Journal: Viruses

Article Title: Zika Virus Antibody Titers Three Years after Confirmed Infection

doi: 10.3390/v13071345

Figure Lengend Snippet: Results from serological assays for ZIKV and DENV antibodies . A : Percentage of positive and negative tested sera with ZIKV VNT and ZIKV NS1 IgG ELISA. B : Correlation between ZIKV NS1 IgG ELISA ratios and titers from the ZIKV VNT. The dotted lines indicate cut-off values for a positive test result. C : ZIKV- and DENV-2 VNT titers from all participants. Lines represent median ± IQR. The dotted line indicates the cut-off value for a positive test result. Statistical differences were tested with the Mann–Whitney test. D : Correlation between DENV-2 VNT titers and ZIKV VNT titers. The dotted lines indicate cut-off values for a positive test result. E : Correlation between ZIKV NS1 IgG ELISA ratios and DENV-2 VNT titers. The dotted lines indicate cut-off values for a positive test result. * P < 0.05.

Article Snippet: Slides were printed with DENV1–4 and ZIKV NS1 proteins (Sino Biological Europe GmbH and Immune Technology Corp., New York, NY) and Equad proteins (DENV envelope proteins containing four amino acid mutations in the highly conserved fusion loop domain to reduce flavivirus cross-reactivity) [ ].

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Viruses

Article Title: Zika Virus Antibody Titers Three Years after Confirmed Infection

doi: 10.3390/v13071345

Figure Lengend Snippet: Heatmap of results from the different serological assays used in this study . A : IgG antibody titers for DENV1–4 Equad and DENV1–4 and ZIKV NS1 antigens determined with a protein microarray. Corresponding ZIKV and DENV-2 VNT titers and ZIKV NS1 IgG ELISA ratios from all participants are shown on the right. Antibody patterns are ranked from highest to lowest ZIKV VNT titer. B : Protein microarray IgG antibody titer patterns for DENV1–4 Equad and DENV1–4 and ZIKV NS1, ZIKV and DENV-2 VNT titers and ZIKV NS1 IgG ELISA ratios from all participants, ranked from highest to lowest ZIKV NS1 ELISA ratio. Numbers on the left Y-axis are the study numbers of the participants in this study. PA; protein microarray, Equad; envelope proteins containing four amino acid mutations in the highly conserved fusion loop domain to reduce flavivirus cross-reactivity, VNT; virus neutralization test.

Article Snippet: Slides were printed with DENV1–4 and ZIKV NS1 proteins (Sino Biological Europe GmbH and Immune Technology Corp., New York, NY) and Equad proteins (DENV envelope proteins containing four amino acid mutations in the highly conserved fusion loop domain to reduce flavivirus cross-reactivity) [ ].

Techniques: Microarray, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests

doi: 10.1093/cid/cix672

Figure Lengend Snippet: Results of Enzyme-Linked Immunosorbent Assays in Different Serum/Plasma Panels

Article Snippet: Purified DENV1-NS1 protein was purchased from the Native Antigen (United Kingdom).

Techniques:

Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests

doi: 10.1093/cid/cix672

Figure Lengend Snippet: Sensitivity and Specificity of Enzyme-Linked Immunosorbent Assays

Article Snippet: Purified DENV1-NS1 protein was purchased from the Native Antigen (United Kingdom).

Techniques:

Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests

doi: 10.1093/cid/cix672

Figure Lengend Snippet: Results of nonstructural protein 1 (NS1) immunoglobulin G (IgG) enzyme-linked immunosorbent assays (ELISAs) for secondary dengue virus (sDENV) and Zika virus (ZIKV) with previous (wp) DENV infection panels. DENV1-NS1 (A) and ZIKV-NS1 (B) IgG ELISAs in convalescent-phase samples from sDENV, ZIKVwpDENV (Nicaragua [Nic]), probable ZIKVwpDENV (Brazil [Bra]) and negative control (NC) panels. C, Relative optical density (rOD) ratio of ZIKV-NS1 to DENV1-NS1. The sensitivity and specificity are shown based on a cutoff rOD ratio at 0.24. DENV1-NS1 (D) and ZIKV-NS1 (E) IgG ELISAs in post–convalescent phase samples (3 months to 2 years post–symptom onset) from sDENV panels. F, Positive rates of DENV1- and ZIKV-NS1 IgG ELISAs in sDENV panels over time. Dotted lines indicate cutoff rOD values for ELISAs. Data are mean of 2 experiments (each in duplicate). Two-tailed Mann-Whitney test was used to compare the 2 groups.

Article Snippet: Purified DENV1-NS1 protein was purchased from the Native Antigen (United Kingdom).

Techniques: Virus, Infection, Negative Control, Two Tailed Test, MANN-WHITNEY

Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests

doi: 10.1093/cid/cix672

Figure Lengend Snippet: Results of nonstructural protein 1 (NS1) immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISAs) in sequential samples from individuals with secondary dengue virus (sDENV) and Zika virus (ZIKV) infection with previous (wp) DENV infections. A, Three cases with sDENV infection, 3–18 months post–symptom onset. B, Four blood donors with sDENV infection, 7 days to 6 months post–index day (PID). C, Five blood donors with ZIKVwpDENV infection, from index day to 3 months PID. Three cases (1, 4, and 7) seroconverted to ZIKV-NS1; 2 cases (15 and 21) had ZIKV-NS1 IgG starting from the index day. Dotted lines indicate cutoff relative optical density (rOD) values for ELISAs. Data are mean of 2 experiments (each in duplicates).

Article Snippet: Purified DENV1-NS1 protein was purchased from the Native Antigen (United Kingdom).

Techniques: Enzyme-linked Immunosorbent Assay, Virus, Infection

Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests

doi: 10.1093/cid/cix672

Figure Lengend Snippet: Proposed algorithm of using 3 serological tests (without neutralization tests) to distinguish different Zika virus (ZIKV) and dengue virus (DENV) infections in dengue- and Zika-endemic regions in the framework of Centers for Disease Control and Prevention guidelines for laboratory diagnosis of ZIKV infection [6, 7]. Only samples tested positive or equivocal by ZIKV or DENV E protein–based immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) are included in the algorithm. The total numbers from each panel and the numbers of positive or negative based on the 3 Nonstructural Protein 1 (NS1) ELISAs are shown in parentheses. Abbreviations: IgG, immunoglobulin G; OD, optical density; pDENV, primary dengue virus infection; pZIKV, primary Zika virus infection; rOD, relative OD; sDENV, secondary dengue virus infection; ZIKVwpDENV, Zika virus infection with previous dengue virus infection.

Article Snippet: Purified DENV1-NS1 protein was purchased from the Native Antigen (United Kingdom).

Techniques: Neutralization, Virus, Infection

Journal: eBioMedicine

Article Title: Yellow fever disease severity and endothelial dysfunction are associated with elevated serum levels of viral NS1 protein and syndecan-1

doi: 10.1016/j.ebiom.2024.105409

Figure Lengend Snippet: Development of quantitative YFV NS1 ELISA. (a) Sigmoidal standard curve of YFV NS1 capture ELISA performed with in-house-produced YFJ19 mAb and thirteen different concentrations of recombinant YFV NS1. The graph depicts mean absorbance values ± standard deviation (SD). (b) Comparison of standard curve of YFV NS1 diluted or not in normal human serum (1:10). The graph depicts mean absorbance values ± SD. (c) Specificity of mAb YFJ19 by direct ELISA performed with NS1 (5 μg/mL) from 12 different flaviviruses as follows: YFV; dengue virus serotypes 1 (DENV1), 2 (DENV2), 3 (DENV2), and 4 (DENV4); Saint Louis encephalitis virus (SLEV); West Nile virus (WNV); Zika virus (ZIKV); Wesselsbron virus (WBV); Usutu virus (USUV); Japanese encephalitis virus (JEV); Tick-borne encephalitis virus (TBEV). (d) Cross-reactivity of anti-flavivirus NS1 mAb (2B7) by direct ELISA performed with NS1 (5 μg/mL) from the 12 different flaviviruses as in c. (e) Western blot analysis showing specificity of mAb YFJ19 for YFV NS1 and cross-reactivity of mAb 2B7.

Article Snippet: Recombinant YFV NS1 (Native Antigen Co.) at 10 μg/mL was used as a positive control.

Techniques: Enzyme-linked Immunosorbent Assay, Produced, Recombinant, Standard Deviation, Comparison, Direct ELISA, Virus, Western Blot

Journal: eBioMedicine

Article Title: Yellow fever disease severity and endothelial dysfunction are associated with elevated serum levels of viral NS1 protein and syndecan-1

doi: 10.1016/j.ebiom.2024.105409

Figure Lengend Snippet: YFV NS1 serum levels in severe and non-severe YF patients. (a) YFV NS1 levels were determined using an in-house sandwich ELISA, as described in Materials and Methods. Studied individuals were classified in three different groups as described in Materials and Methods: Severe YF (n = 39); Non-severe YF (n = 17, missing data for 1 sample); Controls, healthy individuals (n = 5, missing data for 6 samples). Groups were compared by Kruskal–Wallis + Dunn’s multiple comparisons test. (b) YFV NS1 levels in survivors and deceased YFV-infected groups. Groups were compared by Mann–Whitney test. The dashed lines indicate the median of the data, and the error bars represent the interquartile range (IQR) in a and b. (c) YFV NS1 serum levels in individuals with acute YF according to days since symptom onset, visualized using LOESS curves with 95% confidence intervals (shaded areas). Acute disease was defined by detection of viral RNA by RT-qPCR in serum. Samples were run in duplicate.

Article Snippet: Recombinant YFV NS1 (Native Antigen Co.) at 10 μg/mL was used as a positive control.

Techniques: Sandwich ELISA, Infection, MANN-WHITNEY, Quantitative RT-PCR

Journal: eBioMedicine

Article Title: Yellow fever disease severity and endothelial dysfunction are associated with elevated serum levels of viral NS1 protein and syndecan-1

doi: 10.1016/j.ebiom.2024.105409

Figure Lengend Snippet: Relative TEER of human endothelial cells treated with acute YF human serum samples and effects of YFV NS1 treatment on endothelial glycocalyx. (a) Confluent monolayers of human endothelial cells cultured in Transwell inserts were treated or not with 10% serum from three different groups: Severe YF (n = 24, missing data for 15 samples); Non-severe YF (n = 10, missing data for 8 samples); Controls (n = 11): healthy individuals. YFV NS1 (10 μg/mL) was used as positive control. The transendothelial electrical resistance (TEER) was measured from 2 to 10 h post-treatment. Graph shows mean ± SD of relative TEER for each treatment group. (b) Area under the curve (AUC) of negative peaks for the effects of the different treatments on TEER. AUC values represent the sum of the trapezoidal areas (taking into account only the negative peaks) under the curve formed by the data points. Median values of AUC for each group were compared using Kruskal–Wallis’ test. Error bars represent interquartile range (IQR). Outliers were defined according to Tukey’s method, which identifies outliers using the interquartile range (IQR; i.e., the difference between the first and third quartiles [Q3 - Q1]) as any data point below the first quartile (25th percentile) minus 1.5 times the IQR (Q1 - 1.5∗IQR) or above the third quartile (75th percentile) plus 1.5 times the IQR (Q3 + 1.5∗IQR). (c, d) Human endothelial cell monolayers grown on gelatin-coated coverslips were treated with medium only or 10 μg/mL of YFV NS1 for 3 h. After fixation, cell surface syndecan-1 was stained in red and nuclei in blue (Hoechst). (c) Representative figures of cell monolayers stained for syndecan-1 in red and nuclei in blue. (d) Quantification of syndecan-1 protein on the cells surface expressed as mean fluorescence intensity (MFI). NS1 treatment was compared to medium-only treatment by unpaired t-test. Error bars present SD of the mean. (e, f, g) Human endothelial cell monolayers grown on gelatin-coated coverslips were treated with medium only or with different concentrations of YFV NS1 for 6 h. (e) Quantification of syndecan-1 in cell supernatants by ELISA. An in-house-produced mouse anti-YFV NS1 polyserum with or without 2.5 μg/mL of YFV NS1 was used as a control. Treatments were compared with medium-only treatment by one-way ANOVA + Dunnett’s test. Error bars present SD of the mean. (f) Representative figures of cell monolayers stained for sialic acid in red and nuclei in blue. (g) Quantification of sialic acid on the cell surface expressed as MFI. NS1 treatments were compared to medium-only treatment by one-way ANOVA + Dunnett’s multiple comparison test. Error bars present SD of the mean. Significant p -values are shown in the graphs. Acute YF disease was defined by detection of viral RNA by RT-qPCR in serum. Samples were run in duplicate for the TEER assays. IFA and ELISA data are the results of three independent experiments (n = 3).

Article Snippet: Recombinant YFV NS1 (Native Antigen Co.) at 10 μg/mL was used as a positive control.

Techniques: Cell Culture, Positive Control, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Produced, Control, Comparison, Quantitative RT-PCR

Journal: eBioMedicine

Article Title: Yellow fever disease severity and endothelial dysfunction are associated with elevated serum levels of viral NS1 protein and syndecan-1

doi: 10.1016/j.ebiom.2024.105409

Figure Lengend Snippet: Correlation analysis of clinical and laboratory data of study participants during acute YF and viral load according to days of symptoms. (a) Correlation matrix analyzed by Spearman rank correlation. Asterisks indicate significant correlations ( p < 0.05, Spearman r > 0.35). Categorical variables (e.g., sex [male] and death) were entered as coded variables. (b) Correlation between serum levels of YFV NS1 and syndecan-1. (c) Correlation between serum levels of YFV NS1 and TEER AUC values. (d) Correlation between serum levels of syndecan-1 and TEER AUC values. The gray shaded area represents the 95% confidence interval for the fitted LOESS curve in b, c, and d. Abbreviations: TEER, transendothelial electrical resistance; AUC, area under the curve; Hb, hemoglobin; Ht, hematocrit; AST, aspartate transaminase; ALT, alanine aminotransferase; TPI/INR thrombin potential index/international normalized ratio; aPTT, activated partial thromboplastin time; TB, total bilirubin; DB, direct bilirubin; IB, indirect bilirubin. (e) Viral load of YF patients from severe and non-severe cases. Viral load, determined in YF patients serum at the time of admission by RT-qPCR, is expressed as genomic copies per mL. Results were plotted according to days since symptom onset reported at the time of hospital admission. Acute disease was defined by detection of viral RNA by RT-qPCR in serum.

Article Snippet: Recombinant YFV NS1 (Native Antigen Co.) at 10 μg/mL was used as a positive control.

Techniques: Quantitative RT-PCR