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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV240164	2/5	Homo sapiens	HeLa	(d)(U)C DG	Xiong J	2025	56%
Study summary Full title Extracellular vesicles promote the infection and pathogenicity of Japanese encephalitis virus. All authors Xiong J, Yang L, Nan X, Zhu S, Yan M, Xiang S, Zhang L, Li Q, Yang C, Wang X, Wei N, Chen H, Si Y, Cao S, Ye J Journal J Extracell Vesicles Abstract Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to (show more...)Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to public health. Currently, there is no specific therapeutic agent available for JEV infection, primarily due to the complexity of its infection mechanism and pathogenesis. Extracellular vesicles (EVs) have been known to play an important role in viral infection, but their specific functions in JEV infection remain unknown. Here, ultracentrifugation in combination with density gradient centrifugation was conducted to purify EVs from JEV-infected cells. The purified EVs were found to be infectious, with virions observed inside the EVs. Furthermore, our study showed the formation process of virion-containing EVs both in vitro and in vivo, which involved the fusion of multivesicular bodies with the cell membrane, leading to the release of virion-containing intraluminal vesicles into the extracellular space. Further studies revealed that EVs played a crucial role in JEV propagation by facilitating viral entry and assembly-release. Furthermore, EVs assisted JEV in evading the neutralizing antibodies and promoted viral capability to cross the blood-brain and placental barriers. Moreover, in vivo experiments demonstrated that EVs were beneficial for JEV infection and pathogenicity. Taken together, our findings highlight the significant contribution of EVs in JEV infection and provide valuable insights into JEV pathogenesis. (hide) EV-METRIC 56% (89th 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 Japanese encephalitis virus-infected 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 (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD63/ CD81 non-EV: Calnexin Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biomarker/Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS 4h at 150,000g Cell viability (%) 90 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 Swinging bucket Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 20% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 3.5 Orientation Top-down Speed (g) 100,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ CD81 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 150-300 EM EM-type Transmission-EM Image type Wide-field

	EV240164	3/5	Sus scrofa domesticus	PIEC	(d)(U)C DG	Xiong J	2025	56%
Study summary Full title Extracellular vesicles promote the infection and pathogenicity of Japanese encephalitis virus. All authors Xiong J, Yang L, Nan X, Zhu S, Yan M, Xiang S, Zhang L, Li Q, Yang C, Wang X, Wei N, Chen H, Si Y, Cao S, Ye J Journal J Extracell Vesicles Abstract Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to (show more...)Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to public health. Currently, there is no specific therapeutic agent available for JEV infection, primarily due to the complexity of its infection mechanism and pathogenesis. Extracellular vesicles (EVs) have been known to play an important role in viral infection, but their specific functions in JEV infection remain unknown. Here, ultracentrifugation in combination with density gradient centrifugation was conducted to purify EVs from JEV-infected cells. The purified EVs were found to be infectious, with virions observed inside the EVs. Furthermore, our study showed the formation process of virion-containing EVs both in vitro and in vivo, which involved the fusion of multivesicular bodies with the cell membrane, leading to the release of virion-containing intraluminal vesicles into the extracellular space. Further studies revealed that EVs played a crucial role in JEV propagation by facilitating viral entry and assembly-release. Furthermore, EVs assisted JEV in evading the neutralizing antibodies and promoted viral capability to cross the blood-brain and placental barriers. Moreover, in vivo experiments demonstrated that EVs were beneficial for JEV infection and pathogenicity. Taken together, our findings highlight the significant contribution of EVs in JEV infection and provide valuable insights into JEV pathogenesis. (hide) EV-METRIC 56% (89th 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 Japanese encephalitis virus-infected 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 (Differential) (ultra)centrifugation Density gradient Protein markers EV: TSG101/ CD81 non-EV: Calnexin Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biomarker/Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Cell culture supernatant EV-producing cells PIEC EV-harvesting Medium EV-depleted medium Preparation of EDS 4h at 150,000g Cell viability (%) 80 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 Swinging bucket Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 20% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 3.5 Orientation Top-down Speed (g) 100,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins TSG101/ CD81 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 150-300 EM EM-type Transmission-EM Image type Wide-field

	EV240164	4/5	Mesocricetus auratus	BHK-21	(d)(U)C DG	Xiong J	2025	56%
Study summary Full title Extracellular vesicles promote the infection and pathogenicity of Japanese encephalitis virus. All authors Xiong J, Yang L, Nan X, Zhu S, Yan M, Xiang S, Zhang L, Li Q, Yang C, Wang X, Wei N, Chen H, Si Y, Cao S, Ye J Journal J Extracell Vesicles Abstract Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to (show more...)Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to public health. Currently, there is no specific therapeutic agent available for JEV infection, primarily due to the complexity of its infection mechanism and pathogenesis. Extracellular vesicles (EVs) have been known to play an important role in viral infection, but their specific functions in JEV infection remain unknown. Here, ultracentrifugation in combination with density gradient centrifugation was conducted to purify EVs from JEV-infected cells. The purified EVs were found to be infectious, with virions observed inside the EVs. Furthermore, our study showed the formation process of virion-containing EVs both in vitro and in vivo, which involved the fusion of multivesicular bodies with the cell membrane, leading to the release of virion-containing intraluminal vesicles into the extracellular space. Further studies revealed that EVs played a crucial role in JEV propagation by facilitating viral entry and assembly-release. Furthermore, EVs assisted JEV in evading the neutralizing antibodies and promoted viral capability to cross the blood-brain and placental barriers. Moreover, in vivo experiments demonstrated that EVs were beneficial for JEV infection and pathogenicity. Taken together, our findings highlight the significant contribution of EVs in JEV infection and provide valuable insights into JEV pathogenesis. (hide) EV-METRIC 56% (89th 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 Japanese encephalitis virus-infected 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 (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD81/ Alix non-EV: Calnexin Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biomarker/Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Mesocricetus auratus Sample Type Cell culture supernatant EV-producing cells BHK-21 EV-harvesting Medium EV-depleted medium Preparation of EDS 4h at 150,000g Cell viability (%) 80 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 Swinging bucket Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 20% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 3.5 Orientation Top-down Speed (g) 100,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD81/ Alix Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 150-300 EM EM-type Transmission-EM Image type Wide-field

	EV240164	5/5	Mesocricetus auratus	BHK-21	(d)(U)C DG	Xiong J	2025	43%
Study summary Full title Extracellular vesicles promote the infection and pathogenicity of Japanese encephalitis virus. All authors Xiong J, Yang L, Nan X, Zhu S, Yan M, Xiang S, Zhang L, Li Q, Yang C, Wang X, Wei N, Chen H, Si Y, Cao S, Ye J Journal J Extracell Vesicles Abstract Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to (show more...)Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to public health. Currently, there is no specific therapeutic agent available for JEV infection, primarily due to the complexity of its infection mechanism and pathogenesis. Extracellular vesicles (EVs) have been known to play an important role in viral infection, but their specific functions in JEV infection remain unknown. Here, ultracentrifugation in combination with density gradient centrifugation was conducted to purify EVs from JEV-infected cells. The purified EVs were found to be infectious, with virions observed inside the EVs. Furthermore, our study showed the formation process of virion-containing EVs both in vitro and in vivo, which involved the fusion of multivesicular bodies with the cell membrane, leading to the release of virion-containing intraluminal vesicles into the extracellular space. Further studies revealed that EVs played a crucial role in JEV propagation by facilitating viral entry and assembly-release. Furthermore, EVs assisted JEV in evading the neutralizing antibodies and promoted viral capability to cross the blood-brain and placental barriers. Moreover, in vivo experiments demonstrated that EVs were beneficial for JEV infection and pathogenicity. Taken together, our findings highlight the significant contribution of EVs in JEV infection and provide valuable insights into JEV pathogenesis. (hide) EV-METRIC 43% (81st 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 (Differential) (ultra)centrifugation Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biomarker/Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Mesocricetus auratus Sample Type Cell culture supernatant EV-producing cells BHK-21 EV-harvesting Medium EV-depleted medium Preparation of EDS 4h at 150,000g Cell viability (%) 80 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 Swinging bucket Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 20% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 3.5 Orientation Top-down Speed (g) 100,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: speed (g) 100,000 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis None

	EV240164	1/5	Homo sapiens	HeLa	(d)(U)C IAF	Xiong J	2025	13%
Study summary Full title Extracellular vesicles promote the infection and pathogenicity of Japanese encephalitis virus. All authors Xiong J, Yang L, Nan X, Zhu S, Yan M, Xiang S, Zhang L, Li Q, Yang C, Wang X, Wei N, Chen H, Si Y, Cao S, Ye J Journal J Extracell Vesicles Abstract Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to (show more...)Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to public health. Currently, there is no specific therapeutic agent available for JEV infection, primarily due to the complexity of its infection mechanism and pathogenesis. Extracellular vesicles (EVs) have been known to play an important role in viral infection, but their specific functions in JEV infection remain unknown. Here, ultracentrifugation in combination with density gradient centrifugation was conducted to purify EVs from JEV-infected cells. The purified EVs were found to be infectious, with virions observed inside the EVs. Furthermore, our study showed the formation process of virion-containing EVs both in vitro and in vivo, which involved the fusion of multivesicular bodies with the cell membrane, leading to the release of virion-containing intraluminal vesicles into the extracellular space. Further studies revealed that EVs played a crucial role in JEV propagation by facilitating viral entry and assembly-release. Furthermore, EVs assisted JEV in evading the neutralizing antibodies and promoted viral capability to cross the blood-brain and placental barriers. Moreover, in vivo experiments demonstrated that EVs were beneficial for JEV infection and pathogenicity. Taken together, our findings highlight the significant contribution of EVs in JEV infection and provide valuable insights into JEV pathogenesis. (hide) EV-METRIC 13% (34th 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 Japanese encephalitis virus-infected 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 (Differential) (ultra)centrifugation Immunoaffinity capture (non-commercial) Protein markers EV: CD63/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biomarker/Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS 4h at 150,000g Cell viability (%) 90 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 Swinging bucket Pelleting: speed (g) 100000 Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ CD81 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 250-500

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EV-TRACK ID	EV240164
species	Homo sapiens	Sus scrofa domesticus	Mesocricetus auratus	Mesocricetus auratus	Homo sapiens
sample type	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture
cell type	HeLa	PIEC	BHK-21	BHK-21	HeLa
condition	Japanese encephalitis virus-infected	Japanese encephalitis virus-infected	Japanese encephalitis virus-infected	Control condition	Japanese encephalitis virus-infected
separation protocol	dUC/ Density gradient	dUC/ Density gradient	dUC/ Density gradient	dUC/ Density gradient	dUC/ IAF capture (non-commercial)
Exp. nr.	2	3	4	5	1
EV-METRIC %	56	56	56	43	13