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Bovine leukemia virus DNA in human breast tissue.

Buehring GC, Shen HM, Jensen HM, Choi KY, Sun D, Nuovo G - Emerging Infect. Dis. (2014)

Bottom Line: 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.Further research is needed to determine whether BLV may play a direct role in human disease.

View Article: PubMed Central - PubMed

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.

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Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint immunoperoxidase reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb1Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).
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Figure 3: Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint immunoperoxidase reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb1Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).

Mentions: Nested IS-PCR was used to identify the cell type in which the PCR product was localized. Figure 3 shows IS-PCR results obtained by using tax primers for the BLV-negative human sample (no. 143), 1 of the BLV-positive samples (no. 010), the positive and negative cell line controls, and a BLV tax-positive bovine mammary gland sample. The site of the amplified BLV DNA was the secretory mammary epithelium, identified by an anatomic pathologist (H.M.J.). Table 5 summarizes the results for L-PCR and IS-PCR (tax) testing of the 6 tissue samples that were studied in depth.


Bovine leukemia virus DNA in human breast tissue.

Buehring GC, Shen HM, Jensen HM, Choi KY, Sun D, Nuovo G - Emerging Infect. Dis. (2014)

Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint immunoperoxidase reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb1Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).
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Related In: Results  -  Collection

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Figure 3: Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint immunoperoxidase reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb1Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).
Mentions: Nested IS-PCR was used to identify the cell type in which the PCR product was localized. Figure 3 shows IS-PCR results obtained by using tax primers for the BLV-negative human sample (no. 143), 1 of the BLV-positive samples (no. 010), the positive and negative cell line controls, and a BLV tax-positive bovine mammary gland sample. The site of the amplified BLV DNA was the secretory mammary epithelium, identified by an anatomic pathologist (H.M.J.). Table 5 summarizes the results for L-PCR and IS-PCR (tax) testing of the 6 tissue samples that were studied in depth.

Bottom Line: 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.Further research is needed to determine whether BLV may play a direct role in human disease.

View Article: PubMed Central - PubMed

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.

Show MeSH
Related in: MedlinePlus