Multi-color fluorescent reporter dengue viruses with improved stability for analysis of a multi-virus infection.

Reporter virus is a versatile tool to visualize and to analyze virus infections. However, for flaviviruses, it is difficult to maintain the inserted reporter genes on the viral genome, limiting its use in several studies that require homogeneous virus particles and several rounds of virus replication. Here, we showed that flanking inserted GFP genes on both sides with ribosome-skipping 2A sequences improved the stability and the consistency of their fluorescent signals for dengue-virus-serotype 2 (DENV2) reporter viruses. The reporter viruses can infect known susceptible mammalian cell lines and primary CD14+ human monocytes. This design can accommodate several fluorescent protein genes, enabling the generation of multi-color DENV2-16681 reporter viruses with comparable replication capabilities, as demonstrated by their abilities to maintain their fluorescent intensities during co-infections and to exclude superinfections regardless of the fluorescent tags. The reported design of multi-color DENV2 should be useful for high-throughput analyses, single-cell analysis, and characterizations of interference and superinfection in animal models

Isolation of bioactive compounds from medicinal plants used in traditional medicine: Rautandiol B, a potential lead compound against Plasmodium falciparum

Background: Neorautanenia mitis, Hydnora abyssinica, and Senna surattensis are medicinal plants with a variety of traditional uses. In this study, we sought to isolate the bioactive compounds responsible for some of these activities, and to uncover their other potential medicinal properties. Methods: The DCM and ethanol extracts of the roots of N. mitis and H. abyssinica, and the leaves of S. surattensis were prepared and their phytochemical components were isolated and purified using chromatographic methods. These extracts and their pure phytochemical components were evaluated in in-vitro models for their inhibitory activities against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Mycobacterium tuberculosis, α-amylase (AA), and α-glucosidase (AG). Results: Rautandiol B had significant inhibitory activities against two strains of Plasmodium falciparum showing a high safety ratio (SR) and IC50 values of 0.40 ± 0.07 μM (SR - 108) and 0.74 ± 0.29 μM (SR - 133) against TM4/8.2 and K1CB1, respectively. While (−)-2-isopentenyl-3-hydroxy-8-9-methylenedioxypterocarpan showed the highest inhibitory activity against T. brucei rhodesiense with an IC50 value of 4.87 ± 0.49 μM (SR \u3e 5.83). All crude extracts showed inhibitory activities against AA and AG, with three of the most active phytochemical components; rautandiol A, catechin, and dolineon, having only modest activities against AG with IC50 values of 0.28 mM, 0.36 mM and 0.66 mM, respectively. Conclusion: These studies have led to the identification of lead compounds with potential for future drug development, including Rautandiol B, as a potential lead compound against Plasmodium falciparum. The relatively higher inhibitory activities of the crude extracts against AG and AA over their isolated components could be due to the synergistic effects between their phytochemical components. These crude extracts could potentially serve as alternative inhibitors of AG and AA and as therapeutics for diabetes

Infectivity of DENV2-eGFP with the 2x 2A design in human peripheral blood mononuclear cells (PBMC).

<p>PBMCs were infected with DENV2-eGFP in the presence of 4G2 antibody (1 μg/ml). Top row: Monocytes and lymphocytes were gated based on size (FSC-A) and granularity (SSC-A) (far left). The lymphocyte population was then divided into B cells (CD19+CD3-) and T cells (CD19-CD3+). Middle row: Frequency of DENV2-eGFP positive cells in monocyte (CD14+) population (mock infection, left; infection with virus but no antibody, middle; infection with virus and antibody (ADE), right). DENV2-eGFP positive cells in individual populations are boxed in each plot. Bottom row: Frequency of DENV2-positive monocytes infected with wild-type DENV2-16681 (left; infection with virus but no antibody, right; infection with virus and antibody (ADE), right).</p

A panel of multi-color fluorescent DENV2 with comparable replicative abilities.

<p>A) Multi-step replication kinetics of multi-color fluorescent DENV2. Confluent Vero cells were infected with one virus at MOI = 0.01. The culture media was sampled from the flask every 24 hours. Infectious virus titer in the media was assayed by foci-forming assay. B) Focus images of the wild-type (16681) and the multi-color DENV2 (top panel) and the bar graph comparing the sizes of their foci (bottom panel). Focus size was quantified by the number of pixels that it occupied on a digitized image taken on ELISpot reader. Each bar represents the average of the measured focus sizes and the error bars represent standard deviation. Between 143 and 713 foci were used for focus size quantification. C) Representative scatter plots of K562-CD209 infected with DENV2-eGFP and DENV2-mCherry. The first (with DENV2-eGFP) and the second (with DENV2-mCherry) infections were 24 hours apart. The infected cells were analyzed 3 days post infection. In the case of co-infection (right plot), the cells were infected by both viruses at the same time and analyzed 2 days post infection. D) Comparison of infection percentages of the second virus in K562-CD209 between the presence (diagonal-stripe bars) and the absence (empty-bar) of the first virus infections. The infection percentages in the absence of the first virus infections are normalized to 100% for comparing the extent of reduction (or exclusion) conferred by the first virus. E) Histograms of fluorescent intensities for each fluorescent reporter measured from populations of K562-CD209 infected with single virus, and co-infected with two, three, and four reporter DENV2 of different colors.</p

Inability of the reporter DENV2 to replicate in mosquito host.

<p>A) Fluorescent microscopy (top panel) and flow cytometry (bottom panel) of C6/36 cells infected with DENV2-16681 (WT) and DENV2-GFPs with 1x 2A and 2x 2A designs. C6/36 cells were infected with a DENV2 virus at MOI = 0.1 and cultured for 3 days before analyses. Infected cells were identified by immunostaining with 4G2 antibody. The number in each quadrant of the flow cytometry scatter plot represents the percentage of cells in the quadrant. B) Fluorescent microscopy of <i>Aedes aegypti</i> mosquitoes that received DENV2-16681 (WT), DENV2-Clover2-2x 2A, and DENV2-eGFP-2x 2A by intrathoracic injection. The mosquitoes were imaged seven days after injection. C) Propagation of viruses in the mosquitoes injected with DENV2-16681 (WT), DENV2-Clover2-2x 2A, and DENV2-eGFP-2x 2A. The infectious titer (FFU/mosquito) was quantified from the whole-body homogenate of a mosquito by immunostaining foci assay at 4, 7, and 10 days post intrathoracic injection (dpi). The table underneath the plot reports the descriptive statistics of the infectious-titer measurements that include the number of the mosquitoes (n), the minimum (Min), the maximum (Max), the median, the mean, and the standard deviation (SD) of the infectious titers for each condition.</p

Properties of fluorescent proteins, laser and detection filter in flow cytometer.

<p>Properties of fluorescent proteins, laser and detection filter in flow cytometer.</p

Improved consistency of cellular fluorescent distribution after separation of GFP genes from C25 by 2A ribosome skipping.

<p>A) Design of FP reporter gene insertion in the DENV2 genome. P2A = 2A sequence of porcine technovirus-1. T2A = 2A sequence of Thoseasigna virus. 1x 2A denotes the reporter DENV2 design with one 2A sequence at the end of the reporter gene, while 2x 2A denotes the design with two 2A sequences flanking the reporter gene. B) Fluorescent images of Vero, Huh7, and BHK21 cells infected with DENV2-FPs with the 1X 2A design. C) Fluorescent images of Vero, Huh7, and BHK21 cells infected with DENV2-FPs with the 2X 2A design. D) Replication kinetics of DENV2-eGFP (right) and DENV2-clover2 (left) with 1x 2A and 2x 2A designs in Vero cells. Control DENV2 virus without a reporter gene and 2A sequences is denoted as 16681. Confluent Vero cells were infected with one virus at the multiplicity of infection (MOI) = 0.01. Infectious virus titer in the media was quantified by focus-forming assay. Each point is the average of three replicates with error bar representing the standard deviation. E) Focus images of the wild-type and the reporter DENV2-FPs (top panel) and the bar graph comparing the sizes of their foci (bottom panel). The size of each focus was quantified by the number of pixels that it occupied on a digitized image taken on ELISpot reader. Each bar represents the average of the measured focus sizes and error bars represent the standard deviation. At least 227 foci of each virus were used for foci-size quantification.</p

Improved stability of GFP reporter genes on DENV2 genome with 2x 2A design.

<p>A) Fluorescent microscopy of infected Vero cells during passaging. The left diagram outlines the passaging of a reporter virus. MOI of 0.01 was used to infect confluent Vero cells for each passage. Green-fluorescent images of infected Vero cells (right panel) were taken seven days post infection. P1, P2, P3, P4, P5, and P6 designate passage numbers. 1x 2A and 2x 2A denote the design of the reporter viruses. B) Flow-cytometry measurements of Vero cells infected with passaged DENV2-eGFP. Confluent Vero cells were infected with viruses from each passage (culture media collected at day 7) at MOI = 0.1. Infected cells were maintained for two days before whole-cell staining with 4G2 mouse antibody and Alexa-647-conjugated anti-mouse antibody. The percentage of each cell population is shown in each quardrant of the scatter plot. The top row shows the measurements of passaged DENV2-eGFP with the 1x 2A design while the bottom row show those of 2x 2A design. C) Quantification of the stability of GFP reporter genes based on flow-cytometry measurements. The stability was measured as the percentage of GFP-positive cells in the populations of infected cells (4G2-positive cells). % GFP/4G2 = (% cells with GFP)/(% cells with 4G2). D) RT-PCR of passaged DENV2-eGFP. Top diagram shows the locations of the primer binding sites (represented by arrows) on the viral genome of reporter DENV2-eGFP. Bottom is the image of agarose gel electrophoresis of the RT-PCR products of viral RNA extracted from passaged viruses.</p

Sequences of primers and protospacer sequence of gRNAs used in this work.

<p>Sequences of primers and protospacer sequence of gRNAs used in this work.</p