No evidence of bovine leukemia virus proviral DNA and antibodies in human specimens from Japan

Abstract

Background: The potential risk and association of bovine leukemia virus (BLV) with human remains controversial as it has been reported to be both positive and negative in human breast cancer and blood samples. Therefore, establishing the presence of BLV in comprehensive human clinical samples in different geographical locations is essential.

Result: In this study, we examined the presence of BLV proviral DNA in human blood and breast cancer tissue specimens from Japan. PCR analysis of BLV provirus in 97 Japanese human blood samples and 23 breast cancer tissues showed negative result for all samples tested using long-fragment PCR and highly-sensitive short-fragment PCR amplification. No IgG and IgM antibodies were detected in any of the 97 human serum samples using BLV gp51 and p24 indirect ELISA test. Western blot analysis also showed negative result for IgG and IgM antibodies in all tested human serum samples.

Conclusion: Our results indicate that Japanese human specimens including 97 human blood, 23 breast cancer tissues, and 97 serum samples were negative for BLV.

Keywords: Antibody detection; Bovine leukemia virus (BLV); Japanese breast cancers; Japanese human blood; Japanese human sera; PCR.

Fig. 1

Detection of BLV provirus in human blood and breast cancer tissue samples by long- fragment PCR. A Schematic presentation of PCR amplification of larger fragment of BLV proviral gene in human samples. The Numbers on the two sides of the double arrow lines indicate the beginning and ending of the fragment amplified by PCR. The number under the double arrow line shows PCR ID and the length of each PCR product. B Determination of long PCR (LP) sensitivity. Positive control (pc) FLK-BLV DNA were diluted to obtain BLV copy numbers of 100, 10, 5 and 1 copy in each PCR reaction. Distilled water was used as negative control (nc). M indicates molecular weight marker (5`LTR and 3`LTR: 100 bp DNA ladder (MIXELL Inc, Hiroshima, Japan); LP-1 ~ LP-6: 1 kb DNA ladder). C Summary of PCR amplification of long-fragments of BLV genome. *The quality of DNA samples extracted from human samples were assessed by PCR amplification of human KRAS gene

Fig. 2

Detection of BLV provirus in human blood and breast cancer tissue samples by short-fragment PCR. A Schematic representation of PCR amplification of short region of proviral gene in human samples. Numbers on the two sides of double arrow lines indicate the beginning and ending of the fragment amplified by PCR. The number under the double arrow line indicates the BLV proviral gene (LTR; gag; pol; env; tax and tax-3`LTR). B Electrophoresis result of PCR products. Each sample was tested in triplicate. Positive control (pc) DNA obtained from FLK-BLV cells was adjusted to BLV copy numbers of 100, 10, 5 and 1 copy in each PCR and amplified in duplicated. Distilled water was used as the negative control (nc). M indicates molecular weight marker (100 bp DNA ladder, (MIXELL Inc, Hiroshima, Japan)). C Summary of BVL detection in human samples by short range PCR. *The quality of DNA samples extracted from human samples were assessed by PCR amplification of human KRAS gene

Fig. 3

Western blot analysis to test the anti-BLV reactivity of human serum. Human serum samples were used as primary antibody. M indicates protein molecular weight marker; + indicates FLK-BLV cell lysate applied on the SDS-PAGE; − indicates BLV-free FLK cell lysate applied on 12% SDS-PAGE; EBL cattle indicate serum obtained from BLV-infected with EBL; BLV (−) cattle indicates serum from cattle without BLV infection; No 1st Ab control indicates that membrane was incubated with 5% skim milk in PBS without any primary antibody