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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210141	2/5	Homo sapiens	human umbilical vein endothelial cells	IAF Ultrafiltratrion (d)(U)C DG	Zhao F	2023	89%
Study summary Full title Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement. All authors Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G Journal EMBO J Abstract Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide) EV-METRIC 89% (99th 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 ZIKV infected cells Focus vesicles extracellular vesicle 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 Immunoaffinity capture (non-commercial) Ultrafiltratrion (Differential) (ultra)centrifugation Density gradient Protein markers EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9 non-EV: Capsid/ E/ LC3/ Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human umbilical vein endothelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 2,00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0% Highest density fraction 80% Sample volume (mL) 1 Orientation Bottom-up Rotor type P55ST Speed (g) 250000 Duration (min) 1080 Fraction volume (mL) 0,3 Ultra filtration Cut-off size (kDa) 100 kDa Membrane type Regenerated cellulose Immunoaffinity capture Selected surface protein(s) CD9 Other Name other separation method Ultrafiltratrion Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ HSP70/ Alix/ CD81 Detected contaminants Capsid/ E Not detected contaminants LC3/ Calnexin Characterization: RNA analysis RNA analysis Type RT(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM/ Cryo-EM Image type Wide-field Report size (nm) 100

	EV210141	3/5	Homo sapiens	human umbilical vein endothelial cells	Ultrafiltratrion (d)(U)C DG	Zhao F	2023	89%
Study summary Full title Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement. All authors Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G Journal EMBO J Abstract Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide) EV-METRIC 89% (99th 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 ZIKV infected cells Focus vesicles extracellular vesicle 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 Ultrafiltratrion (Differential) (ultra)centrifugation Density gradient Protein markers EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9 non-EV: Capsid/ E/ LC3/ Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human umbilical vein endothelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 2,00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0% Highest density fraction 80% Sample volume (mL) 1 Orientation Bottom-up Rotor type P55ST Speed (g) 250000 Duration (min) 1080 Fraction volume (mL) 0,3 Ultra filtration Cut-off size (kDa) 100 kDa Membrane type Regenerated cellulose Other Name other separation method Ultrafiltratrion Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ HSP70/ Alix/ CD81 Detected contaminants Capsid/ E Not detected contaminants LC3/ Calnexin Characterization: RNA analysis RNA analysis Type RT(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 100

	EV210141	4/5	Homo sapiens	human umbilical vein endothelial cells	Ultrafiltratrion (d)(U)C DG	Zhao F	2023	75%
Study summary Full title Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement. All authors Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G Journal EMBO J Abstract Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide) EV-METRIC 75% (96th 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 ZIKV infected cells Focus vesicles extracellular vesicle 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 Ultrafiltratrion (Differential) (ultra)centrifugation Density gradient Protein markers EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9 non-EV: Capsid/ E Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human umbilical vein endothelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 2,00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0% Highest density fraction 80% Sample volume (mL) 1 Orientation Bottom-up Rotor type P55ST Speed (g) 250000 Duration (min) 1080 Fraction volume (mL) 0,3 Ultra filtration Cut-off size (kDa) 100 kDa Membrane type Regenerated cellulose Other Name other separation method Ultrafiltratrion Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ HSP70/ Syntenin Detected contaminants Capsid/ E Characterization: RNA analysis RNA analysis Type RT(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

	EV210141	1/5	Homo sapiens	human umbilical vein endothelial cells	IAF Ultrafiltratrion (d)(U)C	Zhao F	2023	67%
Study summary Full title Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement. All authors Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G Journal EMBO J Abstract Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide) EV-METRIC 67% (94th 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 Control condition Focus vesicles extracellular vesicle 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 Immunoaffinity capture (non-commercial) Ultrafiltratrion (Differential) (ultra)centrifugation Protein markers EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9 non-EV: Capsid/ E/ LC3/ Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human umbilical vein endothelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 2,00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 100 kDa Membrane type Regenerated cellulose Immunoaffinity capture Selected surface protein(s) CD9 Other Name other separation method Ultrafiltratrion Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ HSP70/ Alix/ CD81 Detected contaminants Capsid/ E Not detected contaminants LC3/ Calnexin Characterization: RNA analysis RNA analysis Type RT(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM/ Cryo-EM Image type Wide-field Report size (nm) 100

	EV210141	5/5	Homo sapiens	Vero 6	IAF Ultrafiltratrion (d)(U)C	Zhao F	2023	44%
Study summary Full title Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement. All authors Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G Journal EMBO J Abstract Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide) EV-METRIC 44% (84th 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 Dengue virus infected cells Focus vesicles extracellular vesicle 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 Immunoaffinity capture (non-commercial) Ultrafiltratrion (Differential) (ultra)centrifugation Protein markers EV: CD9/ Syntenin non-EV: E Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Vero 6 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 2,00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 100 kDa Membrane type Regenerated cellulose Immunoaffinity capture Selected surface protein(s) CD9 Other Name other separation method Ultrafiltratrion Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ Syntenin Detected contaminants E Characterization: RNA analysis RNA analysis Type RT(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

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EV-TRACK ID	EV210141
species	Homo sapiens
sample type	Cell culture
cell type	human umbilical vein endothelial cells	human umbilical vein endothelial cells	human umbilical vein endothelial cells	human umbilical vein endothelial cells	Vero 6
condition	ZIKV infected cells	ZIKV infected cells	ZIKV infected cells	Control condition	Dengue virus infected cells
separation protocol	IAF capture (non-commercial)/ Ultrafiltratrion/ dUC/ Density gradient	Ultrafiltratrion/ dUC/ Density gradient	Ultrafiltratrion/ dUC/ Density gradient	IAF capture (non-commercial)/ Ultrafiltratrion/ dUC	IAF capture (non-commercial)/ Ultrafiltratrion/ dUC
Exp. nr.	2	3	4	1	5
EV-METRIC %	89	89	75	67	44