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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130071	2/2	Bos bovis	Milk	(d)(U)C DG Filtration	Yamada T	2013	33%
Study summary Full title Cell Infectivity in relation to bovine leukemia virus gp51 and p24 in bovine milk exosomes All authors Yamada T, Shigemura H, Ishiguro N, Inoshima Y Journal PLoS One Abstract Exosomes are small membranous microvesicles (40-100 nm in diameter) and are extracellularly released (show more...)Exosomes are small membranous microvesicles (40-100 nm in diameter) and are extracellularly released from a wide variety of cells. Exosomes contain microRNA, mRNA, and cellular proteins, which are delivered into recipient cells via these exosomes, and play a role in intercellular communication. In bovine leukemia virus (BLV) infection of cattle, although it is thought to be a minor route of infection, BLV can be transmitted to calves via milk. Here, we investigated the association between exosomes and BLV in bovine milk. BLV structural proteins, gp51 (Env) and p24 (Gag), were detected in bovine milk exosomes from BLV-infected cattle by Western blot analysis. In cells inoculated with these milk exosomes, BLV DNA was not detected during three serial passages by nested PCR. Purification of exosomes from persistently BLV-infected cells was achieved by immuno-magnetic separation using an antibody against exosomes coupled to magnetic beads. Consistently, BLV gp51 and p24 proteins were detected in purified exosomes. Moreover, reverse transcriptase activity was observed in purified exosomes, meaning that exosomes also contain viral enzyme. However, BLV DNA was not detected in serially passaged cells after inoculation of purified exosomes, indicating that exosomes carrying BLV proteins appeared to be not infectious. These results suggest that BLV proteins are released with milk exosomes and could be transferred into recipient cells of calves via milk exosomes as an alternative route not requiring virus infection. Moreover it is also possible that bovine milk exosomes play a role in clearance of BLV proteins from infected cells. (hide) EV-METRIC 33% (53rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: MFGE8 non-EV: Proteomics no EV density (g/ml) 1.09-1.19 Show all info Study aim Omics Sample Species Bos bovis Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins MFGE8 ELISA Antibody details provided? No Detected EV-associated proteins MFGE8

	EV130071	1/2	Bos bovis	NAY	(d)(U)C Filtration IAF	Yamada T	2013	11%
Study summary Full title Cell Infectivity in relation to bovine leukemia virus gp51 and p24 in bovine milk exosomes All authors Yamada T, Shigemura H, Ishiguro N, Inoshima Y Journal PLoS One Abstract Exosomes are small membranous microvesicles (40-100 nm in diameter) and are extracellularly released (show more...)Exosomes are small membranous microvesicles (40-100 nm in diameter) and are extracellularly released from a wide variety of cells. Exosomes contain microRNA, mRNA, and cellular proteins, which are delivered into recipient cells via these exosomes, and play a role in intercellular communication. In bovine leukemia virus (BLV) infection of cattle, although it is thought to be a minor route of infection, BLV can be transmitted to calves via milk. Here, we investigated the association between exosomes and BLV in bovine milk. BLV structural proteins, gp51 (Env) and p24 (Gag), were detected in bovine milk exosomes from BLV-infected cattle by Western blot analysis. In cells inoculated with these milk exosomes, BLV DNA was not detected during three serial passages by nested PCR. Purification of exosomes from persistently BLV-infected cells was achieved by immuno-magnetic separation using an antibody against exosomes coupled to magnetic beads. Consistently, BLV gp51 and p24 proteins were detected in purified exosomes. Moreover, reverse transcriptase activity was observed in purified exosomes, meaning that exosomes also contain viral enzyme. However, BLV DNA was not detected in serially passaged cells after inoculation of purified exosomes, indicating that exosomes carrying BLV proteins appeared to be not infectious. These results suggest that BLV proteins are released with milk exosomes and could be transferred into recipient cells of calves via milk exosomes as an alternative route not requiring virus infection. Moreover it is also possible that bovine milk exosomes play a role in clearance of BLV proteins from infected cells. (hide) EV-METRIC 11% (30th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration IAF Protein markers EV: Flotilin1/ MFGE8 non-EV: Proteomics no Show all info Study aim Omics Sample Species Bos bovis Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) CD9 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Flotilin1/ MFGE8 ELISA Antibody details provided? No Detected EV-associated proteins MFGE8 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV130071
species	Bos bovis
sample type	Milk	Cell culture
cell type	NA	NAY
medium		serum free
condition	NAY	NAY
separation protocol	(d)(U)C DG Filtration	(d)(U)C Filtration IAF
Exp. nr.	2	1
EV-METRIC %	33	11