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. 2022 May 28;14(6):1182.
doi: 10.3390/v14061182.

Comparative Evaluation of Three Commercial Quantitative Real-Time PCRs Used in Japan for Bovine Leukemia Virus

Syuji Yoneyama  1 , Sota Kobayashi  2 , Towa Matsunaga  3 , Kaoru Tonosaki  4 , Dongze Leng  1 , Yusuke Sakai  1 , Shinji Yamada  1   3 , Atsushi Kimura  3 , Toshihiro Ichijo  3 , Hirokazu Hikono  5 , Kenji Murakami  1   3
Affiliations

Comparative Evaluation of Three Commercial Quantitative Real-Time PCRs Used in Japan for Bovine Leukemia Virus

Syuji Yoneyama et al. Viruses. .

Abstract

Bovine leukemia virus (BLV) is an oncogenic virus belonging to the genus Deltaretrovirus and is the causative agent of enzootic bovine leukosis. Proviral load (PVL) determined by real-time quantitative PCR (qPCR) is now widely used as an indicator of not only BLV infection, but also BLV disease progression. To interpret PVLs determined by different qPCRs used in Japan, we compared a chimeric cycling probe-based qPCR, CY415, targeting the BLV tax region; a TaqMan probe-based qPCR, RC202, targeting the BLV pol region; and a TaqMan probe-based qPCR, CoCoMo, targeting the BLV long terminal repeat (LTR) region. Whole-blood samples collected from 317 naturally BLV-infected cattle (165 Holstein-Friesian and 152 Japanese Black) and tumor tissue samples collected from 32 cattle at a meat inspection center were used. The PVLs determined by each qPCR were strongly correlated. However, the PVL and the proportion of BLV-infected cells determined by RC202 or CoCoMo were significantly higher than those determined by CY415. Genetic analysis of three tumor tissue samples revealed that LTR region mutations or a deletion affected the PVL determined by CoCoMo. These results suggest that the TaqMan-based RC202 or CoCoMo qPCR is better than CY415 for BLV PVL analysis. However, qPCR target region mutations were not rare in tumors and could hamper PVL analysis by using qPCR.

Keywords: BLV; bovine leukemia virus; commercial kit; provirus load; qPCR.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Bovine leukemia virus (BLV) proviral load (PVL) in whole-blood samples, as determined by different qPCRs. (A) BLV PVL. Differences between values obtained were assessed by using Kruskal-Wallis tests with Bonferroni’s multiple comparison as an ad hoc test. Horizontal bars indicate median values. ****, p < 0.0001. (B–D) Correlations between PVLs determined by each qPCR. Spearman’s correlation coefficient (ρ) and the levels of significance (P) regarding values are indicated in the graphs. Diagonal lines are regression lines.
Figure 2
Figure 2
Proportions of bovine leukemia virus (BLV)-infected cells in whole-blood samples, as determined by different qPCRs. (A) Proportion of BLV-infected cells. Differences between values obtained were assessed by using Kruskal-Wallis tests with Bonferroni’s multiple comparison as an ad hoc test. Horizontal bars indicate median values. ****, p < 0.0001). (B–D) Correlations between proviral load (PVL) and the proportion of BLV-infected cells determined by each qPCR. Spearman’s correlation coefficient (ρ) and levels of significance (P) regarding values are indicated in the graphs. Diagonal lines are regression lines.
Figure 3
Figure 3
Comparison of proviral load (PVL) and the proportion of bovine leukemia virus (BLV)-infected cells between Holstein–Friesian (HF) cattle and Japanese Black (JB) cattle. (A) BLV PVL determined by different qPCRs. (B) Proportion of BLV-infected cells determined by different qPCRs. Differences between values obtained were assessed by using the Mann–Whitney U-test (***, p < 0.001; ****; p < 0.0001).
Figure 4
Figure 4
Bovine leukemia virus (BLV) proviral load (PVL) in tumor tissue samples, as determined by different qPCRs. (A) BLV PVL. Differences between values obtained were assessed by using Kruskal–Wallis tests with Bonferroni’s multiple comparison as an ad hoc test. Horizontal bars indicate median values. ****, p < 0.0001. (B–D) Correlations between PVLs determined by each qPCR. Spearman’s correlation coefficient (ρ) and levels of significance (P) regarding values are indicated in the graphs. Diagonal lines are regression lines.
Figure 5
Figure 5
Proportions of bovine leukemia virus (BLV)-infected cells in tumor tissue samples, as determined by different qPCRs. (A) Proportion of BLV-infected cells. Differences between values obtained were assessed by using Kruskal–Wallis tests with Bonferroni’s multiple comparison as an ad hoc test. Horizontal bars indicate median values. ***, p < 0.001. (B–D) Correlation between proviral load (PVL) and the proportion of BLV-infected cells determined by each qPCR. Spearman’s correlation coefficient (ρ) and levels of significance (P) regarding values are indicated in the graphs. Diagonal lines are regression lines.
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