Journal: bioRxiv

Article Title: Phosphorylation of JIP4 at S730 presents anti-viral properties against influenza A virus infection

doi: 10.1101/2021.01.22.427772

Figure Lengend Snippet: A549 cells were transfected with either, control siRNAs or siRNAs targeting JIP4, and infected with PR8 (MOI 5). Cells were lysed every 2 h. Lysates were submitted to Western blot assay and incubated with antibodies against viral proteins PB1 and NS1, as well as against JIP4 for silencing confirmation. Detection of tubulin served as loading control (A) . A549 cells were transfected with either, empty vector (EV) or plasmids encoding JIP4, and infected with PR8 (MOI 5). Cells were lysed every 2 h. Lysates were submitted to Western Blot assay and incubated with antibodies against viral proteins PB1, NS1 and M1, as well as against JIP4 for overexpression confirmation. Detection of tubulin was used as loading control (B) . For analyses of viral mRNA expression, A549 cells were transfected with either siRNAs control or targeting JIP4 and infected with PR8 (MOI 5). Samples were collected every 2 h and progressed for qRT-PCR analyses. Viral mRNAs were normalized over 2 h control group. SPAG9 gene was normalized to MOCK control. Results were statistically analyzed by two-way ANOVA test (C) . **p<0.001; ***p<0.0001 and ****p<0.00001 compared to respective controls. All Western Blot images are representative of three independent experiments. Graphs are a compilation of three different experiments.

Article Snippet: Samples were analyzed for the expression of the viral proteins polymerase basic protein 1 (PB1), non-structural protein 1 (NS1) and matrix protein 1 (M1) (Genetex) or cellular proteins such as tubulin, JIP4, p38 or p-p38 MAPK T180/Y182 (Cell Signaling Technology), pJNK pT183/pY185 (BD Bioscience), JNK (Cell Signaling Technology).

Techniques: Transfection, Control, Infection, Western Blot, Incubation, Plasmid Preparation, Over Expression, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidylcholines Promote Influenza Virus Reproduction through the MAPK/JNK Pathway in PMA-Differentiated THP-1 Macrophages

doi: 10.3390/ijms25126538

Figure Lengend Snippet: Increase in viral RNA and protein levels by LPCs. ( A ) Relative expression levels of IAV genes in THP-1 cells treated with different dosages of LPCs. Color boxes indicated control (□), IAV (■) and IAV with LPC (▒). ( B ) The protein level of IAV proteins in THP-1 cells treated with LPCs. RNA levels were determined using RT-qPCR, and protein expression was detected using Western blotting. Data represent the means ± SDs of three independent experiments. Statistical significance was assessed using an unpaired Student t -test. *** p < 0.001, ** p < 0.005, * p < 0.05 compared with the values obtained for cells infected with IAV. M2: matrix-2; M1: matrix-1; PA: polymerase acidic protein; PB1: polymerase basic 1; NP: nucleoprotein; NS1: non-structural protein; PB2: polymerase basic 2; HA: hemagglutinin; NA: neuraminidase.

Article Snippet: Antibodies against the influenza virus proteins polymerase acidic (PA), nucleoprotein (NP), non-structural protein 1 (NS1), matrix-1 (M1), polymerase basic 1 (PB1), and PB2 were obtained from GeneTex, Inc. (Irvine, CA, USA), and antibodies for JNK, p-JNK, AKT, and p-AKT were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Infection

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidylcholines Promote Influenza Virus Reproduction through the MAPK/JNK Pathway in PMA-Differentiated THP-1 Macrophages

doi: 10.3390/ijms25126538

Figure Lengend Snippet: Differential expression of viral RNAs according to the type of LPCs. The relative expression levels of IAV genes were determined using RT-qPCR. Data represent the means ± SDs of three independent experiments. Statistical significance was assessed using an unpaired Student t -test. *** p < 0.001, ** p < 0.005, * p < 0.05 compared with the values obtained for cells infected with IAV. Color boxes indicated control (□), IAV (■) and IAV with LPCs (▒). M2: matrix-2; M1: matrix-1; PA: polymerase acidic protein; PB1: polymerase basic 1; NP: nucleoprotein; NS1: non-structural protein; PB2: polymerase basic 2; HA: hemagglutinin; NA: neuraminidase.

Article Snippet: Antibodies against the influenza virus proteins polymerase acidic (PA), nucleoprotein (NP), non-structural protein 1 (NS1), matrix-1 (M1), polymerase basic 1 (PB1), and PB2 were obtained from GeneTex, Inc. (Irvine, CA, USA), and antibodies for JNK, p-JNK, AKT, and p-AKT were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Quantitative Proteomics, Expressing, Quantitative RT-PCR, Infection, Control

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidylcholines Promote Influenza Virus Reproduction through the MAPK/JNK Pathway in PMA-Differentiated THP-1 Macrophages

doi: 10.3390/ijms25126538

Figure Lengend Snippet: Attenuation of viral gene expression by MAP kinase inhibitor (SB203580), JNK inhibitor, and PI3K inhibitor (LY294002) in LPC-treated THP-1 cells. Relative expression levels of IAV genes were determined using RT-qPCR. Data represent the means ± SDs of three independent experiments. Statistical significance was assessed using an unpaired Student t -test. *** p < 0.001 compared with values obtained for cells infected with IAV, # p < 0.05 compared with values obtained for cells infected with IAV and treated with LPC. Color boxes indicated control (□), IAV (■) and IAV with LPC and inhibitors (▒). M2: matrix-2; M1: matrix-1; PA: polymerase acidic protein; PB1: polymerase basic 1; NP: nucleoprotein; NS1: non-structural protein; PB2: polymerase basic 2; HA: hemagglutinin; NA: neuraminidase.

Article Snippet: Antibodies against the influenza virus proteins polymerase acidic (PA), nucleoprotein (NP), non-structural protein 1 (NS1), matrix-1 (M1), polymerase basic 1 (PB1), and PB2 were obtained from GeneTex, Inc. (Irvine, CA, USA), and antibodies for JNK, p-JNK, AKT, and p-AKT were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Infection, Control

Journal: PLoS ONE

Article Title: Zika virus as an oncolytic treatment of human neuroblastoma cells requires CD24

doi: 10.1371/journal.pone.0200358

Figure Lengend Snippet: A) Western blot analysis of Zika virus infections in neuroblastoma cells. Total protein analysis was performed for Zika Envelope protein and NS1 (Non-Structural 1) protein compared to GAPDH control. The neuroblastoma cell lines tested included IMR-32, SMS-KAN, SK-N-AS, LA-N-6, SK-N-Be(1), and CHLA-42, using Vero cells as an infection control. All cells were compared to control cells treated with non-infected conditioned media. Samples were assessed 3 days after infection (MOI = 10). These results were representative of the combined data of experiments performed in triplicate. B) Viral Titer (TCID50) assays of IMR-32 and SK-N-AS cells at Day 2 and 3 post-infection. Data is composed of three biological replicates examined in sextuplicate, with error bars representing standard deviation. ** p > 0.05, Student’s t-test. C) Immunofluorescence labeling of Zika viral Envelope protein in IMR-32 and SK-N-AS cells at Day 3 post-infection. Envelope staining is in red (Alexa Fluor 647) and nuclei are stained in blue (DAPI). Samples are also shown together (merged). Cells were scanned using a Nikon A1R VAAS laser point- and resonant-scanning confocal microscope. Images are at a magnification of 40x with a 4x zoom.

Article Snippet: Proteins were transferred to nitrocellulose membranes (0.2 um, BioRad, Cat# 1620112) and probed with the following primary antibodies: Anti-Zika virus NS1 (non-structural protein 1) (One World Lab, Cat# 55964) at 1/200, anti-Zika virus Envelope (Env) (GeneTex, Cat# GTX133314) at 1/1000, and anti-GAPDH (Santa Cruz, FL-335) at 1/2000.

Techniques: Western Blot, Virus, Control, Infection, Standard Deviation, Immunofluorescence, Labeling, Staining, Microscopy

Journal: PLoS ONE

Article Title: Zika virus as an oncolytic treatment of human neuroblastoma cells requires CD24

doi: 10.1371/journal.pone.0200358

Figure Lengend Snippet: A) Western blot analysis of siRNA-mediated knock-down of CD24 expression in IMR-32 cells. Samples include Negative Control siRNA and CD24 siRNA. B) Western blot analysis of the expression of Envelope protein and NS1 (Non-Structural 1) protein in IMR-32 cells after siRNA-mediated knock-down of CD24 expression, 96 hours after Zika infection (MOI = 10). Samples include control cells treated with non-infected conditioned media and infected IMR-32 cells transfected with either Negative Control siRNA or CD24 siRNA. C) Western blot analysis of CD24 expression in the human neuroblastoma cell line SK-N-AS, comparing wild type (WT) to stably selected “Vector Only” (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). D) Western blot analysis of Zika NS1 protein expression 96 hours after Zika infection in CD24-stably expressing SK-N-AS cells, comparing wild type (WT) to stably selected Vector Only (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). GAPDH was used as a load control for all experiments. All results are representative of the combined data of experiments performed in triplicate.

Article Snippet: Proteins were transferred to nitrocellulose membranes (0.2 um, BioRad, Cat# 1620112) and probed with the following primary antibodies: Anti-Zika virus NS1 (non-structural protein 1) (One World Lab, Cat# 55964) at 1/200, anti-Zika virus Envelope (Env) (GeneTex, Cat# GTX133314) at 1/1000, and anti-GAPDH (Santa Cruz, FL-335) at 1/2000.

Techniques: Western Blot, Knockdown, Expressing, Negative Control, Infection, Control, Transfection, Stable Transfection, Plasmid Preparation, Variant Assay

Journal: Pathogens

Article Title: Detection of Anti-Nucleocapsid Antibody in COVID-19 Patients in Bangladesh Is not Correlated with Previous Dengue Infection

doi: 10.3390/pathogens10060637

Figure Lengend Snippet: SARS-CoV-2 RNA levels and correlations with dengue IgG levels. The boxplot corresponds to the 25th and 75th interquartile range (IQR), the horizontal line inside the box to the median and the bars outside the box to the minimum and maximum RNA concentrations (Ct values). The Ct values in n = 19 nasopharyngeal specimens (NP, circles) with a mean of 22.6 (95% CI 22–23) versus n = 29 oropharyngeal specimens (OP, triangles) median of 29 (95% CI 28–30) *** p < 0.0001. The Ct values of NP specimens from COVID-19 patients reporting symptoms (sympt) with a mean of 22 (95% CI 21.8–23) versus no symptoms (none) with a mean of 23.6 (21.5–25.6) p = 0.06. The filled symbols represent severe cases ( A ). The scatter plots represented by open circles and the curve fit of linear regression analysis of Ct values and anti-dengue (DENV) IgG levels are represented by the solid line ( B and C ); n = 19 NP specimens and matched serum; r = −0.11 (95% CI −0.54–0.36), p = 0.65 ( B ) and n = 17 OP specimens and matched serum; r = 0.38 (95% CI −0.73–0.12, p = 0.3.

Article Snippet: The dengue IgM and IgG, the dengue non-structural protein-1 (NS-1) and the SARS CoV-2 IgA and IgG were analyzed by specific ELISA in accordance with the manufacturer’s instructions and recommended cut-off value of NovaTec units (NTU) > 10 for positive results (Novatec Diagnostics, Dietzenbach, Germany); NTU = X *10/QC where X = OD 450nm − OD 620nm of the test sample and QC = OD 450nm − OD 620nm of the quality control equivocal serum sample.

Techniques:

Journal: Pathogens

Article Title: Detection of Anti-Nucleocapsid Antibody in COVID-19 Patients in Bangladesh Is not Correlated with Previous Dengue Infection

doi: 10.3390/pathogens10060637

Figure Lengend Snippet: Kinetics of anti-SARS-CoV-2 N-protein antibodies in rT-PCR positive COVID-19. The IgA ( A ) and IgG ( B ) levels in the course of n = 48 COVID-19 patients. The horizontal dotted line represents the 10 U/L cut-off for positive reactivity in ELISA. Serial measurements of n = 17 patients (open circles) and single time point measurements of n = 12 patients between day 1 to day 8 (open triangles) or n = 19 patients between day 65 to day 177 (open squares). ** p = 0.001 paired student t -test comparing the mean IgA level and the mean IgG level at interval day 65 to day 177.

Article Snippet: The dengue IgM and IgG, the dengue non-structural protein-1 (NS-1) and the SARS CoV-2 IgA and IgG were analyzed by specific ELISA in accordance with the manufacturer’s instructions and recommended cut-off value of NovaTec units (NTU) > 10 for positive results (Novatec Diagnostics, Dietzenbach, Germany); NTU = X *10/QC where X = OD 450nm − OD 620nm of the test sample and QC = OD 450nm − OD 620nm of the quality control equivocal serum sample.

Techniques: Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Pathogens

Article Title: Detection of Anti-Nucleocapsid Antibody in COVID-19 Patients in Bangladesh Is not Correlated with Previous Dengue Infection

doi: 10.3390/pathogens10060637

Figure Lengend Snippet: Antibody positivity in COVID-19 and Dengue Fever in Bangladesh.

Article Snippet: The dengue IgM and IgG, the dengue non-structural protein-1 (NS-1) and the SARS CoV-2 IgA and IgG were analyzed by specific ELISA in accordance with the manufacturer’s instructions and recommended cut-off value of NovaTec units (NTU) > 10 for positive results (Novatec Diagnostics, Dietzenbach, Germany); NTU = X *10/QC where X = OD 450nm − OD 620nm of the test sample and QC = OD 450nm − OD 620nm of the quality control equivocal serum sample.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Pathogens

Article Title: Detection of Anti-Nucleocapsid Antibody in COVID-19 Patients in Bangladesh Is not Correlated with Previous Dengue Infection

doi: 10.3390/pathogens10060637

Figure Lengend Snippet: Levels of anti-SARS-CoV-2 N-protein antibodies and anti-dengue antibodies. The N-protein IgA and IgG and the dengue IgM and IgG in rT-PCR negative COVID-19 ( A ) and in pre-pandemic hospitalized dengue fever ( B ). The difference in mean antibody activities between the two districts were compared by independent student t-tests. In Dhaka city, n = 45 (circles) anti-N-protein specific antibody levels IgA; mean 16 [95% CI 0.8–32], 14% positive and IgG; mean 9 (95% CI 6.7–11.8), 25% positive versus Narayanganj district n = 22 (squares), IgA; mean activity 43 (95% CI 20–66), 73% positive ** p < 0.001 and IgG; mean activity 25 (95% CI 18–32), 73% positive ** p < 0.001. The anti-dengue IgM in Dhaka city mean activity 4.3 (95% CI 3.8–4.9) 2.3%positive versus Narayanganj district; mean activity 5.3 (95% CI 4.4–6.2) 4.5% positive, p = 0.07, and anti-dengue IgG; mean activity 34 [95% CI 28–40] 72% positive versus mean activity 46 (95% CI 39–53), 86% positive, respectively, * p = 0.01. NS = not significant.

Article Snippet: The dengue IgM and IgG, the dengue non-structural protein-1 (NS-1) and the SARS CoV-2 IgA and IgG were analyzed by specific ELISA in accordance with the manufacturer’s instructions and recommended cut-off value of NovaTec units (NTU) > 10 for positive results (Novatec Diagnostics, Dietzenbach, Germany); NTU = X *10/QC where X = OD 450nm − OD 620nm of the test sample and QC = OD 450nm − OD 620nm of the quality control equivocal serum sample.

Techniques: Reverse Transcription Polymerase Chain Reaction, Activity Assay

Journal: Pathogens

Article Title: Detection of Anti-Nucleocapsid Antibody in COVID-19 Patients in Bangladesh Is not Correlated with Previous Dengue Infection

doi: 10.3390/pathogens10060637

Figure Lengend Snippet: Correlations of SARS CoV-2 N protein IgA or IgG and Dengue IgG. The anti-N-protein IgA ( A ) and ( B ) and IgG ( C ) and ( D ) at 14–90 days after COVID-19 symptoms in n = 36 rT−PCR positive ( A ) and ( C ) and at 1–8 days after COVID-19 symptoms in n = 67 rT-PCR negative patients ( B , D ). The values within the area of the box are below the 10 U/L cut-off for positive reactivity in ELISA. The coefficient of correlations between dengue IgG and N-protein IgA in rT-PCR pos COVID-19; r = 0.26 95% CI −0.07–0.54), p = 0.127 and in rT-PCR neg COVID-19; r = −0.28 (95% CI −0.53–0.03), p = 0.07. The coefficient of correlations between dengue IgG and N-protein IgG in rT-PCR pos COVID-19; 0.44 (95% CI 0.03–0.73), p = 0.04 and in rT-PCR neg COVID-19; r = −0.33 (95% CI −0.64–0.08), p = 0.11.

Article Snippet: The dengue IgM and IgG, the dengue non-structural protein-1 (NS-1) and the SARS CoV-2 IgA and IgG were analyzed by specific ELISA in accordance with the manufacturer’s instructions and recommended cut-off value of NovaTec units (NTU) > 10 for positive results (Novatec Diagnostics, Dietzenbach, Germany); NTU = X *10/QC where X = OD 450nm − OD 620nm of the test sample and QC = OD 450nm − OD 620nm of the quality control equivocal serum sample.

Techniques: Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Infectious Diseases

Article Title: Zika Virus Seroprevalence in Urban and Rural Areas of Suriname, 2017

doi: 10.1093/infdis/jiz063

Figure Lengend Snippet: ZIKV IgG ELISA and VNT results in 2017 cohort and pre-ZIKV cohort

Article Snippet: The 44 samples of the pre-ZIKV cohort were tested with a DENV-2 virus particle-based commercial DENV ELISA kit (Euroimmun) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Veterinary clinical pathology

Article Title: Case Report: Adult-onset, chronic, cyclic thrombocytopenia in a Rhesus macaque ( Macaca mulatta ) after dengue virus vaccination and viral challenge

doi: 10.1111/vcp.12497

Figure Lengend Snippet: Anti-dengue virus non-structural protein 1 specific-Elisa results with recombinant antigen from 4 different dengue virus (DENV) serotypes in the serum of the affected macaque during 2 thrombocytopenic episodes (slightly positive IgG titer, red arrow) and a normal clinical phase. Data indicate the presence of anti-DENV1 NS1 and anti-DENV4 NS1 IgG (B), but not anti-DENV2 or DENV3. The control macaques were negative.

Article Snippet: 7 Serum anti-dengue non-structural protein 1 (NS1) IgG antibodies were detected by a commercially-available assay (GRF Diagnostica, Jilin, China; IBL America, Minneapolis, MN) during all 4 different episodes that were tested , though PCR testing of serum for DENV was negative (Zoologix Inc., Chatsworth, CA).

Techniques: Virus, Enzyme-linked Immunosorbent Assay, Recombinant, Control