Bovine Leukemia Virus DNA in Human Breast Tissue

Gertrude Case Buehring

, Hua Min Shen, Hanne M. Jensen, K. Yeon Choi1, Dejun Sun, and Gerard Nuovo

Author affiliations: University of California, Berkeley, California, USA (G.C. Buehring, H.M. Shen, K.Y. Choi, D. Sun); University of California Davis Medical Center, Sacramento, California, USA (H.M. Jensen); Ohio State University Comprehensive Cancer Center, Columbus, Ohio, USA (G. Nuovo)

Abstract

Bovine leukemia virus (BLV), a deltaretrovirus, causes B-cell leukemia/lymphoma in cattle and is prevalent in herds globally. A previous finding of antibodies against BLV in humans led us to examine the possibility of human infection with BLV. We focused on breast tissue because, in cattle, BLV DNA and protein have been found to be more abundant in mammary epithelium than in lymphocytes. In human breast tissue specimens, we identified BLV DNA by using nested liquid-phase PCR and DNA sequencing. Variations from the bovine reference sequence were infrequent and limited to base substitutions. In situ PCR and immunohistochemical testing localized BLV to the secretory epithelium of the breast. Our finding of BLV in human tissues indicates a risk for the acquisition and proliferation of this virus in humans. Further research is needed to determine whether BLV may play a direct role in human disease.

Bovine leukosis (B-cell leukemia/lymphoma), first described in 1871 in Lithuania, was believed to be an infectious disease because it spread through herds of cattle. In 1969, a virus isolated from cultured lymphocytes of cattle in an afflicted herd was identified as the agent of bovine leukosis (1). Since then, bovine leukemia virus (BLV) has been extensively investigated. It is a deltaretrovirus, closely related to human T-cell leukemia virus (HTLV) 1 (2), and has typical retroviral genome regions: LTR (long terminal repeat, promoter region); gag (group-specific antigen, capsid region); pol (polymerase, reverse transcription region, which synthesizes a DNA copy of the BLV RNA genome); and env (envelope). However, unlike other oncogenic retroviruses, deltaretroviruses have an additional region, tax (trans-activating region of the X gene), which has regulatory functions and is oncogenic to host cells. tax causes malignant transformation not through integration and insertional mutagenesis, as many retroviruses do, but by inhibition of DNA repair (base excision pathway) and trans-activating disruption of cellular growth control mechanisms (2).

BLV-infected cattle herds are found worldwide. In the United States, ≈38% of beef herds, 84% of all dairy herds, and 100% of large-scale dairy operation herds are infected (3,4). On average, clinical leukosis develops in <5% of these cattle, which are excluded from the market as a result (1), but BLV-infected lymphocytes are also found in the blood and milk of subclinically infected cows (2). Concerns that this virus might infect humans through exposure to food products from subclinically infected animals prompted 10 studies that used what were then (1975–1979) state-of-the-art immunologic methods to test serum samples from a collective total of 1,761 humans, including cancer patients, farm workers, and veterinarians (5). In these studies no antibodies against BLV were detected, prompting Burridge to conclude in his review article, “There is no epidemiological or serological evidence from human studies to indicate that BLV can infect man” (5).

The advent of immunoblotting, ≈100 times more sensitive than techniques of the 1970s (6), enabled the detection of antibodies reactive with recombinant purified BLV p24 capsid protein in serum samples from 39% of 257 self-selected human volunteers (7). This study could not determine whether the antibodies were a response to infection or merely to heat-inactivated BLV consumed in food products. However, injection of sheep with raw milk from BLV-positive cows stimulated antibody production, whereas injection with pasteurized control milk did not (8,9). The finding of antibodies to BLV in humans prompted us to investigate human tissues for evidence of infection with BLV by using liquid-phase PCR (L-PCR), sequencing, in situ PCR, and immunohistochemical (IHC) testing. We focused on breast tissue because, in cattle, BLV DNA and p24 were detected in mammary tissue, whereas only BLV DNA was detected in lymphocytes (10).