EHV1 shedding data

This data set contains information obtained in three experimental infection studies on horses. The data table consists of 6 columns: (1) “shedding_amount” contains the estimated number of EHV-1 genome copies per mL nasal swab solution, (2) ”individual” contains an index for the identity of each animal, (3) “EHV1_variant” indicates the EHV1 variant that was used for infection, (4) “experiment” indicates to which experiment the data point belongs, (5) “day” describes on which day after infection the sample was collected, and (6) “experiment_day” is a combination of experiment and day

Protocol outline for the siRNA transfection/infection experiments in mice.

a<p>I: infection.</p>b<p><i>control</i>: to evaluate weight loss due to anesthesia.</p

siRNA sequences.

a<p>bold indicates siRNA's that showed significant reduction of EHV-1 replication.</p

siRNA's are effective in reducing viral replication when applied before infection, even in the absence of a transfection reagent.

<p>In a first experiment, Balb/c mice (groups of 12) were transfected intranasally with sigB3 and siOri2, alone or in combination, and mice were infected intranasally with 1×10⁵ PFU of Ab4 0.5 h later (A). In a second experiment, Balb/c mice (groups of 12) were inoculated intranasally with 62.5 pmol sigB3/siOri2, complexed with lipofectamine (closed symbols) or in PBS (open symbols) 6 or 12 h before infection with 1×10⁵ PFU of Ab4 (B). Mice transfected with 75 pmol siLuc were used as positive controls and viral titers were determined in three mice of each group on day 2 p.i. Titers in lungs and standard deviations are shown. Asterisks indicate statistically significant differences (p<0.05) between mice transfected with sigB3 and siOri2, alone or in combination, and mice transfected with the control siRNA siLuc.</p

Effect of siRNA's targeting glycoprotein B (gB) and origin-binding protein (ori) helicase on EHV-1 replication and cell-to-cell spread.

<p>(A). siRNA's targeting gB (sigB3) or Ori (siOri2) were transfected into RK13 cells and cells were infected with 100 PFU of EHV-1 strain rAb4Δgp2 14 h later. Supernatants were collected 24 h p.i. from sigB3- (□) and siOri2-transfected cells (◊), and viral titers were determined with standard plaque assays. Asterisks indicate statistically significant differences (*: p<0.05, **: p<0.01). (B). Cells were fixed with 10% formalin and the average plaque areas were determined. Asterisks indicate statistically significant differences (p<0.05). (C). As controls cells were included, which were either not transfected with siRNA or were transfected with 75 pmol siGFP or siLuc before infection with rAb4Δgp2. Representative pictures were taken with light and fluorescent microscopy (a) and viral titers were determined with standard plaque assays (b).</p

siRNA's efficiently suppresses gB and Ori expression at the mRNA and protein level.

<p>(A). Relative qRT-PCR. RK13 cells were transfected with 37.5 pmol sigB3, 75 pmol siOri2, 75 pmol siLuc or not transfected. Cells were infected 14 h later with 500 PFU of rAb4Δgp2 and at 12 h p.i., RNA was extracted from infected cells. RT-PCR was used to determine relative levels of gB or Ori (white bars) or EHV-1 IR6 (grey bars) mRNA, using rabbit β-actin as the endogenous housekeeping gene. Asterisks indicate statistically significant differences (p<0.05). (B). Western blot analysis. RK13 cells were either not transfected or transfected with 75 pmol of the control siRNA siLuc or 75 pmol sigB3. Cells were infected 14 h later with 500 PFU of rAb4Δgp2. At 24 h p.i., cell lysates were prepared and analyzed by SDS-PAGE under reducing conditions. Anti-gB mAb 3F6 (1/500) and anti-β-actin (1/5000), followed by anti-mouse IgG peroxidase (1/7500) were used. Cell lysates from non-infected RK13 cells were included as a control.</p

Primers and Probes used in the study.

a<p><b>F:</b> Forward primer, <b>R:</b> Reverse primer.</p

Combining siRNA's have an additive effect on reduction of EHV-1 replication and are effective before and after infection.

<p>(A). The siRNA's sigB3 and siOri2 were transfected into RK13 cells, either alone or in different combinations, and cells were infected 14 h later with 500 PFU of rAb4Δgp2. Supernatants were collected 24 h p.i. and viral titers were determined with standard plaque assays. Asterisks indicate statistically significant differences (p<0.05). (B). RK13 cells were transfected with 75 pmol control siRNA (black bars), 37.5 pmol sigB3 (grey bars), 75 pmol siOri2 (hatched bars) or a combination of 6.25 pmol sigB3 and 6.25 pmol siOri2 (white bars). At different times after transfection (ranging form 12 h up to 0 h) cells were infected with 500 PFU of rAb4Δgp2 and supernatants were collected at 24 h p.i. In one set of experiments, cells were first infected with rAb4Δgp2 and transfected with the different siRNA's 1 h later. Viral titers were determined with standard plaque assays. Asterisks indicate statistically significant differences (p<0.05).</p

siRNA's are effective in reducing viral replication when applied after infection.

<p>Balb/c mice (groups of 15) were infected intranasally with 1×10⁵ PFU of Ab4. At 1, 6, 12 or 24 h post-infection, mice were inoculated intranasally with 62.5 pmol sigB3/siOri2, in PBS. (A). Viral titers were determined in lungs of five mice per group on day 3 p.i. by co-cultivation. Titers in lungs and standard deviations are shown. (B). Viral loads in lung tissues were also measured by qPCR and are plotted as EHV-1 genome (IR6 gene) copies per million mouse iNOS gene copies. Asterisks indicate statistically significant differences (p<0.05; Student's t-test) between untreated mice and mice inoculated with 62.5 pmol sigB3/siOri2.</p