© 2016 The Authors. Enzootic bovine leucosis is caused by bovine leukemia virus (BLV) infection, which is highly prevalent in several regions of the world and significantly impacts the livestock industry. In BLV infection, the proviral load in the blood reflects disease progression. Although the BLV genome is highly conserved among retroviruses, genetic variation has been reported. However, the relationship between proviral load and genetic variation is poorly understood. In this study, we investigated the changes in proviral load in BLV-infected cattle in Japan and then identified and analysed a BLV strain pvAF967 that had a static proviral load. First, examining the proviral load in the aleukaemic cattle in 2014 and 2015, cow AF967 showed a static proviral load, while the other cows showed significant increases in proviral load. Sequencing the provirus in cow AF967 showed a deletion of 12 nt located in the G4 gene. An in vitro assay system using BLV molecular clone was set up to evaluate viral replication and production. In this in vitro assay, the deletion mutation in the G4 gene resulted in a significant decrease in viral replication and production. In addition, we showed that the deletion mutation did not affect the viral transcriptional activity of Tax protein, which is also important for virus replication. The emergence of strain pvAF967 that showed a static proviral load, combined with other retrovirus evolutionary traits, suggests that some BLV strains may have evolved to be symbiotic with cattle.