Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Schematic overview of the anti-SARS-CoV-2 drug screening procedure. ( a ) ACE2 targeted inhibitors and compounds from the natural product library were applied prior to infection with SARS-CoV-2 (pre-infection treatment) while compounds from the FDA-approved library and the flavonoid library were applied post infection (post- infection treatment). Out of 2191 compounds tested, 121 displayed viral CPE reductions and 7 were selected for further validations using Vero E6 and HuH7 cell lines and hNEC. ( b ) Uninfected cells, infected cells treated with 0.1% DMSO, and infected cells treated with remdesivir were included as controls in the screen. Following treatment with citicoline, pravastatin sodium, tenofovir alafenamide, imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate, reduced CPE was observed when compared to the DMSO control.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Drug discovery, Infection, Control

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Summary of compounds identified from the primary screen that exhibit potential antiviral effects against SARS-CoV-2 infection.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Infection, Reverse Transcription, Reflux

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Validation of primary hits. For validating compounds with activity pre-infection, Vero E6 cells were first pre-treated with increasing concentrations of ( a ) citicoline, ( b ) pravastatin sodium and ( c ) tenofovir alafenamide prior to infection with SARS-CoV-2. Similarly, Huh7 cells were pre-treated with ( d ) citicoline, ( e ) pravastatin sodium and ( f ) tenofovir alafenamide and subsequently infected with SARS-CoV-2. For post-infection treatment validation, Vero E6 cells were infected with SARS-CoV-2 and treated with increasing concentrations of ( g ) imatinib mesylate, ( h ) calcitriol, ( i ) dexlansoprazole and ( j ) prochlorperazine dimaleate. Similarly, HuH7 cells were also infected then treated with a range of concentrations of ( k ) imatinib mesylate, ( l ) calcitriol, ( m ) dexlansoprazole and ( n ) prochlorperazine dimaleate. The primary and secondary exes correspond to the viral titre and relative cell viability, and the dashed line represents the CC 50 cut-off for cell viability. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for both cell viability and dose-dependent inhibition studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Biomarker Discovery, Activity Assay, Infection, Standard Deviation, Inhibition

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Validation of primary hits in hNECs. Selected primary hits were further validated in hNECs. Cells were either pre-treated with 10 μM citicoline prior to SARS-CoV-2 infection or treated following SARS-CoV-2 infection with 10 μM imatinib mesylate and 10 μM calcitriol. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with ** denoting that p < 0.01. Error bars represent the standard deviation observed from the means of triplicates performed for dose-dependent inhibition studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Biomarker Discovery, Infection, Standard Deviation, Inhibition

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: SARS-CoV-2 and calcitriol modulate vitamin D Receptor (VDR), 24-hydroxylase (24(OH)ase), and cathelicidin (LL-37) in Vero E6 and Huh7 cells. In the first part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media, or SARS-CoV-2 infected and harvested at indicated timepoints in the absence of calcitriol for baseline VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 expression of ( a ) VDR, ( b ) 24(OH)ase and ( c ) LL-37 and Huh7 expression of ( d ) VDR, ( e ) 24(OH)ase and ( f ) LL-37 were as shown. In the second part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media or SARS-CoV-2 for 1 h as above, then treated with 0.01, 0.1, 1, 10 or 20 µM calcitriol following infection. At 48 h post infection, the cells were harvested to check for VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 mRNA expression of ( g ) VDR, ( i ) 24(OH)ase and ( k ) LL-37 and Huh7 expression of ( h ) VDR, ( j ) 24(OH)ase and ( l ) LL-37 were as shown. Data are represented as relative quantity to 0.1% DMSO control, or as relative quantity to mock-infected control. One-way ANOVA and Dunnett’s post-test and paired t -test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for mRNA expression studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Infection, Expressing, Control, Standard Deviation

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Western blot analysis of SARS-CoV-2 spike and VDR proteins in Vero E6 cells. ( a ) Vero E6 cells are either mock infected with media (−) or with SARS-CoV-2 (+) for 1 h. After 1 h post-infection, the cells are then either untreated (−) or treated (+) with 20 µM calcitriol for 6-, 24- and 48- hours before harvesting of total cell lysate. ( b ) Densitometry quantitation of protein expression levels is shown as fold changes to beta-actin. Full-length blots are shown in .

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Western Blot, Infection, Quantitation Assay, Expressing

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: In vivo study of calcitriol treatment. 8-week old K18-hACE2 male and female mice were separated into treatment and non-treatment groups. Treatment study group has been given 3 days pre-treatment and 5 days post-treatment of calcitriol (5 μg/kg) via intraperitoneal injection. The non-treatment group has been given a PBS as a treatment control. Next, 10 3 PFU of SARS-CoV-2 (L, Alpha, and Beta variants) were inoculated through intranasal delivery to all groups after pre-treatment. Physiological parameters: body weight ( a – c ), survival rate ( d – f ), and physiological conditions were monitored after infections. For comparison between individual physiological conditions, 5 dpi infected mice were compared between treatment groups of all three variant infected mice ( g – i ). Scoring of physiological conditions was based on five criteria: appearance of the mouse coat, level of consciousness, activity level, eye condition, and respiratory quality. To compare the severity of damage in mouse tissues after infection, left lung lobes from 4 dpi mice were processed for histological analyses and scored based on six criteria to determine severity ( j – l ). The six criteria are inflammatory cell infiltration, haemorrhage, oedema, bronchial epithelial cell damage, degeneration of alveolar epithelial cells, and parenchymal wall expansion. For viral load determination, right lung, brain, liver, and spleen tissues from the same 4 dpi mice were harvested and homogenised for virus titration ( m – o ). Data is not shown for liver and spleen tissues. Statistical significance is determined with a two-tailed unpaired t -test, with * denoting p < 0.05. Survival group: variant L: n = 6 (treatment and no treatment); Alpha: n = 6 (treatment), n = 4 (no treatment); Beta: n = 6 (treatment), n = 5 (no treatment). 4 dpi group: variant L: n = 7 (treatment and no treatment); Alpha and Beta: n = 5 each (treatment and no treatment). Mock infection, n = 3.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: In Vivo, Injection, Control, Comparison, Infection, Variant Assay, Activity Assay, Virus, Titration, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: Survival of AIR pups increases with decreasing amounts of virus and anti-IFNAR antibody administered to the dam. a Neonates born to AIR, AIR low and IgR treated, ZIKV infected pregnant mice (see Results section for different treatments) were followed for survival out to postnatal day 7 (P7) and the results are presented as Kaplan–Meier survival curves. Each curve indicates the survival of neonates from multiple dams treated with the indicated anti-IFNAR1/normal mouse IgG dose(s) and ZIKV infectious dose. The dash-dot line plotted on the Y-axis indicates median survival as determined by Kaplan–Meier. ** p < 0.01, **** p < 0.0001 indicate a significant increase in survival based on a Log-rank Mantel-Cox curve comparison test. All survival curves were compared relative to the AIR (red) curve. Whole-head H&E-stained sections of an b IgR and c AIR treated neonate at P5 are shown. Notice the diminished brain volume in the ( c , asterisks) AIR animal which corresponds to the only surviving AIR animal at P5 ( a , red line termination). The tissue disruption in the brainstem and cerebellum of ( b ) is due to tissue processing and cutting artifact and is not associated with virus-induced pathology

Article Snippet: Primary antibodies against ZIKV NS2B (rabbit, 1:250, Genetex) or NeuN (rabbit, 1:100, Cell Signaling), or Calbindin-D-28 K (rabbit, 1:2250, Millipore) or Iba1 (rabbit, 1:1000, gift by John Portis (RML)) or GFAP (rabbit, 1:3000, Agilent/Dako) or transmembrane protein 119 (TMEM119, rabbit, 1:1000, Synaptic Systems) were applied for 1 h at 37 °C, except GFAP which was applied for 32 min at 37 °C.

Techniques: Virus, Infection, Comparison, Staining, Disruption

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: ZIKV broadly infect neurons within the CNS of P7 AIR low neonates. P7 neonatal whole brain from naïve, IgR and AIR low pups was evaluated for ( a ) ZIKV RNA by qRT-PCR with virus-specific primers. Each symbol indicates an individual brain. The mean of the individually plotted data points for each group is represented by the horizontal black bars. The dotted line in ( a ) indicated the limit of detection for the assay. Data was analyzed by a One-way ANOVA (F 2, 20 = 12.56, p = 0.0003) with a Tukey Multiple Comparisons Test to determine significance between groups (***). Whole brain sections from b IgR and c AIR low neonates were immunohistochemically labeled for ZIKV NS5 antigen (green) and cell nuclei (grey, Hoechst) and visualized via fluorescence microscopy. Scale bar in ( c ) applies to ( b ). Cerebellum from ( d , h ) naïve, ( e , i ) IgR and ( f , j ) AIR low mice were dual chromogenic labeled for ( d – g ) Calbindin (yellow) and ZIKV NS2B (purple) or ( h – k ) NeuN (yellow) and ZIKV NS2B (purple) to demonstrate cellular organization and neuronal infection. The normal position and shape of the granular layer (GL), Purkinje cell layer (PCL), molecular layer (ML) and external germinal layer (EGL) are shown in a naïve mouse cerebellum labeled with ( d ) Calbindin and ( h ) NeuN at the P7 time point. Calbindin clearly labels Purkinje cell bodies in PCL ( d , lower left inset) and dendrites in the ML ( d , lower left inset, red arrow) and neurons associated with white matter (WM, d, lower right inset, red arrow) while NeuN labels neurons in the granular layer and maturing granular neurons as they emerge from the EGL to populate the GL. The black lines in ( e , i ) highlight the normal-appearing PCL and ML in an IgR mouse. These structures appear similar to those in the naïve mouse ( d , h , PCL and ML). In contrast, the black lines in ( f , j ) highlight the diminished PCL and ML in an AIR low mouse with the distance between each line having decreased. Likewise, notice the disorganization and sparsity of cells in the GL of IgR ( i ) and AIR low ( j ) mice, relative to the ordered GL in the naïve mouse ( h ). The asterisks in ( h – j ) highlight the maturing granular neurons that expression NeuN as they emerge from the EGL to populate the GL in a naïve, IgR and AIR low mouse respectively. The blue boxes in ( f ) and ( j ) corresponds to the higher magnification images in ( g ) and ( k ) respectively. The black arrows in ( g ) and ( k ) demonstrate ZIKV and neuronal marker dual-labeled infected neurons, which result in red-colored cells. In contrast, cerebellar neurons that did not label with Calbindin, but are ZIKV positive are labeled purple (green arrows in g ). The scale bar in ( d ) also applies to ( e , f , h and i )

Article Snippet: Primary antibodies against ZIKV NS2B (rabbit, 1:250, Genetex) or NeuN (rabbit, 1:100, Cell Signaling), or Calbindin-D-28 K (rabbit, 1:2250, Millipore) or Iba1 (rabbit, 1:1000, gift by John Portis (RML)) or GFAP (rabbit, 1:3000, Agilent/Dako) or transmembrane protein 119 (TMEM119, rabbit, 1:1000, Synaptic Systems) were applied for 1 h at 37 °C, except GFAP which was applied for 32 min at 37 °C.

Techniques: Quantitative RT-PCR, Virus, Labeling, Fluorescence, Microscopy, Infection, Expressing, Marker

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: ZIKV infection in the CNS of P7 AIR low neonates results in CNS structural abnormalities. H&E-stained whole brain sections from a IgR and b AIR low mice are shown as representative examples of morphometric measurements taken of cortical thickness (red line in a , b ) and cerebellar area (red polygon in a , b ). Actual recorded values for each section are shown. The scale bar in ( b ) applies to ( a ). Morphometric measurements from each hemisphere of 3 naïve, 10 IgR and 6 AIR low are shown for ( c ) cerebellar area and ( d ) cortical thickness. Each symbol represents the mean measurement from all sections for each hemisphere of each animal. The mean of the individually plotted data points for each group is represented by the horizontal black bars. A One-way ANOVA was used to compare cerebellar area ( c , F 2, 33 = 16.62, p < 0.0001) and cortical thickness ( d , F 2, 33 = 5.54, p = 0.0084) from samples from each group and a Tukey Multiple Comparisons Test was used to determine significance between groups. * p < 0.05, *** p < 0.001, **** p < 0.0001 indicate significance between groups

Article Snippet: Primary antibodies against ZIKV NS2B (rabbit, 1:250, Genetex) or NeuN (rabbit, 1:100, Cell Signaling), or Calbindin-D-28 K (rabbit, 1:2250, Millipore) or Iba1 (rabbit, 1:1000, gift by John Portis (RML)) or GFAP (rabbit, 1:3000, Agilent/Dako) or transmembrane protein 119 (TMEM119, rabbit, 1:1000, Synaptic Systems) were applied for 1 h at 37 °C, except GFAP which was applied for 32 min at 37 °C.

Techniques: Infection, Staining

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: ZIKV infection increases cell death in the CNS of P7 AIR low neonates primarily in the cerebellum, cortex and spinal cord. Representative H&E-stained sections from P7 ( a , c ) naïve and ( b , d ) AIR low cerebellum and spinal cord respectively. Degenerating cerebellar neurons ( b , black arrows) and axonal degeneration ( d , black arrows) were observed in AIR low mice. The scale bar in ( b ) applies to ( a ) and the bar in ( d ) applies to ( c ). Representative immunofluorescence labeled CNS sections from P7 ( e – g ) IgR and ( h – j ) AIR low pups demonstrate ZIKV NS5 (green) and active-Caspase 3 (magenta) positive cells in cortex, hippocampus and cerebellum. Nuclei are shown in grey. Images of active-Caspase 3 and nuclei staining only in AIR low mice are shown in ( k – m ). Red arrows and box insets in ( e – g ) indicate baseline active-Caspase 3 labeling in control animals. Red boxes in ( h – j ) correspond to insets in each specific brain region. Yellow arrows indicate ZIKV/active-Caspase 3 dual positive cells while orange arrows indicate active-Caspase 3 only cells. The scale bar in ( g ) applies to ( e – m )

Article Snippet: Primary antibodies against ZIKV NS2B (rabbit, 1:250, Genetex) or NeuN (rabbit, 1:100, Cell Signaling), or Calbindin-D-28 K (rabbit, 1:2250, Millipore) or Iba1 (rabbit, 1:1000, gift by John Portis (RML)) or GFAP (rabbit, 1:3000, Agilent/Dako) or transmembrane protein 119 (TMEM119, rabbit, 1:1000, Synaptic Systems) were applied for 1 h at 37 °C, except GFAP which was applied for 32 min at 37 °C.

Techniques: Infection, Staining, Immunofluorescence, Labeling, Control

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: ZIKV infection induces glial activation in the CNS of P7 AIR low neonates. Representative sections from P7 ( a , c , e and g ) IgR and ( b , d , f and h ) AIR low mice demonstrating ( a – d ) GFAP and ( e – h ) Iba1 immunofluorescence labeling in the cortex and cerebellum. Specific labeling from each antibody is shown in magenta and cell nuclei (Hoechst) are shown in grey for counterstain. Green boxes in ( g ) and ( h ) correspond to high magnification insets to demonstration cellular morphology. P7 neonatal whole brain from naïve, IgR and AIR low pups was evaluated for RNA expression of glial-specific genes including ( i ) Gfap , ( j ) Aif1 (Iba1) and ( k ) Gpr84 by qRT-PCR with specific primers. Each symbol indicates an individual brain. The mean of the individually plotted data points for each group is represented by the horizontal black bars. A One-way ANOVA was used to compare RNA expression of ( i ) Gfap (F 2, 20 = 44.50, p < 0.0001), ( j ) Aif1 (F 2, 20 = 76.88, p < 0.0001) and ( k ) Gpr84 (F 2, 20 = 103.2, p < 0.0001) from samples from each group and a Tukey Multiple Comparisons Test was used to determine significance between groups. **** p < 0.0001, indicates significance between groups

Article Snippet: Primary antibodies against ZIKV NS2B (rabbit, 1:250, Genetex) or NeuN (rabbit, 1:100, Cell Signaling), or Calbindin-D-28 K (rabbit, 1:2250, Millipore) or Iba1 (rabbit, 1:1000, gift by John Portis (RML)) or GFAP (rabbit, 1:3000, Agilent/Dako) or transmembrane protein 119 (TMEM119, rabbit, 1:1000, Synaptic Systems) were applied for 1 h at 37 °C, except GFAP which was applied for 32 min at 37 °C.

Techniques: Infection, Activation Assay, Immunofluorescence, Labeling, RNA Expression, Quantitative RT-PCR

Journal: Acta Neuropathologica Communications

Article Title: Zika virus vertical transmission in interferon receptor1-antagonized Rag1 −/− mice results in postnatal brain abnormalities and clinical disease

doi: 10.1186/s40478-022-01351-6

Figure Lengend Snippet: Prolonged ZIKV infection in AIR low mice results in sustained CNS structural abnormalities and glial activation leading to eventual clinical neurologic disease. a Neonates born to AIR low (blue line) and IgR (black line) treated, ZIKV infected, pregnant mice (see Results for specific mouse numbers) were followed for survival out to P14 and the results are presented as Kaplan–Meier survival curves. Each curve indicates the survival of neonates from multiple dams treated with the indicated anti-IFNAR1/normal mouse IgG dose(s) and ZIKV infectious dose. The dash-dot line plotted on the Y-axis indicates median survival as determined by Kaplan–Meier. **** p < 0.0001 indicate a significant decrease in survival based on a Log-rank Mantel-Cox curve comparison test where the AIR low curve was compared to the IgR. b P14 naïve, IgR and AIR low pups were evaluated for ZIKV RNA by qRT-PCR with virus-specific primers. The dotted line in ( b ) indicates the limit of detection for the assay. Morphometric measurements from each hemisphere of 3 naïve, 10 IgR and 3 AIR low mice that survived to P14 are shown for ( c ) cerebellar area and ( d ) cortical thickness. Each symbol represents the mean of 3 measured sections from each hemisphere of each animal. The mean of the individually plotted data points for each group is represented by the horizontal black bars. A One-way ANOVA was used to compare cerebellar area ( c , F 2, 28 = 9.64, p = 0.0007) and cortical thickness ( d , F 2, 28 = 1.384, p = 0.2673) from samples from each group and a Tukey Multiple Comparisons Test was used to determine significance between groups. ** p < 0.01, *** p < 0.001, indicate significance between groups. n.s. = not significant. Representative H&E-stained sections from P14 AIR low ( e ) cerebellum and ( f ) spinal cord demonstrate degenerating cerebellar neurons ( e , black arrows) and axonal degeneration ( f , black arrows). The scale bar in ( f ) applies to ( e ). Representative cerebellar sections from ( g ) IgR and ( h ) AIR low pups demonstrating ZIKV NS5 (green) and Iba1 (magenta) immunofluorescence labeling. The scale bar in ( h ) applies to ( g )

Article Snippet: Primary antibodies against ZIKV NS2B (rabbit, 1:250, Genetex) or NeuN (rabbit, 1:100, Cell Signaling), or Calbindin-D-28 K (rabbit, 1:2250, Millipore) or Iba1 (rabbit, 1:1000, gift by John Portis (RML)) or GFAP (rabbit, 1:3000, Agilent/Dako) or transmembrane protein 119 (TMEM119, rabbit, 1:1000, Synaptic Systems) were applied for 1 h at 37 °C, except GFAP which was applied for 32 min at 37 °C.

Techniques: Infection, Activation Assay, Comparison, Quantitative RT-PCR, Virus, Staining, Immunofluorescence, Labeling

Journal: Journal of Biological Engineering

Article Title: Future developments in biosensors for field-ready Zika virus diagnostics

doi: 10.1186/s13036-016-0046-z

Figure Lengend Snippet: Number of peer-reviewed publications on ZIKV related to novel sensors development, topic reviews and commentaries, molecular biology and virology, and epidemiology or clinical evaluations of Zika cases (as of October 15, 2016). Cumulative publications are presented in 5 year increments until 2015 and 1 year increments between 2015 and 2016 ( top ). Publications in 2015–2016 are also presented separately by month ( bottom )

Article Snippet: Lateral flow assay , Chembio Diagnostic Systems , DPP Zika IgM/IgG assay.

Techniques:

Journal: Journal of Biological Engineering

Article Title: Future developments in biosensors for field-ready Zika virus diagnostics

doi: 10.1186/s13036-016-0046-z

Figure Lengend Snippet: Laboratory-based ZIKA assay kits

Article Snippet: Lateral flow assay , Chembio Diagnostic Systems , DPP Zika IgM/IgG assay.

Techniques: Enzyme-linked Immunosorbent Assay, Diagnostic Assay, Virus, Quantitative RT-PCR, Lateral Flow Assay, Immunofluorescence, Multiplex Assay, Luminex, Singleplex Assay, Amplification

Journal: Journal of Biological Engineering

Article Title: Future developments in biosensors for field-ready Zika virus diagnostics

doi: 10.1186/s13036-016-0046-z

Figure Lengend Snippet: a Biocan diagnostic’s Tell Me Fast™ Zika/Dengue/Chikugunya virus IgG/IgM lateral flow assay (reproduced from www.zikatest.com with permission from Biocan Diagnostics, Inc.). b Chemiluminescent particle immunoassay for ZIKV detection by magnetic separation and ultraviolet fluorescence (reproduced from ref. 68 with permission from the authors)

Article Snippet: Lateral flow assay , Chembio Diagnostic Systems , DPP Zika IgM/IgG assay.

Techniques: Virus, Lateral Flow Assay, Fluorescence

Journal: PLoS ONE

Article Title: Evaluation of eight commercial Zika virus IgM and IgG serology assays for diagnostics and research

doi: 10.1371/journal.pone.0244601

Figure Lengend Snippet: Sample panels used in the study.

Article Snippet: The test sensitivities of the ZIKV IgG ELISAs ranged from 52.6% to 84.2%, with Diapro ZIKV IgG ELISA yielding the highest sensitivity ( ).

Techniques: Diagnostic Assay, Infection, Quantitative RT-PCR, Indirect ELISA

Journal: PLoS ONE

Article Title: Evaluation of eight commercial Zika virus IgM and IgG serology assays for diagnostics and research

doi: 10.1371/journal.pone.0244601

Figure Lengend Snippet: List of ZIKV IgM and IgG serologic assays evaluated.

Article Snippet: The test sensitivities of the ZIKV IgG ELISAs ranged from 52.6% to 84.2%, with Diapro ZIKV IgG ELISA yielding the highest sensitivity ( ).

Techniques: Marker, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Evaluation of eight commercial Zika virus IgM and IgG serology assays for diagnostics and research

doi: 10.1371/journal.pone.0244601

Figure Lengend Snippet: (A) Sensitivity and specificity of ZIKV IgM rapid ICT, ELISAs and IIFT using acute samples from ZIKV-confirmed, DENV-confirmed, and non-ZIKV and non-DENV patients (Set A; n = 78). Sensitivity and specificity of ZIKV IgM/IgG rapid ICT, IgM ELISAs/IIFT and IgG ELISAs using (B) 7–14 days’ (Set B1; n = 57) and (C) 23–34 days’ (Set B2; n = 64) convalescent samples from ZIKV-confirmed, DENV-confirmed, and non-DENV patients. Confidence intervals (black bars) were constructed using Wald’s method.

Article Snippet: The test sensitivities of the ZIKV IgG ELISAs ranged from 52.6% to 84.2%, with Diapro ZIKV IgG ELISA yielding the highest sensitivity ( ).

Techniques: Construct

Journal: PLoS ONE

Article Title: Evaluation of eight commercial Zika virus IgM and IgG serology assays for diagnostics and research

doi: 10.1371/journal.pone.0244601

Figure Lengend Snippet: Test performance characteristics (AUROCC) in acute and convalescent patient samples.

Article Snippet: The test sensitivities of the ZIKV IgG ELISAs ranged from 52.6% to 84.2%, with Diapro ZIKV IgG ELISA yielding the highest sensitivity ( ).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Evaluation of eight commercial Zika virus IgM and IgG serology assays for diagnostics and research

doi: 10.1371/journal.pone.0244601

Figure Lengend Snippet: (A) Specificity of ZIKV IgM rapid ICT, ELISAs and IIFT using acute samples from DENV-confirmed (n = 30), and non-ZIKV and non-DENV (n = 30) patients. (B) Specificity of ZIKV IgM/IgG rapid ICT, IgM ELISAs/IIFT and IgG ELISAs using 7–34 days’ convalescent samples from DENV-confirmed (n = 30), and non-DENV patients (n = 30) (Set B). Confidence intervals (black bars) were constructed using Wald’s method. LumiQuick QuickProfile™ ZIKV IgM & IgG rapid ICTs refer to a combo test kit.

Article Snippet: The test sensitivities of the ZIKV IgG ELISAs ranged from 52.6% to 84.2%, with Diapro ZIKV IgG ELISA yielding the highest sensitivity ( ).

Techniques: Construct

Journal: PLoS ONE

Article Title: Evaluation of eight commercial Zika virus IgM and IgG serology assays for diagnostics and research

doi: 10.1371/journal.pone.0244601

Figure Lengend Snippet: Results of ZIKV IgG ELISA using healthy blood donor samples.

Article Snippet: The test sensitivities of the ZIKV IgG ELISAs ranged from 52.6% to 84.2%, with Diapro ZIKV IgG ELISA yielding the highest sensitivity ( ).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Evaluation of eight commercial Zika virus IgM and IgG serology assays for diagnostics and research

doi: 10.1371/journal.pone.0244601

Figure Lengend Snippet: ZIKV seropositivity in convalescent primary and secondary DENV patient samples.

Article Snippet: The test sensitivities of the ZIKV IgG ELISAs ranged from 52.6% to 84.2%, with Diapro ZIKV IgG ELISA yielding the highest sensitivity ( ).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A systematic evaluation of IgM and IgG antibody assay accuracy in diagnosing acute Zika Virus infection in Brazil; lessons relevant to emerging infections

doi: 10.1101/2020.11.25.399386

Figure Lengend Snippet: Zika antibody detected in serum samples collected during the acute (1-6 days after onset), early-convalescent phase (7-13d) and late convalescent-phase (≥14d) from PCR positive Zika cases measured by IgM (A) or IgG (B) NS1 anti-ZIKV ELISAs (Euroimmun). C) IgM NS1 anti-ZIKV ELISA measurements for acute (1-6 days after onset) and convalescent (≥7 days) samples from PCR positive ZIKV cases. D) IgG NS1 anti-ZIKV antibody levels in paired serum samples from PCR positive ZIKV positive cases. Dotted horizontal lines represent the cut-off value used in each assay. Data points above the cut-off are considered positive. Trend-line in C) and D) represent the median antibody levels for acute and convalescent samples. Statistically significant differences between two groups were measured by Mann Whitney U test (*** p=0.0001). Figure shows antibody Ratios* calculated as per manufacturers’ instructions (Antibody Ratio = OD Sample/OD Calibrator).

Article Snippet: Assay sensitivity decreased for specimens collected ≥14 days in the IgM and IgG ZIKV Euroimmun, IgM Novagnost and IgM DENV assay.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: A systematic evaluation of IgM and IgG antibody assay accuracy in diagnosing acute Zika Virus infection in Brazil; lessons relevant to emerging infections

doi: 10.1101/2020.11.25.399386

Figure Lengend Snippet: The change in IgG NS1 Euroimmun anti-ZIKV antibody levels between paired serum samples from PCR positive Zika cases by day of collection (days post symptom onset) of the first (acute) sample. Based on the first sample (collected 0 −7 days) and second sample (median interval between samples was 7 days). The highest fold change (change in antibody level between 1 st and 2 nd samples) was observed among paired samples collected on days 2 and 7 post symptom onset.

Article Snippet: Assay sensitivity decreased for specimens collected ≥14 days in the IgM and IgG ZIKV Euroimmun, IgM Novagnost and IgM DENV assay.

Techniques:

Journal: bioRxiv

Article Title: A systematic evaluation of IgM and IgG antibody assay accuracy in diagnosing acute Zika Virus infection in Brazil; lessons relevant to emerging infections

doi: 10.1101/2020.11.25.399386

Figure Lengend Snippet: Anti-ZIKV antibody levels in sequential serum samples collected from Zika PCR positive cases on different days post symptom onset (0-54 days) measured in A) IgM Euroimmun NS1 and B) IgG Euroimmun NS1 anti-ZIKV ELISAs. Dotted line shows assay cut-offs. The figure shows more consistent detection of anti-Zika antibodies (level above the cut-off) among convalescent samples when measuring IgG compared to IgM. Ratios calculated as per manufacturers’ instruction; 1st collection (acute sample [closed circles]); 2nd collection (convalescent samples [open squares]); 3rd collection (late convalescent samples [closed triangles])

Article Snippet: Assay sensitivity decreased for specimens collected ≥14 days in the IgM and IgG ZIKV Euroimmun, IgM Novagnost and IgM DENV assay.

Techniques:

Journal: bioRxiv

Article Title: A systematic evaluation of IgM and IgG antibody assay accuracy in diagnosing acute Zika Virus infection in Brazil; lessons relevant to emerging infections

doi: 10.1101/2020.11.25.399386

Figure Lengend Snippet: Correlation between anti-ZIKV and anti-DENV antibody levels in individual sera samples. A) IgG anti-DENV ELISA (Panbio) versus IgG anti-ZIKV NS1 (Euroimmun) ELISA. Anti-DENV and anti-ZIKV IgG antibody levels showed a positive correlation. When a patient exhibited a relatively high anti-DENV IgG antibody response they also tended to exhibit a relatively high anti-ZIKV IgG antibody response (p<0.001; r2=0.258; n=168); B) IgM DENV ELISA and IgM ZIKV NS1 ELISA antibody levels. Again the measurements showed a positive correlation (p=0.015; r2=0.015; n=166). Dotted lines show assay cut-offs. Dashed line shows the best-fitting line (Spearman rank correlation [r2]). Correlation was more significant between anti-ZIKV and anti-DENV IgG antibody measurements. The correlation in antibody measurement between ZIKV and DENV ELISAs suggests a degree of overlap in patient responses and/or cross-reaction in antibody detection.

Article Snippet: Assay sensitivity decreased for specimens collected ≥14 days in the IgM and IgG ZIKV Euroimmun, IgM Novagnost and IgM DENV assay.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A systematic evaluation of IgM and IgG antibody assay accuracy in diagnosing acute Zika Virus infection in Brazil; lessons relevant to emerging infections

doi: 10.1101/2020.11.25.399386

Figure Lengend Snippet: Receiver Operating Characteristic (ROC) curve comparing sensitivity and specificity at different cut-off values for the anti-ZIKV IgG NS1 ELISA (n=294 sera); A) ROC Curve; B) Specificity and Sensitivity at each cut-off. The dotted line in B indicates the cut-off recommended by the manufacturer (ratio of 1.1). The accuracy of IgG NS1 ELISA was 77.9% using the manufacturer’s cut-off. Higher accuracy was observed when the cut-off was increased to 1.5 (where the curves intersect on plot B). Using this cut-off, the ELISA had an accuracy of 81.0%. Sensitivity and specificity were 78.9 and 82.2% respectively.

Article Snippet: Assay sensitivity decreased for specimens collected ≥14 days in the IgM and IgG ZIKV Euroimmun, IgM Novagnost and IgM DENV assay.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A systematic evaluation of IgM and IgG antibody assay accuracy in diagnosing acute Zika Virus infection in Brazil; lessons relevant to emerging infections

doi: 10.1101/2020.11.25.399386

Figure Lengend Snippet: Diagram representing the different patterns of anti-viral IgG and IgM antibody responses and viral RNA detection observed in sera over days from symptom onset among (A) virus naïve and (B) previously exposed individuals. The cartoon exhibits a more prominent IgG response compared to IgM among individuals previously exposed to the virus. In our current study, we observed a more prominent anti-ZIKV IgG compared to IgM response (see ).

Article Snippet: Assay sensitivity decreased for specimens collected ≥14 days in the IgM and IgG ZIKV Euroimmun, IgM Novagnost and IgM DENV assay.

Techniques: RNA Detection

Journal: eClinicalMedicine

Article Title: Evaluation of Zika rapid tests as aids for clinical diagnosis and epidemic preparedness

doi: 10.1016/j.eclinm.2022.101478

Figure Lengend Snippet: Chembio Diagnostics DPP® ZCD IgM/IgG (Zika/Chikungunya/Dengue IgM/IgG) System and SD Biosensor (SDB) STANDARD Q Arbo Panel Test. The Chembio ZIKV, CHIKV, and DENV multiplex serology assay system consists of a single cassette where the sample and buffer are placed are placed into a well marked 1 and a second buffer added into well 2. The results are read by placing a RFID microreader over the IgM and IgG windows where result for Zika antibodies can be read in Line 1, chikungunya virus antibodies in Line 2, dengue virus antibodies in Line 3 and C is the control line. For the Chembio Diagnostics DPP® Zika IgM/IgG System, the test cassette is similar to the ZCD IgM/IgG system except that each result window only contains 2 lines, one for the zika antibodies and the other for the control line (picture not shown) . SD Biosensor (SDB) STANDARD Q Arbo Panel Test: the SD Biosensor assay is an immunochromatographic assay for the detection of Dengue NS1 Antigen and IgM antibodies to Zika/Dengue/Chikungunya/Yellow fever virus. It consists of 5 separate cartridges, where each requires specimen and buffer addition. The results can be read visually.

Article Snippet: The Chembio Dual Path Platform (DPP)® Zika IgM/IgG System is a rapid 10–15 min immunochromatographic test for the detection and differentiation of IgM and IgG antibodies to ZIKV in 10 μl fingerstick whole blood, Ethylenediaminetetraacetic acid (EDTA) anticoagulated venous whole blood, serum, or EDTA-anticoagulated plasma samples ( ).

Techniques: Multiplex Assay, Virus, Control, Biosensor Assay, Immunochromatographic Assay

Journal: eClinicalMedicine

Article Title: Evaluation of Zika rapid tests as aids for clinical diagnosis and epidemic preparedness

doi: 10.1016/j.eclinm.2022.101478

Figure Lengend Snippet: ZikaPLAN network of biobank and evaluation sites. The evaluation of Zika virus Rapid Diagnostic Tests (RDTs) submitted under the UNICEF tenders took place across a global network to source needed specimens. Text box on left shows steps in evaluation process. The London School of Hygiene & Tropical Medicine (LSHTM) provided the overall coordination. Triage using the ´Sudden Death´ panel was only performed at IPD.

Article Snippet: The Chembio Dual Path Platform (DPP)® Zika IgM/IgG System is a rapid 10–15 min immunochromatographic test for the detection and differentiation of IgM and IgG antibodies to ZIKV in 10 μl fingerstick whole blood, Ethylenediaminetetraacetic acid (EDTA) anticoagulated venous whole blood, serum, or EDTA-anticoagulated plasma samples ( ).

Techniques: Virus, Diagnostic Assay

Journal: eClinicalMedicine

Article Title: Evaluation of Zika rapid tests as aids for clinical diagnosis and epidemic preparedness

doi: 10.1016/j.eclinm.2022.101478

Figure Lengend Snippet: Summary of the Performance of the Chembio DPP® Zika IgM test.

Article Snippet: The Chembio Dual Path Platform (DPP)® Zika IgM/IgG System is a rapid 10–15 min immunochromatographic test for the detection and differentiation of IgM and IgG antibodies to ZIKV in 10 μl fingerstick whole blood, Ethylenediaminetetraacetic acid (EDTA) anticoagulated venous whole blood, serum, or EDTA-anticoagulated plasma samples ( ).

Techniques: