Single nucleotide polymorphisms in the bovine MHC region of Japanese Black cattle are associated with bovine leukemia virus proviral load
© The Author(s) 2017
Received: 10 January 2017
Accepted: 21 March 2017
Published: 4 April 2017
Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, a malignant B cell lymphoma that has spread worldwide and causes serious problems for the cattle industry. The BLV proviral load, which represents the BLV genome integrated into host genome, is a useful index for estimating disease progression and transmission risk. Here, we conducted a genome-wide association study to identify single nucleotide polymorphisms (SNPs) associated with BLV proviral load in Japanese Black cattle. The study examined 93 cattle with a high proviral load and 266 with a low proviral load. Three SNPs showed a significant association with proviral load. One SNP was detected in the CNTN3 gene on chromosome 22, and two (which were not in linkage disequilibrium) were detected in the bovine major histocompatibility complex region on chromosome 23. These results suggest that polymorphisms in the major histocompatibility complex region affect proviral load. This is the first report to detect SNPs associated with BLV proviral load in Japanese Black cattle using whole genome association study, and understanding host factors may provide important clues for controlling the spread of BLV in Japanese Black cattle.
Bovine leukemia virus (BLV), which infects cattle worldwide [1–6], belongs to the family Retroviridae (genus Deltaretrovirus), together with human T cell leukemia virus types 1 and 2 (HTLV-1 and -2) . Historically, the economic losses caused by BLV infection were thought to be related only to the onset of bovine leukosis, which occurs in only 1–5% of BLV-infected cows within 5 years post-infection . However, recent reports show that BLV infection also reduces milk production [6, 8–11] and causes a high incidence of infectious disease  and reproductive inefficiency, resulting in high culling rates ; thus BLV eradication is of utmost importance.
Previous studies show that the proviral load is an important index for estimating the stage of BLV infection because it is associated with disease progression [14–16], lymphocyte count , viral biokinetics , and virus shedding into saliva and nasal secretions . Indeed, one study shows that cattle with a low proviral load are not a source of BLV transmission . Therefore, determining host factors associated with an increased proviral load is important if we are to develop eradication programs for BLV.
Studies of BLV-associated host factors identified polymorphisms within the bovine major histocompatibility complex (MHC) (BoLA) [21–29]. Recently, Miyasaka et al. revealed that polymorphisms within BoLA class II haplotypes were strongly associated with BLV proviral load in Japanese Black cattle, the main breed of beef cattle in Japan, but less so with that in European breeds . However, no group has undertaken a genome-wide association study (GWAS) to identify such host factors.
SNPs showing a significant association with BLV proviral load
Reference cluster IDb
Minor alleled (case)
Minor alleled (control)
1.91 × 10−7
1.91 × 10−7
5.37 × 10−7
Genes within or near these regions were then analyzed using the UMD3.1 genome assembly tool. Hapmap57616-rs29026690 (27,421,348 bp on BTA23) was located between ENSBTAG00000000580 and ABHD16A (ENSBTAG00000000578) (Additional file 1: Table S1), whereas ARS-BFGL-NGS-113235 (28,223,274 bp on BTA23) was located between the 4th and 5th exons of PRR3 (ENSBTAG00000006914). These SNPs reside within the BoLA class III and class I regions, respectively (Figs. 2b, 3; Additional file 1: Table S1). Therefore, the gene density was much higher than that in other areas of the genome, and a number of candidate genes that could be used to estimate proviral load were present around the detected SNPs . Hapmap33580-BTA-136506 was located on the centromeric side of BTA22, at a distance of 6.5 kb from the CONTACTIN3 (CNTN3) gene (Table 1; Additional file 1: Table S1, Additional file 2: Fig. S1).
To the best of our knowledge, this is the first report to detect SNPs associated with BLV proviral load in Japanese Black cattle using GWAS. Two of the identified SNPs were located in the BoLA region. We found it interesting that these two SNPs were located within the class III and class I regions because a previous study reported involvement of only class II genes . The genome reference sequences for the BoLA region have many gaps, mainly because class I genes were difficult to genotype, making associations with class I genes difficult to determine. Target resequencing of high density SNPs across the MHC region using a next generation sequencer should be undertaken to confirm which genes are truly responsible for regulating the proviral load. Our result showed that the MHC polymorphism is important factor for proviral load. The reason why MHC polymorphisms were associated with proviral load is the polymorphism of classical MHC directly associate with antigen presentation and the difference of antigen presentation in each allele leads to the immunological difference in each host.
Taken together, the results described herein show that MHC genotyping of class III and class I alleles can identify cows with a low proviral load. In the farm with high infection rate, eliminating high proviral load cow is an effective way for eradicating BLV because proviral load is major risk factor for transmitting BLV to other host . Therefore, farmer should frequently check the proviral load because the proviral load is variable, although it is not cost-effective. Taken together with the information of our finding 3 SNPs and our previously report about resistant BoLA class II allele , we can identify the BLV resistant cow. It will be helpful to develop a low cost method of eradicating BLV from farms because we can reduce the frequently measurement of proviral load.
Study conception and design: YA. Data acquisition, analysis, and interpretation: SS, MP and ST. Contribution of reagents/materials/analysis tools: YA and YS. Drafting and revising the manuscript: ST, YA, and SS. Approval of the final version: ST, SS, MP, YS, and YA. All authors read and approved the final manuscript.
We thank the Support Unit at the Biomaterial Analysis, RIKEN BSI Research Resources Center, for help with sequence analysis.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets analyzed herein are available from the corresponding author upon request.
Ethics approval and consent to participate
All protocols for the collection of blood samples were reviewed and approved by the Shirakawa Institute of Animal Genetics Committee on Animal Research (H21-2).
The study was supported by Grants-in-Aid for Scientific Research (A and C) from the Japan Society for the Promotion of Science (JSPS) (Grant Nos. 18255013, 25450405), by a grant from the Integration Research for Agriculture and Interdisciplinary Fields in Japan (Grant No. 14538311). This research was also supported by grants from the Project of the NARO Bio-oriented Technology Research Advancement Institution (Grant No. 16817983) (the special scheme project on regional developing strategy).
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