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Sensitive and specific detection of bovine immunodeficiency virus and bovine syncytial virus by 5′ Taq nuclease assays with fluorescent 3′ minor groove binder-DNA probes
Journal of Virological Methods 116 (2004) 1–9
Sensitive and specific detection of bovine immunodeficiency virus and bovine syncytial virus by 5 Taq nuclease assays with fluorescent 3 minor groove binder-DNA probes Ala E. Lew a,∗ , Russell E. Bock b , John Miles a,1 , Leigh B. Cuttell a,2 , Penelope Steer b , Susan A. Nadin-Davis c a
b
Agency for Food and Fibre Sciences, Queensland Department of Primary Industries, c/o Locked Mail Bag No. 4, Moorooka 4105, Qld, Australia Tick Fever Research Centre, Animal and Plant Health Service, Queensland Department of Primary Industries, 280 Grindle Road, Wacol, Qld, Australia c Animal Diseases Research Institute/Centre for Plant Quarantine Pests, Ottawa Laboratory Fallowfield, 3851 Fallowfield Road, Nepean, Ont., Canada K2H 8P9 Received 13 June 2003; received in revised form 6 October 2003; accepted 13 October 2003
Abstract Sensitive assays are required to detect bovine retroviruses in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5 Taq nuclease assays using 3 minor groove binder DNA probes (TaqMan® MGB) were developed and compared to conventional PCR assays for the sensitive detection of bovine syncytial virus (BSV) and bovine immunodeficiency virus (BIV). Seven beef and dairy herds were screened to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log10 dilutions of plasmids with inserts containing corresponding provirus sequences. Published PCR assays targeting BIV env sequences did not adequately amplify Australian BIV sequences. Pol sequences from Australian strains of BIV and BSV were used to design TaqMan® MGB assays, which improved sensitivity 10-fold (BIV) and 100-fold (BSV), respectively, over conventional PCR tests. This is the first report of Australian sequences of BIV and BSV and the first 5 Taq nuclease assays described to detect these viruses. These methods could be applied to future studies requiring sensitive detection of these two bovine retroviruses. Crown Copyright © 2003 Published by Elsevier B.V. All rights reserved. Keywords: Bovine immunodeficiency virus; Bovine syncytial virus; Bovine retroviruses; 5 Taq nuclease; Minor groove binder; TaqMan®
1. Introduction The bovine retroviruses in the Retroviridae family of viruses include bovine leukemia virus (BLV) (Miller et al., 1969), bovine immunodeficiency virus/bovine immunodeficiency-like virus (BIV) (Van Der Maaten et al., 1972), ∗ Corresponding author. Tel.: +61-7-3362-9502; fax: +61-7-3362-9429. E-mail address: [email protected] (A.E. Lew). 1 Present address: Cellular Immunology, Infectious Diseases & Immunology Division, Comprehensive Cancer Research Centre, Queensland Institute of Medical Research, Royal Brisbane Hospital Post Office, 300 Herston Road, Herston, QLD, 4029, Australia. 2 Present address: Drosophila Genetics Group, Biochemistry and Molecular Biology, School of Pharmacy and Molecular Sciences, James Cook University, Townsville, QLD, Australia.
Jembrana disease virus (JDV) (Wilcox et al., 1992) and bovine syncytial virus (BSV) (Malmquist et al., 1969). Double-stranded DNA copies of these single stranded RNA viruses can integrate into the host genome and cause persistent infections with low viral gene expression but persistent antibody titre in infected hosts. Retroviral infections (BIV, BSV and BLV) of dairy and beef cattle are a major concern to the livestock industry due to the potential for disease ranging from lymphosarcoma to impairment of the immune system, economic and trade effects, and the perception that they may be threats to public health if present in the human food chain (Donham et al., 1987; Evermann and Jackson, 1997; Gradil et al., 1999). The control of these viruses relies on understanding how they are transmitted and on the ability to detect their presence in cattle (Evermann and Jackson, 1997).
0166-0934/$ – see front matter. Crown Copyright © 2003 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2003.10.006
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BIV and JDV are members of the bovine lentivirus group of the Lentivirus genus. BIV causes a subclinical disease syndrome and is found in cattle populations around the world whereas JDV causes an acute, often fatal disease specific to Bali cattle (Bos javanicus) (Chadwick et al., 1995). In Taurean breeds JDV produces a similar disease syndrome to BIV (Soeharsono et al., 1990). Bovine lentiviruses have not been isolated in Australia but there is serological evidence of their presence (Burkala et al., 1999; Forman et al., 1992). The impact of BIV is controversial due to the difficulty in culturing new isolates in vitro and the difficulty in identifying BIV-infected animals (Evermann et al., 2000; Gradil et al., 1999). No reliable diagnostic assay is available, but it appears that PCR assays are more sensitive than serological methods (Gradil et al., 1999). Prevalence studies using PCR have demonstrated a low herd prevalence of BIV in Canada and United States of America (Burger et al., 2000; Gonzalez et al., 2001). Serological detection is limited by low antibody levels (Isaacson et al., 1995), but a promising ELISA has been developed (Abed and Archambault, 2000). BSV of the genus Spumavirinae is generally thought to be non-pathogenic but can be leucotrophic and could exacerbate diseases caused by BIV and/or BLV (Jacobs et al., 1995). BSV is widely distributed and was confirmed in Australian cattle in 1980 (Maker et al., 1980). Generally, diagnosis of BSV infection has been based on crude serological methods and/or virus isolation from cell culture (Johnson et al., 1983; Malmquist et al., 1969). Recently, a PCR method was developed allowing rapid and sensitive detection of this virus (Pamba et al., 1999). Tick Fever is an endemic disease of cattle in northern Australia caused by the organisms Babesia bovis, B. bigemina or Anaplasma marginale. The Queensland Department of Primary Industries has distributed vaccines against Tick fever in Australia for over a century. Since 1964 vaccines containing attenuated strains of B. bovis, B. bigemina and A. centrale grown in splenectomised calves have been used to immunise cattle against the disease in Australia (Callow et al., 1997). Since an incidence of transmission of BLV through the use of a batch of contaminated vaccine was reported (Rogers et al., 1988), a rigorous testing programme for BLV has been initiated. Reports of concomitant infections involving BIV, BLV and BSV (Jacobs et al., 1995; Meas et al., 2002), prompted our investigation into methods for the detection of BIV and BSV as part of our vaccine donor calf screening procedures. Real time PCR has engendered wider acceptance of PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination compared with sensitive nested PCR assays (Mackay et al., 2002). 5 Taq nuclease assays successfully detecting BIV and BSV have not been reported. The aim of this study is to develop sensitive tests for BSV and BIV to enable the selection of uninfected donor cattle for vaccine production.
2. Materials and methods 2.1. Animal samples Originally, serum and peripheral blood mononuclear cells (PBMCs) were submitted for the evaluation of DNA extraction methods and various PCR assays for BLV (Lew et al., in press). These same samples were used for the evaluation of BSV and BIV tests. These were obtained from three BLV endemic dairy herds in north Queensland (Herds A, B and F); three BLV endemic dairy herds in south east Queensland (Herds C–E); and from 202 beef heifers assembled at TFRC from seven western Queensland properties for possible enrolment in a specific pathogen free breeding herd (Herd H). Whole blood was collected from the jugular vein into vacutainer tubes containing EDTA (Terumo Europe, Belgium). Serum samples were collected either from the coccygeal artery or jugular vein, allowed to clot at room temperature and the serum separated for subsequent analysis. 2.1.1. DNA purification methods DNA from animals from Herds A–C were prepared from 0.5 ml whole blood using a commercial QIAamp DNA blood mini kit (Qiagen Pty. Ltd., Australia). DNA from remaining herds were prepared from purified PBMCs instead of whole blood. Ten millilitre of whole blood in EDTA and 12.5 ml PBS were added to a LymphoprepTM tube (Nycomed Pharma AS, Diagnostics, Oslo, Norway) to isolate PBMCs from each animal as per the manufacturer’s instructions. PBMCs resuspended in 1.0 ml PBS were then counted using an Animal Blood Counter according to the manufacturer’s instructions (ABX Hematologie, France). DNA was prepared from 5 × 106 cells and stored at −20 ◦ C until used for PCR testing. DNA yields were measured spectrophotometrically. 2.1.2. Identification of BIV positive animals In 1999, prior to this study, DNA was prepared from LymphoprepTM extracts of separated blood samples from a herd identified to be seropositive for BIV/JDV by a Western blot assay (Burkala et al., 1999). The DNA from these samples were stored and used to identify Australian BIV and BSV sequences using conventional PCR methods in this study (Section 2.2). 2.2. Conventional PCR methods All primer sequences are summarised in Table 1. Oligonucleotide primers for conventional PCR assays were synthesised through Genset Oligos Pty Ltd. (now merged with Proligo, CO, USA). All conventional PCR assays were carried out in a Perkin-Elmer 480 (Applied Biosystems, CA, USA). For new PCR methods described in this study, conditions for reactions and cycling are described below otherwise published protocols were implemented.
A.E. Lew et al. / Journal of Virological Methods 116 (2004) 1–9
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Table 1 Primers and probes utilised in this study PCR assay
Primers and probe setsa
Sequence targetb
Sequence 5 → 3
Target gene/references
BIV nested PCR 1
POL3 POL4
4252–4272 4641–4662
aca acg ggc cgt gct tta ctg cct ctt cct cta tta ctg ctg c
pol gene/Nadin-Davis (1995)
POL5 POL6
4284–4306 4570–4591
gar aat cta tgt aag tat ctg gg ctg tty ctt acg taa cac cac t
Unpublished data (Nadin-Davis)
P09 P06
1805–1825 2529–2510
cac tgg acg aga tga ggt agt tgg tag tct gat aaa tgg ca
pol gene/Suarez and Whetstone (1998)
P04 P11
2133–2152 2498–2478
cag gct ctt aag gaa att gt cca tcc ttg tgg tag aac att
P06 P04
5413–5432 7095–7114
cta tgg atc agg acc tag ac gcc agc aca agc agg aat at
P01 P45
5532–5551 5965–5946
atc aat aac ggt gag atc ca tct atg gta tct ctg gct gc
BIVJDVF BIVJDVR
1703–1724c 2455–2433
wga stt ctg tga ccc crt ctg c cct tct cka tty acm ggg acy ac
BIVJDVFnest BIVJDVRnest
1806–1827 2348–2327
ayw ggr gca gat gag gta gtg c aca tts cyk aat tcc tgg rgg
BIV1
MGB probe 1
4338–4354
6FAM cca caa tcc cag gga gtd
TaqMan® MGB
BIVF1 BIVR1
4311–4335 4384–4359
aca aaa act acg gga ata ccc tac a tct ttt aga tct ctg tgg gct ctt tc
BIV2
MGB probe 2
4366–4382
6FAM ccc aca gag atc taa aad
TaqMan® MGB
BIVF2 BIVR2
4337–4355 4418–4395
ccc aca atc cca ggg agt t ggt ttc aca atc tcc ctg ata agc
BSV gag PCR
Gag-1 Gag-2
2551–2568 2793–2773
gac gca aca acc aac cac gtt ctt gtc cgt atc gtt gtg
gag gene/Pamba et al. (1999)
BSV pol env PCR
Pol/Env-1 Pol/Env-2
6528–6548 6737–6721
caa ata tca tca acc gca gtg cac ccg gtg gat caa ag
pol/env region/Pamba et al. (1999)
BSV
MGB probe
6561–6579
6FAM tga aga tcg ttg tgg gca td
pol/env region/this study
TaqMan® MGB
BSVF BSVR
6528–6552 6582–6599
caa ata tca tca acc gca gtg tac a ttc ccg cac tcc ttc caa
BIV nested PCR 2
BIV nested PCR 3
BIV/JDV conserved nested PCR
env gene/Suarez and Whetstone (1998)
pol gene/this study
pol gene/this study
pol gene/this study
a
The bracket indicates forward and reverse primer sets. The primer positions are based on GenBank accession no. M32690 for BIV and accession no. NC 001831 for BSV. c Positions of primers based on BIV, positions based on JDV accession no. U21603 are 1497–1518 for BIVJDVF; 2249–2247 for BIVJDVR; 1602–1620 for BIVJDVFnest; and 2142–2121 for BIVJDVRnest. d All probes were 5 labelled with 6-carboxyfluorescein (6-FAM) phoshoramidite as the 5 reporter dye and MGB probes use a non-fluorescent quencher attached to the 3 minor groove binder moiety (AB). b
2.2.1. BIV PCR Four sets of primers were used to attempt to amplify Australian BIV from the seropositive herd pooled sample (Table 1). The cycling conditions described by Gradil et al. (1999) were used for the env and pol nested PCR assays originally described by Suarez and Whetstone (1998) (BIV nested PCR 2 & 3, Table 1). An additional set of primers for nested amplification of conserved regions of both BIV and JDV was designed. An additional pol nested PCR employed primers POL 3 and 4 which were described previously (Nadin-Davis, 1995) whilst the internal primers, POL 5 and 6, which have not been reported previously, were designed using a Canadian BIV sequence (unpublished data, BIV
nested PCR 1, Table 1). A BIV plasmid imported from Canada was used as a positive control for this PCR assay (see Section 2.3) For assays described in this study, 1 unit of Taq polymerase, 1× PCR buffer with MgCl2 (Roche Molecular Biochemicals, Germany), 50 pmol of each primer and 100 M each of deoxynucleotide triphosphate were used to amplify 1 g DNA (whole blood or lymphocyte DNA) in 50 l PCR reactions. Cycles consisted of 3 min denaturing at 94 ◦ C followed by 35 cycles of 94 ◦ C 30 s, annealing 55 ◦ C 30 s, 72 ◦ C 1 min. Two microliters of the first reaction was transferred to the nested PCR reaction using new primers under identical reaction conditions.
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2.2.2. BSV PCR DNA from bovine syncytial virus from Canada was imported into Australia to use as a positive control for the optimisation of the BSV PCR. The virus had been cultivated and purified from foetal lamb spleen cells. Single PCR assays using primers targeting the pol/env region and the gag region were used to amplify Australian BSV from sequences from Herd A, as described by Pamba et al. (1999) without using southern hybridisation. 2.3. PCR product cloning for sequence analysis and positive controls BIV and BSV PCR products generated from Australian animal samples were cloned into the TA vector as recommended by the manufacturer (Invitrogen Corporation, CA, USA). Plasmids were purified using standard alkaline lysis techniques. Plasmids with inserts were sequenced using standard M13 primers and BigDye Terminator Mix following methods recommended by the manufacturer (Applied Biosystems, CA, USA). Sequences were analysed by the Griffith University DNA Sequencing Facility (School of Biomolecular and Biomedical Science, Griffith University Nathan QLD 4111 Australia) on an ABI 377 DNA Sequencer and sequence identity was confirmed using BLASTn (Altschul et al., 1990) via the NCBI website (http://www.ncbi.nlm.nih.gov/). Australian BIV and BSV sequences were submitted to the GenBank database (Benson et al., 2000) and assigned accession numbers AF548462 and AF548463 and AF548464 and AF548465, respectively. A BIV plasmid, pUC118-BIVKp3, was prepared from a 3.0 Kb-Kpn I fragment of the BIV genome (originally from a lambda library) and was imported from Canada (ADRI) for BIV PCR optimisation in Australia. This BIV fragment contains most of the pol ORF including the RT domain. Fragments of both BIV and BSV sequences that were amplified from Australian cattle (as described above) were used as TaqMan® assay controls. The concentration of plasmid stocks were calculated on the original undiluted plasmid preparation by electrophoretic comparison to DNA MASSTM Ladder (Life Technologies) in agarose gels according to the manufacturer’s instructions. Plasmid copy numbers were then determined by calculating molecular weight of the plasmid and subsequent log dilutions (660 Da for each bp, 1 mol = 6 × 1023 molecules). A BLV pUC8 plasmid clone (Coulston et al., 1990) was used as a negative control for BIV and BSV PCR assays. 2.4. TaqMan® PCR assays 2.4.1. TaqMan® probes and primers Oligonucleotide sequences for TaqMan® assays were confirmed by aligning sequences for each virus accessed through the following GenBank accessions: M32690 bovine immunodeficiency-like virus, complete proviral genome (Garvey et al., 1990); U21603 Jembrana disease virus Ta-
banan/87 strain, complete genome (Chadwick et al., 1995); NC 001831 bovine syncytial virus, complete genome (Renshaw and Casey, 1994). Sequences were aligned through PileUp which was accessed through the Australian National Genomic Information Service (ANGIS) (Littlejohn et al., 1996). Primers and probes were designed using Primer Express Version 2 (Applied Biosystems, Applera Corporation, CA, USA) and were all purchased through Applied Biosystems (AB). Sequences for primers and probes used are summarised in Table 1. 2.4.2. TaqMan® assay conditions The TaqMan® PCR Universal Master Mix (ABI Prism® 7700 Sequence Detection System Users Manual), and Platinum Quantitative PCR SuperMix-UDG (Invitrogen Corp.), were used with assay conditions as recommended except that the number of cycles was extended up to 50. Assays were carried out on the ABI Prism® 7700 Sequence Detector (AB) and the Corbett RotorGene 2000 (Corbett Research, Sydney, Australia). All samples were tested in duplicate and an average cycle threshold (CT ) value used to define the result. A threshold of 0.1 fluorescence units was applied to enable comparison of results between experiments. The parameter CT is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. Occasionally low copy number samples (in plasmid dilutions) yielded consistently positive and negative results in duplicates. These were considered to be ‘inconsistently positive’ due to pipetting of low copy numbers of target. Ranges of plasmid dilutions were included as controls to ensure comparable detection levels. An amplification plot is the plot of fluorescence signal versus cycle number and these were generated by data analysis at the end of each experiment according to the manufacturer’s instructions (Applied Biosystems; Corbett Research).
3. Results 3.1. BIV detection 3.1.1. Amplification of Australian BIV sequences using conventional PCR Pooled DNA from seropositive animals was used to amplify BIV sequences for subsequent development of a BIV TaqMan® assay. Results from initial attempts to amplify BIV using nested PCR assays 2 & 3 (Suarez and Whetstone, 1998) were poor, with no amplification observed using the env primers and inconsistent, very faint positive results, using the pol set (not shown). Many variations to the published procedures were attempted including changes to buffers and PCR cycling temperatures with no detectable amplification products (not shown). The BIV/JDV conserved primers also did not successfully amplify a specific product from the pooled DNA sample (positive herd used for BIV screening) showing a ladder of faint bands (not shown). The pol primers designed by Nadin-Davis (BIV nested PCR 1, Table 1)
A.E. Lew et al. / Journal of Virological Methods 116 (2004) 1–9
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Fig. 1. Real-time fluorescence and conventional PCR detection of BIV plasmid control. BIV2 TaqMan® MGB assay, copy numbers are indicated (50 cycles). NTC: no template control. Plasmid copy numbers as shown.
Table 2 Summary of comparative sensitivities of PCR assays by comparing the amplification of plasmid dilutions for each respective virus Virus
PCR test and (reference)
Plasmid concentrationa
Copy number
BIV
Nested amplification (Nadin-Davis pol nested test) BIV2 TaqMan® MGB assay (this study)b
1.5 fg 15–150 ag
450 4.5c –45
BSV
PCR amplification (pol/env primers, Pamba et al., 1999) BSV TaqMan® MGB assay (this study)b
3 fg 30 ag
900 9
a Plasmid concentration was calculated from undiluted and diluted plasmid preparations through Agarose gel electrophoresis comparisons to a DNA MASSTM Ladder (Life Technologies). Plasmids were diluted in sterile distilled water. b Detection of BIV plasmid consistent at 150 ag when plasmid diluted in 1 g PBMCs in BIV2 TaqMan® assay; detection limit of 30 pg of DNA from cell culture derived BSV in the BSV TaqMan® assay. c Consistent amplification of 4.5 copies of BIV provirus in one of two duplicates at cycle threshold of 39.
amplified BIV from the pooled sample resulting in the isolation of two clones which were sequenced and aligned with published sequences prior to TaqMan® MGB assay design (Fig. 2A). 3.1.2. Sensitivity and specificity of BIV PCR assays The Australian BIV sequences were aligned with published BIV sequences and two BIV TaqMan® MGB assays were designed within this region (Table 1, Fig. 2A). BIV1 TaqMan® MGB assay did not perform well, emitting low fluorescence levels for both positive and negative controls (not shown). BIV2 TaqMan® MGB assay produced acceptable fluorescence levels and differentiated relevant positive and negative controls (Fig. 1). Based on the amplification of plasmid controls diluted in water, the BIV2 TaqMan® MGB test improved BIV detection compared to the nested pol (BIV nested PCR 1) PCR assay 10–100-fold (Table 2, Fig. 1) to detect approximately 4.5–45 copies. As few as 4.5 copies were detected consistently in one of two duplicates, whereas 45 copies were consistently amplified in both duplicates (Fig. 1). The BIV2 TaqMan® MGB assay did not amplify working stocks of BLV or BSV plasmids (not shown). The detection limit of the BIV TaqMan® MGB assay using BIV plasmid diluted in
BIV negative DNA sample (1 g) was consistent at 45 copies (Table 3). 3.1.3. BIV PCR testing on animal herds The BIV2 TaqMan® MGB assay was subsequently applied to screen Herds A–C, D–F (Table 3) and H. No TaqMan® MGB positive samples were identified in Herds A–C (Table 3) possibly due to lower sensitivity when analysing DNA prepared from whole blood. Table 3 Number of samples positive per herd in BIV and BSV proviral TaqMan® assays Herd
BIVa
BSVa
A B C D E F H
0/15 0/8 0/22 9/9b 17/30b 8/12 0/202
6/15 6/8 12/22 11/11 27/32 11/12 1/202
a Results presented as: the number of positive samples/total number of samples tested. b Not all samples were available for testing—D7, D10, E18 and E20 were not tested in the BIV TaqMan® assay.
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Nine of 9 (Herd D, two not tested), 17 of 29 (Herd E) and 8 of 11 (Herd F) animals were TaqMan® MGB positive for BIV using DNA prepared from purified PBMCs (Table 3). Two of these positives were confirmed by BIV nested PCR 1 with all remaining samples from Herds D–F negative in the conventional PCR (not shown). All samples from Herd H were negative in both nested PCR (BIV nested PCR 1) and TaqMan® MGB BIV2 assay. All positive animal samples were positive in both duplicate samples tested. 3.2. BSV detection 3.2.1. Isolation of Australian BSV sequences using conventional PCR Australian BSV was successfully amplified by the published BSV pol/env PCR assay but not by the gag primer set (not shown). Two BSV products (from Herd A) from the pol/env amplification were cloned and sequenced and aligned with published sequences prior to TaqMan® MGB assay design (Fig. 2B).
3.2.2. Sensitivity and specificity of BSV PCR assays The TaqMan® MGB test detected approximately nine copies of BSV and improved the sensitivity of the BSV assay over the pol/env PCR by 100-fold (Table 2). This assay did not amplify working stocks of BIV or BLV plasmids (not shown). The TaqMan® MGB assay detected 30 pg of DNA prepared from cell culture derived BSV (Table 2). 3.2.3. BSV PCR testing on animal herds Herds A–F were tested using the BSV TaqMan® MGB assay (Table 3). Six of 15, 6 of 8, 12 of 22, 11 of 11, 26 of 31 and 11 of 12 animals were TaqMan® MGB positive for BSV from Herds A–C (whole blood DNA extracts) and D–F (PBMCs extracts), respectively (Table 3). A higher prevalence of BSV was detected in Herds D–F than Herds A–C, possibly due to refinements to the DNA purification methods. The TaqMan® MGB assay identified one positive animal in Herd H (breeder herd) not detected by the conventional PCR applied here. Overall, BSV was more prevalent than BIV.
(A)
BIV M32690 4287 BIV AF548462 0 BIV AF548463 0
AATCTATGTA AGTATCTGGG GATCACAAAA ACTACGGGAA TACCCTACAA ---------- ---------- ---------- ---------- ------------------- ---------- ---------- ---------- ---------G
BIV M32690 4337 BIV AF548462 50 BIV AF548463 50
CCCACAATCC CAGGGAGTTG TAGAAAGAGC CCACAGAGAT CTAAAAGACA ---------- ---------- -T-------- ---------- ------------------- ---------- ---------- ---------- ----------
BIV M32690 4387 BIV AF548462 100 BIV AF548463 100
GATTGGCAGC TTATCAGGGA GATTGTGAAA CCGTAGAAGC AGCCCTTAGC ------T--- ---------- ---------- ---------- ------------------- ---------- ---------- ---C------ ----------
BIV M32690 4437 BIV AF548462 150 BIV AF548463 150
CTCGCATTAG TTTCTTTAAA TAAAAAAAGA GGGGGAATAG GGGGCCATAC ---------- ---------- -------GA- ---------- ------------------- ---------- -------G-- ---------- ---T------
(B)
BSV NC_001831 6528 CAAATATCAT CAACCGCAGT GTACATGGTC CTGTGAAGAT CGTTGTGGGC BSV AF548464 0 ---------- ---------- ---------- ---------- ---------BSV AF548465 0 ---------- ---------- ---------- ---------- ---------BSV NC_001831 6578 ATCGTTGGAA GGAGTGCGGG AATTGTATTC CACAGGATGG CTCCTCCGAT BSV AF548464 50 ---------- ---------- ---------- ---------- ---------BSV AF548465 50 ---------- ---------- ---------- ---------- ---------BSV NC_001831 6628 GACGCTTCAG CAGTGGCTGC AGTGGAGATA TAACCTAGAG ACGACCAACC BSV AF548464 100 ---------- ---------- ---------- ---------- --A------BSV AF548465 100 ---------- ---------- ---------- ---------- --A------BSV NC_001831 6678 TCTTACAGAT GAACCCTAAA ATGGAGAGTG BSV AF548464 150 ---------- ---------- ---------BSV AF548465 150 ---------- ---------- ---------Fig. 2. Alignment of Australian sequences (AF548462–AF548465) with sequences accessed from GenBank (M32690, NC 001831). Common bases are indicated by a dash and base differences are indicated as shown. (A) BIV sequences. BIV1 TaqMan® MGB assay primers and probe sequences are indicated by bold font, BIV2 TaqMan® MGB assay primers and probe sequences are underlined. (B) BSV sequences TaqMan® MGB assay primers and probe sequences are indicated by bold font. See also Table 1.
A.E. Lew et al. / Journal of Virological Methods 116 (2004) 1–9
4. Discussion We report the development of two 5 Taq nuclease assays using fluorescent 3 minor groove binder DNA probes (TaqMan® MGB) to detect BIV and BSV provirus respectively. Taq nuclease assays are increasingly being applied in viral diagnostics (Drosten et al., 2002; Kearns et al., 2001; Leutenegger et al., 1999; Locatelli et al., 2000; Mackay et al., 2002; Smith et al., 2001; Warrilow et al., 2002). Limited sequence data from Australian strains of both BIV and BSV was adequate for the development of 5 Taq nuclease assays employing MGB probes which exhibit minimal background fluorescence (Salmon et al., 2002). The sensitivity and specificity of these assays were superior to the traditional PCR tests examined here. The sensitivity of Taq nuclease assays has been found to be equal to that of nested PCR assays and capable of detecting approximately 10 copies of virus in clinical samples (Mackay et al., 2002). The limits of detection here were calculated using pure plasmid preparations and equal sensitivity is not likely in clinical samples where unknown numbers of provirus copies are present in a mixed DNA sample. Additionally, the calculation of copy number relies on the accuracy of determining DNA concentration, which may vary between laboratories. For this study we relied upon the comparison of the same plasmid dilution as a means of comparing methods used to amplify each provirus species. Pamba et al. (1999) demonstrated that southern hybridisation of PCR products improved the PCR detection limits 100-fold and the virus detection limits were equivalent to those obtained using the Taq nuclease assay reported here. Although serological evidence for the presence of BIV in Australia has been reported (Burkala et al., 1999; Forman et al., 1992), virus has never been isolated and, until this study, no Australian BIV sequences had been reported. Nested PCR assays, capable of detecting 10 copies of provirus, have been reported as the most reliable method of detecting BIV, (Gonzalez et al., 2001; Gradil et al., 1999; Suarez et al., 1995). However, prolonged difficulties were experienced in amplifying Australian BIV sequences, with no amplification using the BIV env primers and only poor results with the pol primers described by Suarez and Whetstone (1998) which were used with greater success by Gradil et al. (1999). The diversity of BIV env sequences has been confirmed in other studies (Kalvatchev et al., 2000; Meas et al., 2001) and for this reason should not be considered a potential target when developing a diagnostic assay. A JDV/BIV PCR, based on the same region utilised for the Suarez and Whetstone (1998) pol primers (3 region), was also unsuccessful. The primers designed to target the 5 region of the pol gene successfully amplified Australian sequences by conventional PCR and for this reason was the target of choice for the development of sensitive BIV-specific 5 Taq nuclease (TaqMan® ) assays. While the first BIV TaqMan® MGB assay did not perform well, possibly due to unusual folding at the primer or probe
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target regions or within the primer sequences, the second TaqMan® assay significantly improved the detection of Australian BIV. The purification of DNA from PBMCs appears critical for the detection of BIV in this study. This study appeared to demonstrate a high prevalence of BIV in samples taken from BLV endemic dairy herds. There were no BIV positive samples from the BLV negative beef breeding herd tested here (Herd H). An overall low herd prevalence of BIV has been confirmed in studies undertaken in other countries (Burger et al., 2000; Gonzalez et al., 2001; Scobie et al., 2001), however BLV status of the samples was not confirmed. If the correlation to BLV status is a valid observation, the overall prevalence of BIV in Australia would also be low due to the very low prevalence in beef herds in Queensland (Ward, 1995) and an extended BLV eradication program in Queensland dairy herds. There is some evidence towards the co-infection of BLV positive animals with BIV (Meas et al., 2002), however some seroprevalence studies provide evidence contrary to this (Jacobs et al., 1995; Cho et al., 1999; Meas et al., 1998). The ideal regime for BIV testing would probably be to combine a reliable serologic assay with a specific PCR test, as is currently the case with BLV (Evermann et al., 2000). We could not duplicate the western blot assay used by Burkala et al. (1999) (not shown) and other serological tests such as the IFA are no longer commercially available (Orr et al., 2003), thus currently a serologic assay is not accessible. Further testing will reveal whether BLV endemicity is linked to the presence of other bovine retroviruses. Evidence of JDV has not been found in Australia and based on sequence comparisons it is unlikely that the BIV TaqMan® assay applied here would detect JDV. The conserved JDV/BIV PCR assay did not amplify products that could be analysed by sequencing, thus it would appear that either the 5 pol regions of both BIV and JDV are variable among strains or possess unusual secondary structures somehow interfering with optimal extension. The degeneracy of JDV/BIV primers may have compromised the sensitivity of detection in this instance. No further attempts were made in this study to specifically isolate JDV. This is the first report of Australian BSV sequences obtained by amplification using the pol/env primers, which were originally reported as being more sensitive than the gag primers (Pamba et al., 1999). The TaqMan® assay described here further improved the sensitivity of detection as expected. Detection of BSV also improved when using DNA purified from PBMCs as opposed to whole blood extracts. As confirmed by BSV serological surveys in Australia and other countries (Jacobs et al., 1995; Johnson et al., 1988), a higher prevalence of BSV compared to BLV (not shown) and BIV was detected using the methods described here. The ability to identify proviral BSV allows differentiation of this virus from BIV or BLV, the induced-cytopathic effects of which are similar (Pamba et al., 1999). The difficulty here in applying published BIV PCR assays used successfully in other countries confirms the need
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to compare sequences from a range of strains when designing virus specific assays (Marsolais et al., 1994). Less sequence data is available for BIV and BSV compared with BLV, although genetic diversity of BIV env genes has been identified (Kalvatchev et al., 2000; Meas et al., 2001). Few base differences were found when comparing the small regions of Australian BIV and BSV with respective sequences available in sequence databases. Generally, the pol region was the most suitable target for the assays applied here as demonstrated by the ability of BIV (pol) and BSV (pol/env) tests to amplify Australian sequences. It is also essential to adequately concentrate and purify provirus to enable the detection of low numbers of virus copies in PCR, particularly for BIV. The assays described here can be applied to future prevalence and virus pathogenicity studies for BSV and BIV.
Acknowledgements The authors acknowledge Evan Burkala for the assistance and preliminary screening of animals to assist to identify Australian BIV positive animals. The authors are thankful to Andrew Masel, David Rutter (Applied Biosystems) and Bob Simpson (Real Time PCR Facility, University of Queensland) for their support and assistance in developing all TaqMan® assays; and Adam Spurway from Corbett Research for the use of the RotorGene.
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A.E. Lew et al. / Journal of Virological Methods 116 (2004) 1–9 Maker, D., Coackley, W., Smith, V.W., 1980. Isolation of bovine syncytial virus. Aust. Vet. J. 56, 153–154. Malmquist, W., Van Der Maaten, M.J., Boothe, A.D., 1969. Isolation, immunodifusion, immunofluorescence, and electron microscopy of a syncytial virus of lymphosarcomatous and apparently normal cattle. Cancer Res. 29, 188–200. Marsolais, G., Dubuc, R., Bergeron, J., Morrey, J.D., Kelly, E.J., Jackson, M.K., 1994. Importance of primer selection in the application of PCR technology to the diagnosis of bovine leukemia virus. J. Vet. Diagn. Invest. 6, 297–301. Meas, S., Kabeya, H., Yoshihara, S., Ohashi, K., Matsuki, S., Mikami, Y., Sugimoto, C., Onuma, M., 1998. Seroprevalence and field isolation of bovine immunodeficiency virus. J. Vet. Med. Sci. 60, 1195– 1202. Meas, S., Ohashi, K., Sugimoto, C., Onuma, M., 2001. Phylogenetic relationships of bovine immunodeficiency virus in cattle and buffaloes based on surface envelope gene sequences. Brief report. Arch. Virol. 146, 1037–1045. Meas, S., Usui, T., Ohashi, K., Sugimoto, C., Onuma, M., 2002. Vertical transmission of bovine leukemia virus and bovine immunodeficiency virus in dairy cattle herds. Vet. Microbiol. 84, 275–282. Miller, J.M., Miller, L.D., Olsen, C., Gillette, K.G., 1969. Virus-like particles in phytohemagglutinin-stimulated lymphocyte cultures with reference to bovine lymphosarcoma. J. Natl. Cancer Inst. 43, 1297– 1305. Nadin-Davis, S., 1995. Detection of bovine immunodeficiency-like virus by the polymerase chain reaction. In: Becker, Y., Darai, G. (Eds.), PCR: Protocols for Diagnosis of Human and Animal Virus Diseases, Springer-Verlag, Berlin, pp. 491–498. Orr, K.A., O’Reilly, K.L., Scholl, D.T., 2003. Estimation of sensitivity and specificity of two diagnostic tests for bovine immunodeficiency virus using Bayesian techniques. Prev. Vet. Med. 61, 79–89. Pamba, R., Jeronimo, C., Archambault, D., 1999. Detection of bovine retrospumavirus by the polymerase chain reaction. J. Virol. Meth. 78, 199–208. Renshaw, R.W., Casey, J.W., 1994. Transcriptional mapping of the 3 end of the bovine syncytial virus. J. Virol. 68 (2), 1021–1028.
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Rogers, R.J., Dimmock, C.K., De vos, A.J., Rodwell, B.J., 1988. Bovine Leucosis virus contamination of a vaccine produced in vivo against bovine babesiosis and anaplasmosis. Aust. Vet. J. 65, 285–287. Salmon, M.A., Vendrame, M., Kummert, J., Lepoivre, P., 2002. Detection of apple chlorotic leaf spot virus using a 5 nuclease assay with a fluorescent 3 minor groove binder-DNA probe. J. Virol. Meth. 104, 99–106. Scobie, L., Venables, C., Sayers, A., Weightman, S., Jarrett, O., 2001. Prevalence of bovine immunodeficiency virus infection in cattle in Great Britain. Vet. Rec. 149, 459–460. Smith, I.L., Halpin, K., Warrilow, D., Smith, G.A., 2001. Development of a fluorogenic RT-PCR assay (TaqMan) for the detection of Hendra virus. J. Virol. Meth. 98 (1), 33–40. Soeharsono, S., Hartaningsih, N., Soetrisno, M., Kertayadnya, G., Wilcox, G., 1990. Studies of experimental Jembrana disease in Bali cattle. I. Transmission and persistence of the infectious agent in ruminants and pigs, and resistance of recovered cattle to re-infection. J. Comp. Pathol. 103, 49–59. Suarez, D.L., Whetstone, C.A., 1998. PCR diagnosis of the bovine immunodeficiency-like virus. In: Meltzer, S.J. (Ed.), Methods in Molecular biology, vol. 92. PCR in Bioanalysis, Human Press, Totowa, NJ, pp. 67–79. Suarez, D.L., Van Der Maaten, M.J., Whetstone, C.A., 1995. Improved early and long-term detection of bovine lentivirus by a nested polymerase chain reaction test in experimentally infected calves. Am. J. Vet. Res. 56, 579–586. Van Der Maaten, M.J., Boothe, A.D., Seger, C.L., 1972. Isolation of a virus from cattle with persistent lymphocytosis. J. Natl. Cancer Inst. 49, 1649–1657. Ward, M.P., 1995. Serological studies of bovine leukaemia virus infection in Queensland beef cattle. Aust. Vet. J. 72, 71–72. Warrilow, D., Northill, J., Pyke, A., Smith, G., 2002. Single rapid TaqMan fluorogenic probe based PCR assay that detects all four dengue serotypes. J. Med. Virol. 66, 524–528. Wilcox, G., Kertayadnya, G., Hartaningsih, N., Dharma, D., Soeharsono, S., Robertson, T., 1992. Evidence for a viral aetiology of Jembrana disease in Bali cattle. Vet. Microbiol. 33, 367–374.
Sensitive and specific detection of bovine immunodeficiency virus and bovine syncytial virus by 5 Taq nuclease assays with fluorescent 3 minor groove binder-DNA probes Ala E. Lew a,∗ , Russell E. Bock b , John Miles a,1 , Leigh B. Cuttell a,2 , Penelope Steer b , Susan A. Nadin-Davis c a
b
Agency for Food and Fibre Sciences, Queensland Department of Primary Industries, c/o Locked Mail Bag No. 4, Moorooka 4105, Qld, Australia Tick Fever Research Centre, Animal and Plant Health Service, Queensland Department of Primary Industries, 280 Grindle Road, Wacol, Qld, Australia c Animal Diseases Research Institute/Centre for Plant Quarantine Pests, Ottawa Laboratory Fallowfield, 3851 Fallowfield Road, Nepean, Ont., Canada K2H 8P9 Received 13 June 2003; received in revised form 6 October 2003; accepted 13 October 2003
Abstract Sensitive assays are required to detect bovine retroviruses in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5 Taq nuclease assays using 3 minor groove binder DNA probes (TaqMan® MGB) were developed and compared to conventional PCR assays for the sensitive detection of bovine syncytial virus (BSV) and bovine immunodeficiency virus (BIV). Seven beef and dairy herds were screened to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log10 dilutions of plasmids with inserts containing corresponding provirus sequences. Published PCR assays targeting BIV env sequences did not adequately amplify Australian BIV sequences. Pol sequences from Australian strains of BIV and BSV were used to design TaqMan® MGB assays, which improved sensitivity 10-fold (BIV) and 100-fold (BSV), respectively, over conventional PCR tests. This is the first report of Australian sequences of BIV and BSV and the first 5 Taq nuclease assays described to detect these viruses. These methods could be applied to future studies requiring sensitive detection of these two bovine retroviruses. Crown Copyright © 2003 Published by Elsevier B.V. All rights reserved. Keywords: Bovine immunodeficiency virus; Bovine syncytial virus; Bovine retroviruses; 5 Taq nuclease; Minor groove binder; TaqMan®
1. Introduction The bovine retroviruses in the Retroviridae family of viruses include bovine leukemia virus (BLV) (Miller et al., 1969), bovine immunodeficiency virus/bovine immunodeficiency-like virus (BIV) (Van Der Maaten et al., 1972), ∗ Corresponding author. Tel.: +61-7-3362-9502; fax: +61-7-3362-9429. E-mail address: [email protected] (A.E. Lew). 1 Present address: Cellular Immunology, Infectious Diseases & Immunology Division, Comprehensive Cancer Research Centre, Queensland Institute of Medical Research, Royal Brisbane Hospital Post Office, 300 Herston Road, Herston, QLD, 4029, Australia. 2 Present address: Drosophila Genetics Group, Biochemistry and Molecular Biology, School of Pharmacy and Molecular Sciences, James Cook University, Townsville, QLD, Australia.
Jembrana disease virus (JDV) (Wilcox et al., 1992) and bovine syncytial virus (BSV) (Malmquist et al., 1969). Double-stranded DNA copies of these single stranded RNA viruses can integrate into the host genome and cause persistent infections with low viral gene expression but persistent antibody titre in infected hosts. Retroviral infections (BIV, BSV and BLV) of dairy and beef cattle are a major concern to the livestock industry due to the potential for disease ranging from lymphosarcoma to impairment of the immune system, economic and trade effects, and the perception that they may be threats to public health if present in the human food chain (Donham et al., 1987; Evermann and Jackson, 1997; Gradil et al., 1999). The control of these viruses relies on understanding how they are transmitted and on the ability to detect their presence in cattle (Evermann and Jackson, 1997).
0166-0934/$ – see front matter. Crown Copyright © 2003 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2003.10.006
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BIV and JDV are members of the bovine lentivirus group of the Lentivirus genus. BIV causes a subclinical disease syndrome and is found in cattle populations around the world whereas JDV causes an acute, often fatal disease specific to Bali cattle (Bos javanicus) (Chadwick et al., 1995). In Taurean breeds JDV produces a similar disease syndrome to BIV (Soeharsono et al., 1990). Bovine lentiviruses have not been isolated in Australia but there is serological evidence of their presence (Burkala et al., 1999; Forman et al., 1992). The impact of BIV is controversial due to the difficulty in culturing new isolates in vitro and the difficulty in identifying BIV-infected animals (Evermann et al., 2000; Gradil et al., 1999). No reliable diagnostic assay is available, but it appears that PCR assays are more sensitive than serological methods (Gradil et al., 1999). Prevalence studies using PCR have demonstrated a low herd prevalence of BIV in Canada and United States of America (Burger et al., 2000; Gonzalez et al., 2001). Serological detection is limited by low antibody levels (Isaacson et al., 1995), but a promising ELISA has been developed (Abed and Archambault, 2000). BSV of the genus Spumavirinae is generally thought to be non-pathogenic but can be leucotrophic and could exacerbate diseases caused by BIV and/or BLV (Jacobs et al., 1995). BSV is widely distributed and was confirmed in Australian cattle in 1980 (Maker et al., 1980). Generally, diagnosis of BSV infection has been based on crude serological methods and/or virus isolation from cell culture (Johnson et al., 1983; Malmquist et al., 1969). Recently, a PCR method was developed allowing rapid and sensitive detection of this virus (Pamba et al., 1999). Tick Fever is an endemic disease of cattle in northern Australia caused by the organisms Babesia bovis, B. bigemina or Anaplasma marginale. The Queensland Department of Primary Industries has distributed vaccines against Tick fever in Australia for over a century. Since 1964 vaccines containing attenuated strains of B. bovis, B. bigemina and A. centrale grown in splenectomised calves have been used to immunise cattle against the disease in Australia (Callow et al., 1997). Since an incidence of transmission of BLV through the use of a batch of contaminated vaccine was reported (Rogers et al., 1988), a rigorous testing programme for BLV has been initiated. Reports of concomitant infections involving BIV, BLV and BSV (Jacobs et al., 1995; Meas et al., 2002), prompted our investigation into methods for the detection of BIV and BSV as part of our vaccine donor calf screening procedures. Real time PCR has engendered wider acceptance of PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination compared with sensitive nested PCR assays (Mackay et al., 2002). 5 Taq nuclease assays successfully detecting BIV and BSV have not been reported. The aim of this study is to develop sensitive tests for BSV and BIV to enable the selection of uninfected donor cattle for vaccine production.
2. Materials and methods 2.1. Animal samples Originally, serum and peripheral blood mononuclear cells (PBMCs) were submitted for the evaluation of DNA extraction methods and various PCR assays for BLV (Lew et al., in press). These same samples were used for the evaluation of BSV and BIV tests. These were obtained from three BLV endemic dairy herds in north Queensland (Herds A, B and F); three BLV endemic dairy herds in south east Queensland (Herds C–E); and from 202 beef heifers assembled at TFRC from seven western Queensland properties for possible enrolment in a specific pathogen free breeding herd (Herd H). Whole blood was collected from the jugular vein into vacutainer tubes containing EDTA (Terumo Europe, Belgium). Serum samples were collected either from the coccygeal artery or jugular vein, allowed to clot at room temperature and the serum separated for subsequent analysis. 2.1.1. DNA purification methods DNA from animals from Herds A–C were prepared from 0.5 ml whole blood using a commercial QIAamp DNA blood mini kit (Qiagen Pty. Ltd., Australia). DNA from remaining herds were prepared from purified PBMCs instead of whole blood. Ten millilitre of whole blood in EDTA and 12.5 ml PBS were added to a LymphoprepTM tube (Nycomed Pharma AS, Diagnostics, Oslo, Norway) to isolate PBMCs from each animal as per the manufacturer’s instructions. PBMCs resuspended in 1.0 ml PBS were then counted using an Animal Blood Counter according to the manufacturer’s instructions (ABX Hematologie, France). DNA was prepared from 5 × 106 cells and stored at −20 ◦ C until used for PCR testing. DNA yields were measured spectrophotometrically. 2.1.2. Identification of BIV positive animals In 1999, prior to this study, DNA was prepared from LymphoprepTM extracts of separated blood samples from a herd identified to be seropositive for BIV/JDV by a Western blot assay (Burkala et al., 1999). The DNA from these samples were stored and used to identify Australian BIV and BSV sequences using conventional PCR methods in this study (Section 2.2). 2.2. Conventional PCR methods All primer sequences are summarised in Table 1. Oligonucleotide primers for conventional PCR assays were synthesised through Genset Oligos Pty Ltd. (now merged with Proligo, CO, USA). All conventional PCR assays were carried out in a Perkin-Elmer 480 (Applied Biosystems, CA, USA). For new PCR methods described in this study, conditions for reactions and cycling are described below otherwise published protocols were implemented.
A.E. Lew et al. / Journal of Virological Methods 116 (2004) 1–9
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Table 1 Primers and probes utilised in this study PCR assay
Primers and probe setsa
Sequence targetb
Sequence 5 → 3
Target gene/references
BIV nested PCR 1
POL3 POL4
4252–4272 4641–4662
aca acg ggc cgt gct tta ctg cct ctt cct cta tta ctg ctg c
pol gene/Nadin-Davis (1995)
POL5 POL6
4284–4306 4570–4591
gar aat cta tgt aag tat ctg gg ctg tty ctt acg taa cac cac t
Unpublished data (Nadin-Davis)
P09 P06
1805–1825 2529–2510
cac tgg acg aga tga ggt agt tgg tag tct gat aaa tgg ca
pol gene/Suarez and Whetstone (1998)
P04 P11
2133–2152 2498–2478
cag gct ctt aag gaa att gt cca tcc ttg tgg tag aac att
P06 P04
5413–5432 7095–7114
cta tgg atc agg acc tag ac gcc agc aca agc agg aat at
P01 P45
5532–5551 5965–5946
atc aat aac ggt gag atc ca tct atg gta tct ctg gct gc
BIVJDVF BIVJDVR
1703–1724c 2455–2433
wga stt ctg tga ccc crt ctg c cct tct cka tty acm ggg acy ac
BIVJDVFnest BIVJDVRnest
1806–1827 2348–2327
ayw ggr gca gat gag gta gtg c aca tts cyk aat tcc tgg rgg
BIV1
MGB probe 1
4338–4354
6FAM cca caa tcc cag gga gtd
TaqMan® MGB
BIVF1 BIVR1
4311–4335 4384–4359
aca aaa act acg gga ata ccc tac a tct ttt aga tct ctg tgg gct ctt tc
BIV2
MGB probe 2
4366–4382
6FAM ccc aca gag atc taa aad
TaqMan® MGB
BIVF2 BIVR2
4337–4355 4418–4395
ccc aca atc cca ggg agt t ggt ttc aca atc tcc ctg ata agc
BSV gag PCR
Gag-1 Gag-2
2551–2568 2793–2773
gac gca aca acc aac cac gtt ctt gtc cgt atc gtt gtg
gag gene/Pamba et al. (1999)
BSV pol env PCR
Pol/Env-1 Pol/Env-2
6528–6548 6737–6721
caa ata tca tca acc gca gtg cac ccg gtg gat caa ag
pol/env region/Pamba et al. (1999)
BSV
MGB probe
6561–6579
6FAM tga aga tcg ttg tgg gca td
pol/env region/this study
TaqMan® MGB
BSVF BSVR
6528–6552 6582–6599
caa ata tca tca acc gca gtg tac a ttc ccg cac tcc ttc caa
BIV nested PCR 2
BIV nested PCR 3
BIV/JDV conserved nested PCR
env gene/Suarez and Whetstone (1998)
pol gene/this study
pol gene/this study
pol gene/this study
a
The bracket indicates forward and reverse primer sets. The primer positions are based on GenBank accession no. M32690 for BIV and accession no. NC 001831 for BSV. c Positions of primers based on BIV, positions based on JDV accession no. U21603 are 1497–1518 for BIVJDVF; 2249–2247 for BIVJDVR; 1602–1620 for BIVJDVFnest; and 2142–2121 for BIVJDVRnest. d All probes were 5 labelled with 6-carboxyfluorescein (6-FAM) phoshoramidite as the 5 reporter dye and MGB probes use a non-fluorescent quencher attached to the 3 minor groove binder moiety (AB). b
2.2.1. BIV PCR Four sets of primers were used to attempt to amplify Australian BIV from the seropositive herd pooled sample (Table 1). The cycling conditions described by Gradil et al. (1999) were used for the env and pol nested PCR assays originally described by Suarez and Whetstone (1998) (BIV nested PCR 2 & 3, Table 1). An additional set of primers for nested amplification of conserved regions of both BIV and JDV was designed. An additional pol nested PCR employed primers POL 3 and 4 which were described previously (Nadin-Davis, 1995) whilst the internal primers, POL 5 and 6, which have not been reported previously, were designed using a Canadian BIV sequence (unpublished data, BIV
nested PCR 1, Table 1). A BIV plasmid imported from Canada was used as a positive control for this PCR assay (see Section 2.3) For assays described in this study, 1 unit of Taq polymerase, 1× PCR buffer with MgCl2 (Roche Molecular Biochemicals, Germany), 50 pmol of each primer and 100 M each of deoxynucleotide triphosphate were used to amplify 1 g DNA (whole blood or lymphocyte DNA) in 50 l PCR reactions. Cycles consisted of 3 min denaturing at 94 ◦ C followed by 35 cycles of 94 ◦ C 30 s, annealing 55 ◦ C 30 s, 72 ◦ C 1 min. Two microliters of the first reaction was transferred to the nested PCR reaction using new primers under identical reaction conditions.
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2.2.2. BSV PCR DNA from bovine syncytial virus from Canada was imported into Australia to use as a positive control for the optimisation of the BSV PCR. The virus had been cultivated and purified from foetal lamb spleen cells. Single PCR assays using primers targeting the pol/env region and the gag region were used to amplify Australian BSV from sequences from Herd A, as described by Pamba et al. (1999) without using southern hybridisation. 2.3. PCR product cloning for sequence analysis and positive controls BIV and BSV PCR products generated from Australian animal samples were cloned into the TA vector as recommended by the manufacturer (Invitrogen Corporation, CA, USA). Plasmids were purified using standard alkaline lysis techniques. Plasmids with inserts were sequenced using standard M13 primers and BigDye Terminator Mix following methods recommended by the manufacturer (Applied Biosystems, CA, USA). Sequences were analysed by the Griffith University DNA Sequencing Facility (School of Biomolecular and Biomedical Science, Griffith University Nathan QLD 4111 Australia) on an ABI 377 DNA Sequencer and sequence identity was confirmed using BLASTn (Altschul et al., 1990) via the NCBI website (http://www.ncbi.nlm.nih.gov/). Australian BIV and BSV sequences were submitted to the GenBank database (Benson et al., 2000) and assigned accession numbers AF548462 and AF548463 and AF548464 and AF548465, respectively. A BIV plasmid, pUC118-BIVKp3, was prepared from a 3.0 Kb-Kpn I fragment of the BIV genome (originally from a lambda library) and was imported from Canada (ADRI) for BIV PCR optimisation in Australia. This BIV fragment contains most of the pol ORF including the RT domain. Fragments of both BIV and BSV sequences that were amplified from Australian cattle (as described above) were used as TaqMan® assay controls. The concentration of plasmid stocks were calculated on the original undiluted plasmid preparation by electrophoretic comparison to DNA MASSTM Ladder (Life Technologies) in agarose gels according to the manufacturer’s instructions. Plasmid copy numbers were then determined by calculating molecular weight of the plasmid and subsequent log dilutions (660 Da for each bp, 1 mol = 6 × 1023 molecules). A BLV pUC8 plasmid clone (Coulston et al., 1990) was used as a negative control for BIV and BSV PCR assays. 2.4. TaqMan® PCR assays 2.4.1. TaqMan® probes and primers Oligonucleotide sequences for TaqMan® assays were confirmed by aligning sequences for each virus accessed through the following GenBank accessions: M32690 bovine immunodeficiency-like virus, complete proviral genome (Garvey et al., 1990); U21603 Jembrana disease virus Ta-
banan/87 strain, complete genome (Chadwick et al., 1995); NC 001831 bovine syncytial virus, complete genome (Renshaw and Casey, 1994). Sequences were aligned through PileUp which was accessed through the Australian National Genomic Information Service (ANGIS) (Littlejohn et al., 1996). Primers and probes were designed using Primer Express Version 2 (Applied Biosystems, Applera Corporation, CA, USA) and were all purchased through Applied Biosystems (AB). Sequences for primers and probes used are summarised in Table 1. 2.4.2. TaqMan® assay conditions The TaqMan® PCR Universal Master Mix (ABI Prism® 7700 Sequence Detection System Users Manual), and Platinum Quantitative PCR SuperMix-UDG (Invitrogen Corp.), were used with assay conditions as recommended except that the number of cycles was extended up to 50. Assays were carried out on the ABI Prism® 7700 Sequence Detector (AB) and the Corbett RotorGene 2000 (Corbett Research, Sydney, Australia). All samples were tested in duplicate and an average cycle threshold (CT ) value used to define the result. A threshold of 0.1 fluorescence units was applied to enable comparison of results between experiments. The parameter CT is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. Occasionally low copy number samples (in plasmid dilutions) yielded consistently positive and negative results in duplicates. These were considered to be ‘inconsistently positive’ due to pipetting of low copy numbers of target. Ranges of plasmid dilutions were included as controls to ensure comparable detection levels. An amplification plot is the plot of fluorescence signal versus cycle number and these were generated by data analysis at the end of each experiment according to the manufacturer’s instructions (Applied Biosystems; Corbett Research).
3. Results 3.1. BIV detection 3.1.1. Amplification of Australian BIV sequences using conventional PCR Pooled DNA from seropositive animals was used to amplify BIV sequences for subsequent development of a BIV TaqMan® assay. Results from initial attempts to amplify BIV using nested PCR assays 2 & 3 (Suarez and Whetstone, 1998) were poor, with no amplification observed using the env primers and inconsistent, very faint positive results, using the pol set (not shown). Many variations to the published procedures were attempted including changes to buffers and PCR cycling temperatures with no detectable amplification products (not shown). The BIV/JDV conserved primers also did not successfully amplify a specific product from the pooled DNA sample (positive herd used for BIV screening) showing a ladder of faint bands (not shown). The pol primers designed by Nadin-Davis (BIV nested PCR 1, Table 1)
A.E. Lew et al. / Journal of Virological Methods 116 (2004) 1–9
5
Fig. 1. Real-time fluorescence and conventional PCR detection of BIV plasmid control. BIV2 TaqMan® MGB assay, copy numbers are indicated (50 cycles). NTC: no template control. Plasmid copy numbers as shown.
Table 2 Summary of comparative sensitivities of PCR assays by comparing the amplification of plasmid dilutions for each respective virus Virus
PCR test and (reference)
Plasmid concentrationa
Copy number
BIV
Nested amplification (Nadin-Davis pol nested test) BIV2 TaqMan® MGB assay (this study)b
1.5 fg 15–150 ag
450 4.5c –45
BSV
PCR amplification (pol/env primers, Pamba et al., 1999) BSV TaqMan® MGB assay (this study)b
3 fg 30 ag
900 9
a Plasmid concentration was calculated from undiluted and diluted plasmid preparations through Agarose gel electrophoresis comparisons to a DNA MASSTM Ladder (Life Technologies). Plasmids were diluted in sterile distilled water. b Detection of BIV plasmid consistent at 150 ag when plasmid diluted in 1 g PBMCs in BIV2 TaqMan® assay; detection limit of 30 pg of DNA from cell culture derived BSV in the BSV TaqMan® assay. c Consistent amplification of 4.5 copies of BIV provirus in one of two duplicates at cycle threshold of 39.
amplified BIV from the pooled sample resulting in the isolation of two clones which were sequenced and aligned with published sequences prior to TaqMan® MGB assay design (Fig. 2A). 3.1.2. Sensitivity and specificity of BIV PCR assays The Australian BIV sequences were aligned with published BIV sequences and two BIV TaqMan® MGB assays were designed within this region (Table 1, Fig. 2A). BIV1 TaqMan® MGB assay did not perform well, emitting low fluorescence levels for both positive and negative controls (not shown). BIV2 TaqMan® MGB assay produced acceptable fluorescence levels and differentiated relevant positive and negative controls (Fig. 1). Based on the amplification of plasmid controls diluted in water, the BIV2 TaqMan® MGB test improved BIV detection compared to the nested pol (BIV nested PCR 1) PCR assay 10–100-fold (Table 2, Fig. 1) to detect approximately 4.5–45 copies. As few as 4.5 copies were detected consistently in one of two duplicates, whereas 45 copies were consistently amplified in both duplicates (Fig. 1). The BIV2 TaqMan® MGB assay did not amplify working stocks of BLV or BSV plasmids (not shown). The detection limit of the BIV TaqMan® MGB assay using BIV plasmid diluted in
BIV negative DNA sample (1 g) was consistent at 45 copies (Table 3). 3.1.3. BIV PCR testing on animal herds The BIV2 TaqMan® MGB assay was subsequently applied to screen Herds A–C, D–F (Table 3) and H. No TaqMan® MGB positive samples were identified in Herds A–C (Table 3) possibly due to lower sensitivity when analysing DNA prepared from whole blood. Table 3 Number of samples positive per herd in BIV and BSV proviral TaqMan® assays Herd
BIVa
BSVa
A B C D E F H
0/15 0/8 0/22 9/9b 17/30b 8/12 0/202
6/15 6/8 12/22 11/11 27/32 11/12 1/202
a Results presented as: the number of positive samples/total number of samples tested. b Not all samples were available for testing—D7, D10, E18 and E20 were not tested in the BIV TaqMan® assay.
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Nine of 9 (Herd D, two not tested), 17 of 29 (Herd E) and 8 of 11 (Herd F) animals were TaqMan® MGB positive for BIV using DNA prepared from purified PBMCs (Table 3). Two of these positives were confirmed by BIV nested PCR 1 with all remaining samples from Herds D–F negative in the conventional PCR (not shown). All samples from Herd H were negative in both nested PCR (BIV nested PCR 1) and TaqMan® MGB BIV2 assay. All positive animal samples were positive in both duplicate samples tested. 3.2. BSV detection 3.2.1. Isolation of Australian BSV sequences using conventional PCR Australian BSV was successfully amplified by the published BSV pol/env PCR assay but not by the gag primer set (not shown). Two BSV products (from Herd A) from the pol/env amplification were cloned and sequenced and aligned with published sequences prior to TaqMan® MGB assay design (Fig. 2B).
3.2.2. Sensitivity and specificity of BSV PCR assays The TaqMan® MGB test detected approximately nine copies of BSV and improved the sensitivity of the BSV assay over the pol/env PCR by 100-fold (Table 2). This assay did not amplify working stocks of BIV or BLV plasmids (not shown). The TaqMan® MGB assay detected 30 pg of DNA prepared from cell culture derived BSV (Table 2). 3.2.3. BSV PCR testing on animal herds Herds A–F were tested using the BSV TaqMan® MGB assay (Table 3). Six of 15, 6 of 8, 12 of 22, 11 of 11, 26 of 31 and 11 of 12 animals were TaqMan® MGB positive for BSV from Herds A–C (whole blood DNA extracts) and D–F (PBMCs extracts), respectively (Table 3). A higher prevalence of BSV was detected in Herds D–F than Herds A–C, possibly due to refinements to the DNA purification methods. The TaqMan® MGB assay identified one positive animal in Herd H (breeder herd) not detected by the conventional PCR applied here. Overall, BSV was more prevalent than BIV.
(A)
BIV M32690 4287 BIV AF548462 0 BIV AF548463 0
AATCTATGTA AGTATCTGGG GATCACAAAA ACTACGGGAA TACCCTACAA ---------- ---------- ---------- ---------- ------------------- ---------- ---------- ---------- ---------G
BIV M32690 4337 BIV AF548462 50 BIV AF548463 50
CCCACAATCC CAGGGAGTTG TAGAAAGAGC CCACAGAGAT CTAAAAGACA ---------- ---------- -T-------- ---------- ------------------- ---------- ---------- ---------- ----------
BIV M32690 4387 BIV AF548462 100 BIV AF548463 100
GATTGGCAGC TTATCAGGGA GATTGTGAAA CCGTAGAAGC AGCCCTTAGC ------T--- ---------- ---------- ---------- ------------------- ---------- ---------- ---C------ ----------
BIV M32690 4437 BIV AF548462 150 BIV AF548463 150
CTCGCATTAG TTTCTTTAAA TAAAAAAAGA GGGGGAATAG GGGGCCATAC ---------- ---------- -------GA- ---------- ------------------- ---------- -------G-- ---------- ---T------
(B)
BSV NC_001831 6528 CAAATATCAT CAACCGCAGT GTACATGGTC CTGTGAAGAT CGTTGTGGGC BSV AF548464 0 ---------- ---------- ---------- ---------- ---------BSV AF548465 0 ---------- ---------- ---------- ---------- ---------BSV NC_001831 6578 ATCGTTGGAA GGAGTGCGGG AATTGTATTC CACAGGATGG CTCCTCCGAT BSV AF548464 50 ---------- ---------- ---------- ---------- ---------BSV AF548465 50 ---------- ---------- ---------- ---------- ---------BSV NC_001831 6628 GACGCTTCAG CAGTGGCTGC AGTGGAGATA TAACCTAGAG ACGACCAACC BSV AF548464 100 ---------- ---------- ---------- ---------- --A------BSV AF548465 100 ---------- ---------- ---------- ---------- --A------BSV NC_001831 6678 TCTTACAGAT GAACCCTAAA ATGGAGAGTG BSV AF548464 150 ---------- ---------- ---------BSV AF548465 150 ---------- ---------- ---------Fig. 2. Alignment of Australian sequences (AF548462–AF548465) with sequences accessed from GenBank (M32690, NC 001831). Common bases are indicated by a dash and base differences are indicated as shown. (A) BIV sequences. BIV1 TaqMan® MGB assay primers and probe sequences are indicated by bold font, BIV2 TaqMan® MGB assay primers and probe sequences are underlined. (B) BSV sequences TaqMan® MGB assay primers and probe sequences are indicated by bold font. See also Table 1.
A.E. Lew et al. / Journal of Virological Methods 116 (2004) 1–9
4. Discussion We report the development of two 5 Taq nuclease assays using fluorescent 3 minor groove binder DNA probes (TaqMan® MGB) to detect BIV and BSV provirus respectively. Taq nuclease assays are increasingly being applied in viral diagnostics (Drosten et al., 2002; Kearns et al., 2001; Leutenegger et al., 1999; Locatelli et al., 2000; Mackay et al., 2002; Smith et al., 2001; Warrilow et al., 2002). Limited sequence data from Australian strains of both BIV and BSV was adequate for the development of 5 Taq nuclease assays employing MGB probes which exhibit minimal background fluorescence (Salmon et al., 2002). The sensitivity and specificity of these assays were superior to the traditional PCR tests examined here. The sensitivity of Taq nuclease assays has been found to be equal to that of nested PCR assays and capable of detecting approximately 10 copies of virus in clinical samples (Mackay et al., 2002). The limits of detection here were calculated using pure plasmid preparations and equal sensitivity is not likely in clinical samples where unknown numbers of provirus copies are present in a mixed DNA sample. Additionally, the calculation of copy number relies on the accuracy of determining DNA concentration, which may vary between laboratories. For this study we relied upon the comparison of the same plasmid dilution as a means of comparing methods used to amplify each provirus species. Pamba et al. (1999) demonstrated that southern hybridisation of PCR products improved the PCR detection limits 100-fold and the virus detection limits were equivalent to those obtained using the Taq nuclease assay reported here. Although serological evidence for the presence of BIV in Australia has been reported (Burkala et al., 1999; Forman et al., 1992), virus has never been isolated and, until this study, no Australian BIV sequences had been reported. Nested PCR assays, capable of detecting 10 copies of provirus, have been reported as the most reliable method of detecting BIV, (Gonzalez et al., 2001; Gradil et al., 1999; Suarez et al., 1995). However, prolonged difficulties were experienced in amplifying Australian BIV sequences, with no amplification using the BIV env primers and only poor results with the pol primers described by Suarez and Whetstone (1998) which were used with greater success by Gradil et al. (1999). The diversity of BIV env sequences has been confirmed in other studies (Kalvatchev et al., 2000; Meas et al., 2001) and for this reason should not be considered a potential target when developing a diagnostic assay. A JDV/BIV PCR, based on the same region utilised for the Suarez and Whetstone (1998) pol primers (3 region), was also unsuccessful. The primers designed to target the 5 region of the pol gene successfully amplified Australian sequences by conventional PCR and for this reason was the target of choice for the development of sensitive BIV-specific 5 Taq nuclease (TaqMan® ) assays. While the first BIV TaqMan® MGB assay did not perform well, possibly due to unusual folding at the primer or probe
7
target regions or within the primer sequences, the second TaqMan® assay significantly improved the detection of Australian BIV. The purification of DNA from PBMCs appears critical for the detection of BIV in this study. This study appeared to demonstrate a high prevalence of BIV in samples taken from BLV endemic dairy herds. There were no BIV positive samples from the BLV negative beef breeding herd tested here (Herd H). An overall low herd prevalence of BIV has been confirmed in studies undertaken in other countries (Burger et al., 2000; Gonzalez et al., 2001; Scobie et al., 2001), however BLV status of the samples was not confirmed. If the correlation to BLV status is a valid observation, the overall prevalence of BIV in Australia would also be low due to the very low prevalence in beef herds in Queensland (Ward, 1995) and an extended BLV eradication program in Queensland dairy herds. There is some evidence towards the co-infection of BLV positive animals with BIV (Meas et al., 2002), however some seroprevalence studies provide evidence contrary to this (Jacobs et al., 1995; Cho et al., 1999; Meas et al., 1998). The ideal regime for BIV testing would probably be to combine a reliable serologic assay with a specific PCR test, as is currently the case with BLV (Evermann et al., 2000). We could not duplicate the western blot assay used by Burkala et al. (1999) (not shown) and other serological tests such as the IFA are no longer commercially available (Orr et al., 2003), thus currently a serologic assay is not accessible. Further testing will reveal whether BLV endemicity is linked to the presence of other bovine retroviruses. Evidence of JDV has not been found in Australia and based on sequence comparisons it is unlikely that the BIV TaqMan® assay applied here would detect JDV. The conserved JDV/BIV PCR assay did not amplify products that could be analysed by sequencing, thus it would appear that either the 5 pol regions of both BIV and JDV are variable among strains or possess unusual secondary structures somehow interfering with optimal extension. The degeneracy of JDV/BIV primers may have compromised the sensitivity of detection in this instance. No further attempts were made in this study to specifically isolate JDV. This is the first report of Australian BSV sequences obtained by amplification using the pol/env primers, which were originally reported as being more sensitive than the gag primers (Pamba et al., 1999). The TaqMan® assay described here further improved the sensitivity of detection as expected. Detection of BSV also improved when using DNA purified from PBMCs as opposed to whole blood extracts. As confirmed by BSV serological surveys in Australia and other countries (Jacobs et al., 1995; Johnson et al., 1988), a higher prevalence of BSV compared to BLV (not shown) and BIV was detected using the methods described here. The ability to identify proviral BSV allows differentiation of this virus from BIV or BLV, the induced-cytopathic effects of which are similar (Pamba et al., 1999). The difficulty here in applying published BIV PCR assays used successfully in other countries confirms the need
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to compare sequences from a range of strains when designing virus specific assays (Marsolais et al., 1994). Less sequence data is available for BIV and BSV compared with BLV, although genetic diversity of BIV env genes has been identified (Kalvatchev et al., 2000; Meas et al., 2001). Few base differences were found when comparing the small regions of Australian BIV and BSV with respective sequences available in sequence databases. Generally, the pol region was the most suitable target for the assays applied here as demonstrated by the ability of BIV (pol) and BSV (pol/env) tests to amplify Australian sequences. It is also essential to adequately concentrate and purify provirus to enable the detection of low numbers of virus copies in PCR, particularly for BIV. The assays described here can be applied to future prevalence and virus pathogenicity studies for BSV and BIV.
Acknowledgements The authors acknowledge Evan Burkala for the assistance and preliminary screening of animals to assist to identify Australian BIV positive animals. The authors are thankful to Andrew Masel, David Rutter (Applied Biosystems) and Bob Simpson (Real Time PCR Facility, University of Queensland) for their support and assistance in developing all TaqMan® assays; and Adam Spurway from Corbett Research for the use of the RotorGene.
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