Recently, we developed the BLV-CoCoMo-qPCR system to detect various BLV strains with broad geographical origins. Proviral load determined in this manner was found to correlate not only with BLV infection capacity as assessed by syncytium formation, but also with BLV-induced disease progression. The analyses described here show that the BLV-CoCoMo-qPCR method is a useful tool for evaluating BLV infection status. Conventional serological techniques, including AGID, PHA, and ELISA, are commonly used to diagnose BLV infection in Japan. Especially, the AGID test is currently a “golden standard” for determining BLV infection in Japan. We demonstrated that animals that were BLV-positive, as determined by the serological test (46.9% for PHA, 15.3% for AGID, and 62.3% for ELISA), were negative for proviral load, as determined by BLV-CoCoMo-qPCR (Table 2 and Figure 2). Furthermore, the sensitivity and reproducibility of BLV-CoCoMo-qPCR were greater than those of two previously developed real-time PCR methods (the TaqMan MGB assay developed by Lew et al. and the commercial TaKaRa cycleave PCR kit), using an infectious full-length molecular clone of BLV, pBLV-IF . Moreover, the proportion of 370 cattle that were positive for anti-BLV antibody by the three serological tests was partially correlated with the proviral load determined by BLV-CoCoMo-qPCR. This result was confirmed by the finding that the kinetics of the proviral load did not quite correlate with changes in anti-BLV antibody production in two cattle experimentally infected with BLV. Collectively, these results suggest that the quantitative measurement of proviral load by BLV-CoCoMo-qPCR is a useful for monitoring the spread of BLV. In addition, the results show that serological and genomic tests complement each other and result in correct detection of BLV-infected cattle.
Whereas the positive detection ratefor nested PCR correlated well with the proviral load determined by BLV-CoCoMo-qPCR, the rates of animals that were positive for anti-BLV antibody by the three serological test did not correlate with the proviral load (Figure 2A). This finding indicates an inconsistency between the proviral load determined by BLV-CoCoMo-qPCR and the detection of BLV infection by serological tests. High positive rates for each serotest were observed in animals that were negative in BLV-CoCoMo-qPCR (Table 2 and Figure 2A). One possible explanation is that SK577, which was experimentally infected with BLV, produced antibodies against BLV but had a very low copy number of BLV throughout the experimental period (Figure 3). We previously reported that BLV-infected cattle retain a full-length proviral genome throughout the course of the disease . Another in vivo dynamic study indicated that the turnover rate of infected cells is higher in BLV-infected sheep . Based on the present data and previous results, we speculate that BLV-infected cells that express viral genes are eliminated by a strong anti-viral immune response; however, this would allow the cells that escape host immunity to survive, resulting in persistence of the virus in those cells throughout lifespan of the animal. Alternatively, it may be that BLV does not accumulate only in the peripheral blood used for BLV-CoCoMo-qPCR, but also in organs. We observed that numerous cattle that were negative for anti-BLV antibody by each serotest (72 animals for PHA, 26 animals for AGID, and 2 animals for ELISA) were positive for the provirus as determined by BLV-CoCoMo-qPCR (Figure 2). This result indicates that it is difficult to detect BLV infection by using the serological test alone. BLV infections without the detection of BLV antibodies by serological tests have been observed previously [32–36]. Thus, this result showed that, when viral gene expression is very low in BLV-infected cells, the infected cells can escape the immune response and survive, without evoking an immune response by viral protein production.
An advantage of the BLV-CoCoMo-qPCR method is that it uses degenerate primers designed from 52 individual BLV LTR sequences identified from 356 BLV sequences in GenBank. It also uses the CoCoMo algorithm that was developed specifically for the detection of multiple viral species . It is possible that the degeneracy of the CoCoMo primers could be too high, which would reduce the concentration of primers specific for a particular target sequence and decrease the sensitivity of the assay. However, this issue did not arise in the present study: despite the use of degenerate primers, the sensitivity and reproducibility of BLV-CoCoMo-qPCR were greater than that of two previously developed real-time PCR methods (i.e., TaqMan MGB which was developed by Lew et al. and TaKaRa cycleave PCR) (Table 1 and Figure 1), as follow reasons. 1) To improve the sensitivity of our assay, we selected BLV-LTR (which is present at two copies per provirus) as the target of CoCoMo-qPCR. In contrast, TaqMan MGB developed by Lew et al. and TaKaRa cycleave PCR target the BLV pol and tax gene, respectively, which are present at only one copy per provirus. 2) The TaqMan probe was used to improve the sensitivity and specificity and to counter any drawbacks associated with high degeneracy. Importantly, the sequence of the BLV TaqMan probe, located between positions corresponding to two of the CoCoMo primers, was completely conserved among the 52 BLV variants. 3) A preliminary experiment demonstrated that the primer annealing regions of the TaqMan MGB assay developed by Lew et al. were variable in 10% of the 78 pol sequences selected from GenBank (on 2nd October, 2010). This finding indicates that it is difficult to detect all of these variants. By contrast, use of degenerate primers allows for the detection of BLV sequence variants, including those that arise from mutations. Indeed, we previously demonstrated that this method can detect various BLV strains of broad geographical origins, including Japan, Peru, Bolivia, Chile, and the U.S.A. .
In the present study, we found that the propagation of and immunoresponses to BLV were different in two cattle that carried different BoLA-DRB3 genotypes. This experiment may help direct future research into examining whether or not progression of BLV-induced diseases correlates with not only viral propagation, but also with host factors that are associated with the immune response. We previously reported that, in sheep experimentally infected with BLV, quantitative and/or qualitative aspects of the immunoresponse, production of neutralizing antibodies against BLV, and elimination of BLV depended on the particular allelic forms of the MHC class II molecules expressed by an individual and, in particular, on certain polymorphic amino acid residues in class II molecules [37, 38]. Additional studies are required to define the mechanism of association between BLV-induced disease progression and MHC polymorphism.
Using BLV-CoCoMo-qPCR, we previously found an increase in the proviral load during disease progression . This result strongly suggests that proviral load may be an excellent indicator for monitoring disease progression and for implementing segregation programs to minimize BLV transmission. One advantage of the proviral load measurement is that it can be used to follow the dynamics of BLV-infected cells in vivo. However, in the current study, proviral load was only determined in cell populations from peripheral blood. Because transformed phenotype of target lymphocytes for BLV is CD5+-B cells , it is easy to imagine that the proviral load in peripheral blood increases with disease progression. However, BLV can infect not only B cells, but also many other cell populations. It is still unknown which peripheral blood or organs maintain the BLV proliferation. In cattle harboring anti-BLV antibodies but lacking detectable BLV provirus in the blood, BLV may accumulate not only in the peripheral blood but also in organs. On the other hand, in cattle showing detectable BLV provirus in peripheral blood but lacking anti-BLV antibodies, BLV gene expression may be strongly suppressed. The BLV-CoCoMo-qPCR method can be used to investigate the mechanism by which BLV persists in vivo, by analyzing which organ is the key component in the maintenance of BLV proliferation.