BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm
© Jimba et al; licensee BioMed Central Ltd. 2010
Received: 21 May 2010
Accepted: 2 November 2010
Published: 2 November 2010
Bovine leukemia virus (BLV) is closely related to human T-cell leukemia virus (HTLV) and is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly extended course that often involves persistent lymphocytosis and culminates in B-cell lymphomas. BLV provirus remains integrated in cellular genomes, even in the absence of detectable BLV antibodies. Therefore, to understand the mechanism of BLV-induced leukemogenesis and carry out the selection of BLV-infected animals, a detailed evaluation of changes in proviral load throughout the course of disease in BLV-infected cattle is required. The aim of this study was to develop a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in clinical animals.
Degenerate primers were designed from 52 individual BLV long terminal repeat (LTR) sequences identified from 356 BLV sequences in GenBank using the CoCoMo algorithm, which has been developed specifically for the detection of multiple virus species. Among 72 primer sets from 49 candidate primers, the most specific primer set was selected for detection of BLV LTR by melting curve analysis after real-time PCR amplification. An internal BLV TaqMan probe was used to enhance the specificity and sensitivity of the assay, and a parallel amplification of a single-copy host gene (the bovine leukocyte antigen DRA gene) was used to normalize genomic DNA. The assay is highly specific, sensitive, quantitative and reproducible, and was able to detect BLV in a number of samples that were negative using the previously developed nested PCR assay. The assay was also highly effective in detecting BLV in cattle from a range of international locations. Finally, this assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression.
Using our newly developed BLV-CoCoMo-qPCR assay, we were able to detect a wide range of mutated BLV viruses. CoCoMo algorithm may be a useful tool to design degenerate primers for quantification of proviral load for other retroviruses including HTLV and human immunodeficiency virus type 1.
Many viruses mutate during evolution, which can lead to alterations in pathogenicity and epidemic outbreaks [1, 2]. The development of molecular techniques, especially those applications based on the polymerase chain reaction (PCR), has revolutionized the diagnosis of viral infectious diseases [3, 4]. Degenerate oligonucleotide primers, which allow the amplification of several possible mutated versions of a gene, have been successfully used for cDNA cloning and for the detection of sequences that are highly variable due to a high rate of mutation . Degenerate primers are useful for the amplification of unknown genes, and also for the simultaneous amplification of similar, but not identical, genes . The use of degenerate primers can significantly reduce the cost and time spent on viral detection. The "Coordination of Common Motifs" (CoCoMo) algorithm has been developed especially for the detection of multiple virus species (Endoh D, Mizutani T, Morikawa S, Hamaguchi I, Sakai K, Takizawa K, Osa Y, Asakawa M, Kon Y, Hayashi M: CoCoMo-Primers: a web server for designing degenerate primers for virus research, submitted). This program uses an extension of the COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CodeHop) technique , which is based on multiple DNA sequence alignments using MAFFT multiple sequence alignment program . The CoCoMo selects common gap tetranucleotide motifs (GTNM), which include codons from the target sequences. It then selects amplifiable sets of common GTNMs using a database-based method and constructs consensus oligonucleotides at the 5' end of each common amplifiable GTNM. The consensus degenerate sequence is then attached to the designed degenerate primers. Thus, the CoCoMo algorithm is very useful in the design of degenerate primers for highly degenerate sequences.
Bovine leukemia virus (BLV) is closely related to human T-cell leukemia virus types 1 and 2 (HTLV-1 and -2) and is the etiological agent of enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle . Infection with BLV can remain clinically silent, with cattle in an aleukemic state. It can also emerge as a persistent lymphocytosis (PL), characterized by an increased number of B lymphocytes, or more rarely, as a B-cell lymphoma in various lymph nodes after a long latent period .
In addition to the structural and enzymatic Gag, Pol, and Env proteins, BLV encodes at least two regulatory proteins, namely Tax and Rex, in the pX region located between the env gene and the 3' long terminal repeat (LTR) . Moreover, BLV contains several other small open reading frames in the region between the env gene and the tax/rex genes in the pX region. These encode products designated as R3 and G4 . BLV has two identical LTRs, which possess a U3 region, an unusually long R region, and a U5 region; these LTRs only exert efficient transcriptional promoter activity in cells productively infected with BLV . BLV can integrate into dispersed sites within the host genome  and appears to be transcriptionally silent in vivo . Indeed, transcription of the BLV genome in fresh tumor cells or in fresh peripheral blood mononuclear cells (PBMCs) from infected individuals is almost undetectable by conventional techniques [12, 13]. In situ hybridization has revealed the expression of viral RNA at low levels in many cells, and at a high level in a few cells in populations of freshly isolated PBMCs from clinically normal BLV-infected animals . It appears that BLV provirus remains integrated in cellular genomes, even in the absence of detectable BLV antibodies. Therefore, in addition to the routine diagnosis of BLV infection using conventional serological techniques such as the immunodiffusion test [15–18] and enzyme-linked immunosorbent assay (ELISA) [17–20], diagnostic BLV PCR techniques that aim to detect the integrated BLV proviral genome within the host genome are also commonly used [17–19, 21–23]. However, real-time quantitative PCR for BLV provirus of all known variants has not been developed, largely due to differences in amplification efficiency caused by DNA sequence variations between clinical samples.
BLV infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry [16–20, 24–26]. Recent studies on the genetic variability of the BLV env gene have shown genetic variations among BLV isolates from different locations worldwide [24, 27]. Therefore, in this study, we used the CoCoMo algorithm to design degenerate primers addressing BLV diversity and used these primers to develop a new quantitative real-time PCR method to measure the proviral load of all BLV variants. To normalize the viral genomic DNA, the BLV-CoCoMo-qPCR technique amplifies a single-copy host gene [bovine leukocyte antigen (BoLA)-DRA gene] in parallel with the viral genomic DNA. The assay is specific, sensitive, quantitative and reproducible, and is able to detect BLV strains from cattle worldwide, including those for which previous attempts at detection by nested PCR failed. Interestingly, we succeeded in confirming that the BLV copy number in PBMC clearly increased with disease progression.
Principle of absolute quantification for determination of BLV proviral copy number
Use of the CoCoMo algorithm to construct a primer set with the ability to amplify all BLV strains
Primer sequences for amplification of BLV LTR candidate regions by the Coordination of Common Motif (CoCoMo) algorithm
Primer annealing position in the BLV LTR sequence1
43-67, 68-92, 130-154
8232-56, 8257-81, 8319-43
124-148, 169-193, 170-194
8313-37, 8358-82, 8359-83
26-50, 91-115, 247-271
8215-39, 8280-8304, 8436-60
123-147, 128-152, 211-235
8312-36, 8317-41, 8400-24
202-226, 360-384, 375-399
8391-8415, 8549-73, 8564-88
4-28, 90-114, 105-129, etc
8610-8634, 8619-8643, etc
279-303, 421-445, 430-454
8468-8492, 8610-34, 8619-43
378-402, 421-445, 428-452
8567-8591, 8610-34, 8617-41
Selection of the primer set and probe for amplification of the BLV LTR region
To determine whether the CoCoMo primer sets amplified the BLV LTR region, touch-down PCR was performed with 72 candidate primer sets (Figure 2B) using genomic DNA extracted from BLV-infected BLSC-KU-17 cells. As shown in Figure 2A, we identified 16 sets of primers, 1-6, 9, 15-17, 20, 21, 24, 33, 43 and 46, which successfully amplified the BLV LTR region.
The specificity of the 16 selected primer sets was evaluated by melting-curve analysis of amplification using genomic DNA extracted from BLSC-KU-17 cells or PBMCs from BLV-free normal cattle Ns118, with reagent-only as the negative control. Figure 2C shows the four typical melting-curves. Amplicons consisting of a single PCR product with a single melting temperature exhibited a single peak, while amplicons consisting of two or more products exhibited multiple peaks. The amplicon generated using primer set ID15 of CoCoMo 6 and CoCoMo 81 had a single melting temperature using BLSC-KU-17 genomic DNA. Using these primers, no amplicons were generated using genomic DNA from PBMCs in BLV-free normal cattle Ns118 or using the reagent-only control. In contrast, other primer sets, such as ID3, ID16 and ID17 generated amplicons from genomic DNA extracted from PBMCs from BLV-free normal cattle Ns118 or in reagent only, as well as from genomic DNA extracted from BLSC-KU-17 cells. Therefore, we proceeded to optimize the amplification conditions using primer sets CoCoMo 6 and CoCoMo 81, which were the best pair for the detection of the BLV LTR region (Figure 2D). Under these optimized conditions, amplification melting-curve analysis using genomic DNAs extracted from 56 BLV-infected cattle and from 3 BLV-free normal cattle showed the same patterns as seen in BLSC-KU-17 cells and BLV-free normal cattle Ns118 (data not shown).
The internal BLV TaqMan probe was constructed from a region of low variability located between positions corresponding to the CoCoMo 6 and CoCoMo 81 primers in the LTR regions of the BLV genome (Figure 1), and was labeled with carboxyfluorescein (FAM) dye, non-fluorescent quencher (NFQ) and minor groove binder (MGB) probe for enhancing the probe melting temperature. The probe was designated as FAM-BLV.
Construction of the primer set and probe for quantification of the BoLA-DRA gene
For normalization of the genomic DNA used as the PCR template, we designed primers and a probe for quantification of the BoLA-DRA gene (Figure 1). We obtained sequences from an MR1 cDNA clone [DDBJ: No. D37956] and selected the exon 4 region of the BoLA-DRA gene as the target for amplification. We designed the amplification primer set DRA643 and DRA734 and the internal BoLA-DRA TaqMan probe using the Primer Express 3.0 (Applied Biosystems, Tokyo, Japan). The probe was labeled with VIC dye, NFQ and an MGB probe for enhancing the probe melting temperature, and was designated as VIC-DRA.
Quantification of plasmid DNA copy number to create standard curves for absolute quantitative PCR
To obtain standards for quantification of BLV proviral DNA and cellular DNA, pBLV-LTR/SK, which includes a full-length LTR of BLV, and pBoLA-DRA/SK, which includes a full-length bovine DRA gene, were prepared at 103.1 ng/μl (pBLV-LTRconc) and 125.0 ng/μl (pBoLA-DRAconc), respectively. The copy numbers of these plasmids were calculated by the serial dilution method: each plasmid was diluted 10-fold, and the target DNA was detected by nested PCR. For example, at a 10-11 dilution of pBLV-LTRconc, PCR amplification failed to detect any PCR product, including the BLV LTR. The PCR reaction was then replicated 10 times at the 10-11 dilution, and the success rate was found to be 5/10. This result showed that 5 of 10 PCR solutions did not contain the LTR gene, expressed in equation form as: f (x = 0) = 5/10. Finally, the average copy number of the target gene (λ) was calculated as -loge (5/10) = 0.231 corresponding to a copy number for pBLV-LTRconc of 2.31 × 1010/μl. Using the same strategy, the copy number of pBoLA-DRAconc was determined to be 2.54 × 1010/μl. For confirmation of the reliability of estimated copy numbers, we also calculated draft copy numbers from the DNA weight and obtained a very similar result (2.35 × 1010 for pBLV-LTRconc, and 2.85 × 1010 for pBoLA-DRAconc).
Final procedure for the optimization of BLV-CoCoMo-qPCR
To construct the standard curve, the following dilutions of pBLV-LTRconc and pBoLA-DRAconc were created: 0.1 copy/μl, 1 copy/μl, 1,000 copies/μl and 1,000,000 copies/μl. A 168-bp amplicon from the BLV LTR region was amplified in a total volume of 20 μl of 1 × TaqMan Gene Expression Master Mix containing 500 nM CoCoMo 6 primer, 50 nM CoCoMo 81 primer, 150 nM FAM-BLV probe (5'-FAM-CTCAGCTCTCGGTCC-NFQ-MGB-3'), and 30 ng of template DNA. In addition, a 57-bp amplicon of the BoLA-DRA region was amplified in a total volume of 20 μl of 1 × TaqMan Gene Expression Master Mix containing 50 nM of DRA643 primer (5'-CCCAGAGACCACAGAGAATGC-3'), 50 nM of DRA734 primer (5'-CCCACCAGAGCCACAATCA-3'), 150 nM of VIC-DRA probe (5'-VIC-TGTGTGCCCTGGGC-NFQ-MGB 3'), and 30 ng of template DNA. PCR amplification was performed with the ABI 7500 Fast Real-time PCR system according to the following program: Uracil-DNA Glycosylase (UDG) enzyme activation at 50°C for 2 min followed by AmpliTaq Gold Ultra Pure (UP) enzyme activation at 95°C for 10 min, and then 85 cycles of 15 sec at 95°C and 1 min at 60°C. Copy numbers obtained for the BLV LTR and BoLA-DRA were used to calculate BLV proviral load per 100,000 cells, as shown in Equation (A).
Reproducibility of BLV-CoCoMo-qPCR
Intra- and inter-assay reproducibility of BLV-CoCoMo-qPCR
1998 ± 385
2107 ± 296
1581 ± 150
3951 ± 691
3751 ± 529
3049 ± 150
20388 ± 222
23484 ± 1854
28375 ± 1381
30155 ± 6184
27247 ± 1454
34954 ± 3021
57236 ± 6127
59375 ± 3195
53225 ± 2514
90947 ± 0
73002 ± 3228
61388 ± 4779
87377 ± 7434
94934 ± 5891
Evaluation of the specificity of BLV-CoCoMo-qPCR primers using various retroviruses
Evaluation of the sensitivity of BLV-CoCoMo-qPCR compared with nested PCR
Comparison of BLV-CoCoMo-qPCR and Serial dilution nested PCR
Correlation of BLV-CoCoMo-qPCR and syncytium formation assay
BLV provirus detection in cattle from different geographic locations by BLV-CoCoMo-qPCR and nested PCR
Comparison of BLV detection by BLV-CoCoMo-qPCR and nested PCR in cattle from Japan, Peru, Bolivia, Chile and the U.S.A.
Results of BLV detection by BLV-CoCoMo-qPCR/nested PCR
Correlation analysis of disease progression and BLV proviral load
In this study, we describe the successful development of a highly specific, accurate, and sensitive method for the quantification of BLV proviral load from infected animals.
The BLV-CoCoMo-qPCR system is able to detect various BLV strains from a broad geographical origin, including Japan, Peru, Bolivia, Chile and the U.S.A. Although early studies on genetic variability of the BLV env gene identified very little variation among isolates , recent studies based on restriction fragment length polymorphism analysis and on analysis of the full-length BLV env gene have revealed at least seven different BLV genotypes in circulation worldwide [24, 27, 29]. This novel classification suggests that BLV divergence has increased worldwide. From a total of 356 BLV sequences in GenBank, we were able to obtain 52 distinct BLV LTR nucleotide sequences. This indicates that many BLV variants exist, and our results suggest that detection of all of these variants is possible using BLV-CoCoMo-qPCR. Thus, we have clearly demonstrated that the CoCoMo algorithm is a useful tool for designing degenerate primers corresponding to multiple BLV variants. In fact, Tong et al.  indicated that semi-nested or nested PCR assays with consensus-degenerate hybrid oligonucleotide primers for Paramyxoviridae could be developed to be either highly specific or more broadly inclusive, enabling targeting at the subfamily or genus level. Using this type of approach, there is a risk that the degeneracy of the CoCoMo primers could be too high, thereby reducing the concentration of primer specific to the target sequence and decreasing the assay's sensitivity. This issue did not arise in our study since BLV-CoCoMo-qPCR was highly sensitive and gave superior results to nested PCR amplification (Figure 6). In addition, the BLV-CoCoMo-qPCR system was very effective in detecting virus in BLV-infected cattle from a range of geographic locations (Table 3). The TaqMan probe was used to improve sensitivity and specificity and acts to counter any drawbacks associated with high degeneracy. It is important to note that the sequence of the BLV TaqMan probe, located between positions corresponding to the CoCoMo 6 and CoCoMo 81 primers, was completely conserved among the 52 BLV variants. The ELISA and immunodiffusion screening methods for BLV in cattle are also highly sensitive but they act by detecting antibodies against BLV, in contrast to the direct detection of integrated provirus by PCR . The ELISA and immunodiffusion methods suffer from a high rate of false positives, and they are also ineffective in determining whether calves are infected since circulating maternal antibodies from BLV-infected dams can interfere with the assay. Reliable detection of BLV-infection in cattle therefore requires a high-sensitivity method for the detection of provirus. To this end, we developed BLV-CoCoMo-qPCR.
Several approaches were used to confirm the high specificity of BLV quantification using CoCoMo primers. First, the CoCoMo 6 and CoCoMo 81 primers yielded a single peak by melting curve analysis in cells infected with BLV, but did not amplify a product in uninfected cells or in the reagent-only negative control. Second, PCR amplification was detected in BLV-positive cattle, but was negative in all 120 BLV-negative cattle tested. Third, infectious molecular clones including several non-BLV retroviral LTRs were not amplified by our system.
A previous study  reported a method to quantify BLV provirus using real-time PCR. This method targeted the BLV env and pol genes, which are present at only one copy per provirus, and the primer annealing regions were potentially susceptible to mutation. The BLV LTR target of BLV-CoCoMo-qPCR is present at two copies per provirus, which contributes to the improved sensitivity of our assay. Indeed, using the quantitative PCR method described by Lew et al. , we could not detect provirus at less than 18 copies/105 cells, a concentration that was readily detectable by BLV-CoCoMo-qPCR (data not shown). Our method also has the advantage that the use of degenerate primers allows for the detection of BLV sequence variants, including those that arise from mutations.
The BLV-CoCoMo-qPCR method was also accurate. The provirus copy number obtained using real-time PCR with CoCoMo primers correlated closely with the result from serial dilution nested PCR. Because we aimed to use BLV-CoCoMo-qPCR to quantify cell-associated BLV provirus, we performed a parallel quantitation of the single-copy cellular gene BoLA-DRA. This measurement allowed adjustment for variations in amplification efficiency between samples. Using this strategy, we observed sufficient intra- and inter-assay reproducibility for the diagnosis of infected animals. The assay CV range was 0.0% to 20.5%, which was markedly better than that reported for quantification of HTLV-1 proviral load (8.2% to 31.4%  or 49% to 55% ). The high reproducibility of our assay enabled its use for the quantitation of proviral load during disease progression. The accuracy of BLV-CoCoMo-qPCR was also confirmed by sequencing analysis. We selected nine samples in which BLV provirus could be detected by BLV-CoCoMo-qPCR, but not by nested PCR, and sequenced the amplicons. Using this strategy, we confirmed that the nine samples were infected by BLV, and this highlights the ability of the BLV-CoCoMo-qPCR method to detect provirus in samples that were negative by nested PCR. In two of the nine samples, sequence mismatches were detected at the annealing region for the nested PCR primers, thereby suggesting an explanation for the failure of nested PCR to detect BLV in these samples.
The syncytia assay is a common strategy for detecting viable BLV virus particles . However, this method requires cell culture, is time consuming and often difficult, and also has low sensitivity. We tested whether the proviral copy number obtained with our assay correlated with the syncytium formation assay, since this would suggest that our assay could be used for diagnosis at the BLV infection stage. Syncytia formation correlated strongly with a proviral load of over 10,000 copies/105 cells, as calculated by BLV-CoCoMo-qPCR. In BLV-infected cattle with a low proviral load detected by BLV-CoCoMo-qPCR, syncytia formation could hardly be detected. Thus, BLV-CoCoMo-qPCR appears to be capable of correctly determining the level of BLV infection in animals with low viral loads.
The pre-leukemic phase of BLV infection, called as PL, is characterized by the expansion of infected surface immunoglobulin M-positive B-cells with proviral insertion at multiple sites. On the other hand, a unique integration site is characteristic of malignant of malignant B-cells found in BLV-infected individuals after the onset of overt leukemia/lymphoma [13, 23, 35]. According to this model, proviral load should increase during disease progression but this has not been formally demonstrated. Using BLV-CoCoMo-qPCR, we were able to detect an increase in proviral load during disease progression. This result strongly suggests that proviral load may be an excellent indicator for monitoring the progression of disease, but may also be useful for implementing segregation programs to minimize BLV transmission. Previous experiments also identified host factors or genetic backgrounds that correlate with disease progression. For example, tumor-associated c143 antigens have been identified that are serine phosphorylated specifically in cattle with EBL, and genetic polymorphisms in cancer-associated genes such as p53 and tumor necrosis factor (TNF) have also been linked with EBL [35–41]. Proviral load may also be a valuable measure for identifying markers that influence progression to the lymphoma stage. Our assay may be valuable for estimating the effectiveness of vaccination and may also be capable of detecting changes in proviral load in BLV-infected cattle with the TNF and BoLA alleles that have previously been associated with resistance or susceptibility to BLV-induced lymphoma. Finally, since our assay detected all BLV variants, the CoCoMo algorithm appears to be a useful tool for designing degenerate primers for the quantification of proviral loads of other retroviruses, including HTLV and HIV-1.
Using CoCoMo primers, we have developed a new quantitative real-time PCR method to measure the proviral load of known and novel BLV variants. Our method is highly specific, sensitive, quantitative and reproducible for detection of the BLV LTR region in infected animals. The method was effective in detecting BLV in cattle from a range of geographical locations, and detected BLV in a broader range of samples than the previously developed nested PCR. Finally, we have shown for the first time that the proviral load correlates well with the stage of disease progression.
Clinical samples, cell lines and DNA extraction
Blood samples were obtained from 117 healthy cattle with negative BLV serology, 163 BLV-infected cattle that were clinically and hematologically normal, 16 clinically normal BLV-infected cattle with PL, and 89 BLV-infected cattle with EBL. These cattle were all maintained in Japan. A further 116 cattle that were maintained in Bolivia, Peru, Chile and the U.S.A were included in the study. BLV-infected cattle were classified according to previously established criteria  and the genomic integrations of the BLV provirus. PBMCs were separated from blood by the method of Miyasaka and Trnka .
The BLV-infected B lymphoma cell line BLSC-KU-17  was maintained in Dulbecco's modified Eagle's medium (Life Technologies Japan, Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Sigma Aldrich Chemie Gmbh, Steinem, Germany), penicillin and streptomycin. CC81, a cat cell line transformed by mouse sarcoma virus, was maintained in RPMI 1640 medium (Sigma-Aldrich Co. Ltd., Ayrshire, UK) supplemented with 10% heat-inactivated FCS, penicillin and streptomycin.
Genomic DNA was extracted from (a) whole blood by the DNA Wizard Genomic DNA purification Kit (Promega, Madison, WI), (b) PBMCs by the procedure described by Hughes et al.  and (c) 40 μl of whole blood spotted on FTA elute cards (Whatman, Tokyo, Japan), using standard procedures.
Design of primers and probes
The 52 variants of individual BLV LTR sequences were selected from 356 BLV sequences in GenBank as shown in Additional files 1-3. The target sequences were subjected to a BLV LTR modified version of the CoCoMo-primer-design algorithm (http://www.geneknot.jp/cocomo; Endoh D, Mizutani T, Morikawa S Hamaguchi I, Sakai K, Takizawa K, Osa Y, Asakawa M, Kon Y, Hayashi M,: CoCoMo-Primers: a web server for designing degenerate primers for virus research. Submitted), which was developed for designing degenerate primers to detect multiple strains of viruses.
To detect specific PCR products, we used the TaqMan™probe system (Applied Biosystems, Tokyo, Japan), with probe sequences designed using Primer Express software, version 2.0 (Applied Biosystems).
To obtain pBLV-LTR/SK, which included a full-length LTR from BLV, we used PCR with the primers BLV-LTR/XhoI (5'-CCCGCTCGAGTGTATGAAAGATCATGCCGA-3'; positions 1 to 20) and BLV-LTRR/BamHI (5'-CGGGATCCTGTTTGCCGGTCTCTCCTGG-3'; positions 511 to 531) and genomic DNA extracted from BLSC-KU-17 cells as a template. PCR products were cloned into pBluescript II SK (+) (Stratagene, La Jolla, CA). The numbering of nucleotides corresponds to positions in the sequences determined by Derse et al. . To generate pBoLA-DRA/SK, which includes a full-length bovine DRA gene, we digested MR1 from the mammalian expression vector pCDM8  with Xba I. The Xba I-Xba I fragment including the MR1 sequence was then subcloned into pBluescript II SK (+) (Stratagene). Additional clones used included a BLV infectious clone, pBLV-IF ; a HTLV-1 infectious clone, pK30 ; a HIV-1 infectious clone, pNL4-3 ; a SIV infectious clone, SIVmac239/WT ; a hybrid MMTV provirus plasmid ; a M-MLV infectious clone, pL-4  and plasmids including pUC18 (Takara Bio Inc., Tokyo, Japan), pUC19 (Takara Bio Inc.), and pBR322 (Promega).
Calculation of copy number by the serial dilution method
pBLV-LTR/SK and pBoLA-DRA/SK were digested with Sca I and purified using a Sephadex G-50 column (GE Healthcare Japan, Tokyo, Japan). The genomic DNA was digested with Xho I in the presence of 5 mM spermidine for 24 h. The samples were diluted to serial ten-fold dilutions with TE buffer [10 mM Tris-HCl (pH 8.0) with 1 mM ethylenediamine tetraacetic acid ] on-ice. To avoid DNA adsorption to the microtubes, super smooth processed tubes (BM4015 Platinum super polypropylene; BM bio, Tokyo, Japan) were used for the preparation of template for the standard curve.
Limiting dilutions were performed for linearized pBLV-LTR/SK, pBoLA-DRA/SK and genomic DNA. Detection of the BLV LTR gene and the BoLA-DRA gene from plasmid DNA was performed by real-time PCR with the CoCoMo primers 6 and 81, or the primers DRA643 and DRA734, respectively. The BLV LTR gene was also detected in genomic DNA using nested PCR. At the dilution point at which amplification products were unable to be detected, PCRs were repeated 10 times and the frequency of negative results was calculated (f(x = 0)). The copy numbers of the target genes were calculated according to a Poisson distribution model: λ = -loge(f(x = 0)), where λ = average copy number of the target gene.
PCR conditions for candidate CoCoMo primer sets for the amplification of the BLV LTR
Touch-down PCR amplifications were carried out in a 20 μl volume of 1 × buffer for rTaq DNA Polymerase (TOYOBO, Tokyo, Japan) containing 1.0 unit rTaq, 0.2 mM dNTPs, 1.0 mM MgCl2, 500 nM of forward and reverse primers, and 1 μl of 1:1000 diluted BLV LTR amplicon, which had been amplified from genomic DNA by nested PCR. PCR amplification was performed with a TGRADIENT thermocycler (Biometra, Göttingen, Germany) according to the following program: an initial denaturation at 95°C for 10 min, followed by 40 cycles of 15 s at 95°C, 10 s at 60°C to 52°C (annealing temperature was gradually decreased from 60°C to 52°C, by 0.2°C every three cycles) and 10 s at 72°C. Five μl of PCR products was used for 2% agarose gel electrophoresis and amplification products were detected by ethidium bromide staining.
Melting curve analysis for evaluating PCR specificity
PCR amplifications took place in a total volume of 20 μl of 1 × LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics GmbH, Basel, Switzerland) containing 500 nM of each of the CoCoMo primers, 3 mM of MgCl2, and 30 ng of genomic DNA. PCR amplifications were performed with a Light Cycler 2.0 (Roche Diagnostics GmbH) according to the following program: an initial denaturation at 95°C for 10 min, followed by 75 cycles of 15 s at 95°C, 5 s at 65°C, and 9 s at 72°C. The melting process was monitored by fluorescence of the DNA-binding SYBR Green I dye for the detection of double-stranded DNA.
Detection of BLV LTR by nested PCR
The first PCR amplification was done using the primers BLTRF-YR (5'-TGTATGAAAGATCATGYCGRC-3' LTR 1-21) and BLTRR (5'- AATTGTTTGCCGGTCTCTC-3' LTR 515-533). The amplifications were carried out in a total volume of 20 μl of 1 × buffer for rTaq DNA Polymerase (TOYOBO) containing 250 nM of BLTRF-YR primer and BLTRR primer, 0.5 units of rTaq polymerase, 0.2 mM dNTPs, 2.5 mM MgCl2, and 30 ng of template DNA. PCR amplification was performed with a TGRADIENT thermocycler (Biometra) according to the following program: an initial denaturation at 94°C for 2 min, followed by 35 cycles of 30 sec at 94°C, 30 sec at 58°C and 30 s at 72°C, and a final cycle of 5 min at 72°C.
The second set of PCR amplifications was performed in a total volume of 20 μl of 1 × buffer for rTaq DNA Polymerase (TOYOBO) containing 250 nM of 256 primer and 453 primer (see below), 0.5 units of rTaq polymerase, 0.2 mM of dNTPs, 2.5 mM of MgCl2, and 1 μl of first-round PCR product. The oligonucleotide sequences used in the second PCR were 256 (5'-GAGCTCTCTTGCTCCCGAGAC-3', LTR 256-276) and 453 (5'-GAAACAAACGCGGGTGCAAGCCAG-3', LTR 430-454), and have been described previously . PCR amplification was performed with a TGRADIENT thermocycler (Biometra) according to the following program: an initial denaturation at 94°C for 2 min, followed by 35 cycles of 30 s at 94°C, 30 s at 58°C and 30 s at 72°C, and a final cycle of 5 min at 72°C. Five μl of PCR products was used for 2% agarose gel electrophoresis and the PCR products were detected by ethidium bromide staining.
Amplification, Cloning, and DNA sequencing of BLV LTR regions
To analyze the nucleotide sequence of samples that were positive by BLV-CoCoMo-qPCR but negative by nested PCR, genomic DNA from BLV-infected cattle was subjected to amplification by PCR using MightyAmp DNA polymerase Ver.2 (TAKARA), KOD plus Neo (TOYOBO), and TaqMan Universal Master Mix II system (AB). BLV LTR specific oligonucleotide primers BLTR56F (5'-AACGTCAGCTGCCAGAAA-3'), BLTR134F (5'-AAAATCCACACCCTGAGCTG-3'), CoCoMo6, CoCoMo81, and BLTR544R (5'- ACGAGCCCCCAATTGTTT-3') were designed by reference to the LTR regions of BLV proviral sequences. The PCR products were subcloned into pGEM-T Easy vector (Promega) by TA cloning and the nucleotide sequence was determined by cycle sequencing using standard procedures.
Syncytium formation assay
The assay was performed according to a previously described procedure [15, 22]. CC81 cells were grown for 72 h in a 6-cm-diameter dish and incubated with 4 × 105 PBMC from BLV-infected cattle in RPMI 1640 medium. Cells were fixed in May-Grunwald solution for 2 min and stained with Giemsa solution for 15 min. After washing with water, cells were examined under a light microscope. Cells containing more than five nuclei were counted as syncytia.
Statistical analysis was conducted using R 2.10.1 statistical computing software. For multiple testing, a pairwise t test was used for calculating pairwise comparisons between group levels with corrections. P-values of less than 0.05 were considered significant. Regression analysis was used to examine the correlation between the serial dilution method and the BLV-CoCoMo-qPCR method, and between the syncytium count and the provirus copy number.
We thank Dr. Shingo Katoh for useful discussion; Mr. Keisuke Fukumoto, Miss. Yuki Matsumoto, Dr. Taku Miyasaka, Dr. Mariluz Arainga-Ramirez, Dr. Naohiko Kobayashi, Dr. Tamako Matsuhashi, Mrs. Konomi Kuramochi, Mrs. Etsuko Saito, Dr. Jiyun Kim, Mr. Kazunori Yamada and Mr. Tomoyuki Murakami for excellent technical assistance, kind help and suggestions; and Dr. Misao Onuma, Dr. Guangai Xue, Dr. Verónica Gabriela de la Barra Díaz, Dr. Guillermo Giovambattista, Dr. Enrique J. Pofcher, Dr. Hermelinda Rivera-Geronimo, Dr. Hideki Saito, Dr. Tomas J. Acosta, Dr. Misao Kanemaki, Dr. Manuel L. Ortiz and Dr. Jorge Oltra for helpful sampling; We also thank Dr. Tomoyuki Miura, Dr. Akinori Ishimoto and Dr. Akio Adachi for kindly providing various provirus plasmids; We thank the NIH AIDS research and reference reagent program for kindly providing HTLV-1 provirus plasmid; We are grateful to the Support Unit for Bio-material Analysis, RIKEN BSI Research Resources Center, for help with sequence analysis. This work was supported by Grants-in-Aid for Scientific Research (A and B) and by a Grant from the Program for the Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry.
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