using real-time RT-PCR in RNA extracted from the bursae of experimentally infected 1-day-old chickens. Groups of 20 chickens were inoculated with either (a) STC strain IBDV or (b) variant E IBDV, and five chickens were sampled at time points 0, 12, 24 and 48 h after infection. IBDV log genomic copy number in the bursa of each chicken is illustrated.
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Using the classical sequence-specific probe, there was no signal detected from control RNA samples from chickens inoculated with DMEM. IBDV STC RNA was detected in bursal tissue at all time points following inoculation with STC strain virus (Fig. 3). Isolates of the D78 classical strain were also detected in RNA samples using the probe based on conserved classical strain sequence (Table 1). A weak signal was detected for RNA samples containing variant E virus using the classical sequence-specific probe, however, the amplitude of the signal was consistently lower. Therefore, raising the threshold for signal detection allowed differentiation of classical and variant isolates (Table 1). The classical sequence-specific probe did not amplify the Holland very virulent strain sequence. 3.4. Real-time RT-PCR assay for IBDV very virulent strains A standard curve was derived from the Holland vvIBDV.pCR3.1 plasmid standard (Fig. 2). The R2 value for the Holland vvIBDV standard curve was 0.999, the Rn significance threshold was 0.01, and the Ct threshold range was cycles 3–35. The linear range of the standard curve was between 2 × 102 and 2 × 1010 template copies. The vvIBDV-sequence-specific probe detected Holland strain virus in RNA purified from infected bursal samples, but did not amplify the STC or D78 classical strains, nor the variants A, D and G and GLS-5 variant strains. 3.5. 28S internal standard Standard curves were derived for the real-time RT-PCR assays of the 28S internal standard, based on the method described by Moody et al. (2000) (Kaiser et al., 2000). The 28S gene was amplified by real-time RT-PCR from a 10-fold dilution series of sample RNA. The coefficient of determination (R2 value) for the standard curve was 0.997, the Rn significance threshold was 0.05, and the Ct threshold range was cycles 10–40. The 28S standard assay was used to standardize sample differences in cell and RNA content. 3.6. Differentiation of classical, variant and very virulent subgroups of IBDV The real-time RT-PCR assays developed can be used to differentiate between isolates of classical, variant and very virulent strains of IBDV. Classical sequence-specific probe detects all IBDV classical viruses tested, with a weak sub-threshold signal for variant isolates. Variant sequencespecific probe detects and is specific to variant virus isolates. The very virulent sequence-specific probe detects and is specific to very virulent virus isolates tested. There was no significant difference observed when quantifying both the plasmid standards or the RNA samples from IBDV isolates, when using either the single-step, real-time, RT-PCR or the two-step method from cDNA (data not shown).
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4. Discussion In this study, we report the development of an IBDV diagnostic assay using dual-labeled fluorescent probes and realtime RT-PCR to differentiate IBDV isolates into classical and variant strains. The assay is highly sensitive and can detect as little as 3 × 103 (in the case of classical sequence-specific probes), 3 × 102 (for variant sequence-specific probes) or 2 × 102 (for very virulent sequence-specific probes) genomic templates in tissue samples. The variant sequence-specific probe detected all 130 variant isolates tested, including both field and experimental isolates of variants A, D, E and G and GLS-5. The variant sequence-specific probe did not detect the 88 classical isolates tested. The 88 classical isolates, field and experimental isolates, were specifically detected by the classical sequence-specific probe. A probe with very virulent strain-specific sequence was designed for the purpose of extending the application of the assay to the diagnosis of very virulent IBDV where this strain is prevalent. Testing of the very virulent strain-specific probe was limited to a single isolate as vvIBDV is currently not found in the USA and due to the difficulty of importing vvIBDV into the USA. The real-time RT-PCR assay has considerable advantages for application to flock diagnosis of IBDV. The speed and ease of the reaction facilitates its application to the analysis of multiple samples. Because the fluorescence signal is detected in real-time, results are obtained rapidly and without further analysis, such as separation of PCR products by electrophoresis for visualization. The assay has a broad range of between 7 and 8 logs in which viral genomic template can be accurately quantified. Very low amounts of viral genome can be detected, as little as 300 copies. Therefore, changes in viral titers occurring during the progression of infection can be accurately assessed in flock samples. Assessment of titer as well as viral strain is valuable in tracking the status of flock infections and pinpointing the beginning of disease outbreaks. The planning of effective vaccination schedules requires knowledge of the strain of IBDV currently in the flock, as the majority of vaccines in commercial use are strain-specific. The utility of the assay in charting the progression of infection is demonstrated by the time-course study of experimental infection of 1-day-old chickens with either classical or variant strains of IBDV (Fig. 3). Viral titer in the bursa was assayed at 12, 24 and 48 h after oral infection of 1-day-old birds. The real-time RT-PCR assay detected a peak average STC strain titer of 9 × 1017 genomic copies as early as 12 h after infection, and titers declined to an average of 4 × 1016 copies by 48 h. A peak variant E strain titer of 2 × 1019 genomic copies was observed 24 h after infection, and as with STC strain infection, titers declined by 48 h to an average of 1 × 1017 copies. These two patterns of infection were evident for all chickens in the respective treatment groups. The results point to an early acute infection with classical strain viruses, and a slightly longer latent period for variant strain viruses. The higher levels of titer observed for variant E than for STC
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strain suggests a greater capacity of variant viruses for replication in the bursa of newborn chickens. Variant viruses are reported to cause rapid atrophy of the bursa without bursal haemorrhage and inflammation or high mortality (Snyder et al., 1988a). The application to flock surveillance of sensitive assays that can detect not only strain but also viral titer will lead to the accumulation of data that clearly relates viral titer to the incidence of clinical disease and immune suppression for the different strains of IBDV. It is anticipated that these studies be extended to the analysis of mixed IBDV infection, both with different IBDV strains and mixed pathogen infections. The variant sequence-specific and the very virulent sequence-specific probes were highly specific to variant and very virulent isolates, respectively, and no signals were detected for other subgroup templates. The classical sequencespecific probe was also specific to sequences of classical isolates, however, in some assays of variant isolates a lowamplitude, sub-threshold, fluorescence signal was detected. The amplitude of the fluorescence signal is dependent on the affinity of the probe for the template during PCR amplification, which in turn leads to the release of the fluorophore from the vicinity of the quencher by the 3 -exonuclease activity of the polymerase. A weak sub-threshold signal is indicative of the classical probe 1 having a low affinity for the variant isolate template. This does not interfere with the ability of the assay to specifically identify classical isolates, as raising the threshold for detection eliminates signal from amplification of variant isolates. Neither are false negatives likely to occur with increased template concentration. Cycle time required for the fluorescence signal to cross the threshold is a function of template concentration, and signal amplitude is not dependent on concentration. The real-time RT-PCR assay developed for IBDV is unique in that it targets sequence from the VP4 gene. Differential diagnostic assays have been largely directed to the hypervariable region of VP2 due to the abundance of sequence variability within this region, however, this variability exists both within and between strains and the sequence in this region does not serve as a consistent marker for the differentiation of variant, classical and very virulent strains. While heterogeneity between vaccine and laboratory strains is low for this region of VP2, heterogeneity between field strains is marked and can be problematic, as evident by RFLP patterns from field strains in epidemiological surveys which consistently do not fit into the established molecular groupings, and differences between studies in the findings as to the utility of particular restriction endonuclease sites as group specific markers. Jackwood et al. in (Jackwood and Sommer, 1999) found as many as 19 field strains with RFLP patterns that could not be classified into the established groups using this system. The VP4 gene is highly conserved in IBDV most probably due to its essential function in the processing of the VP243 polyprotein. No association has been identified between VP4 sequence variation and isolate virulence, VP4 is
a non-structural protein, and does not contain major protective antigens. Therefore, it is expected that there is minimal selective pressure on the gene due to host factors or immune selection, and mutations present in VP4 are expected to be restricted to conservative mutations that do not disrupt protease function. The VP4 gene is targeted as a marker of strains of classical, very virulent, and variant viruses, and VP4 sequence alignments identified a sequence region that is highly conserved within these strains, but varies between strains. There are several factors that support the use of conserved VP4 sequence as a marker of virus strains. Sequence analysis of isolates of either classical, antigenic variant or very virulent strains indicate that viruses within these strains are more closely related in sequence similarity to each other, than to isolates from different strains. A close phylogenetic relationship within either the classical, variant or virulent strains is further suggested by the emergence of variant viruses at a single time-point in the history of IBDV infection, and also of very virulent strains simultaneously in Europe, Asia and Africa. The availability of VP4 sequence data from a greater number of IBDV isolates is required to further validate its usage as a marker.
Acknowledgement The authors thank Dr. Y.M. Saif, The Ohio State University, for kindly supplying the Holland strain vvIBDV RNA.
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