In Vitro Characterization of a Nineteenth-Century Therapy for Smallpox

In the nineteenth century, smallpox ravaged through the United States and Canada. At this time, a botanical preparation, derived from the carnivorous plant Sarracenia purpurea, was proclaimed as being a successful therapy for smallpox infections. The work described characterizes the antipoxvirus activity associated with this botanical extract against vaccinia virus, monkeypox virus and variola virus, the causative agent of smallpox. Our work demonstrates the in vitro characterization of Sarracenia purpurea as the first effective inhibitor of poxvirus replication at the level of early viral transcription. With the renewed threat of poxvirus-related infections, our results indicate Sarracenia purpurea may act as another defensive measure against Orthopoxvirus infections

The effect of <i>S. purpurea</i> extracts on VACV induced CPE and protein synthesis.

<p>A and C) HeLa cells were infected with VACV at an MOI = 10 followed by the addition of 25 microL <i>S. purpurea</i> extract/ml media immediately (0 min) or at 15, 30, 60 or 120 min post infection. ‘No treatment’ cells received ethanol/glycerol carrier only. For (A), at 6 HPI, the cell monolayers were photographed. For (C), at 3 HPI, cell lysates were prepared and the VACV E3L protein or total VACV proteins detected by Western blot. * indicate the position of VACV proteins and the VACV-E3L protein. Duplicate experiments were done at 6 HPI (not shown) B) Hela cells were infected with a VACV construct expressing cyan fluorescent protein fused to the viral A5 core protein. In the first panel, the infection was maintained at 4°C. For the middle and last panel, the infection was done at 37°C, in the absence or presence of <i>S. purpurea</i>, respectively. D) HeLa cells were mock infected or infected with VACV at an MOI = 10 followed by the addition of 25 microL <i>S. purpurea</i> extract/ml media or ethanol/glycerol carrier. At 4 HPI, the cell monolayers were radiolabelled with [³⁵S]-methionine, cell lysates prepared, proteins separated by SDS-PAGE, and visualized by autoradiography. * indicate the position of VACV proteins.</p

Mechanism of action of poxvirus therapeutics.

<p>Illustration indicates the general replication cycle of VACV. The previously shown targets of known antipoxvirus compounds, cidofovir and ST-246, are shown, as well as the presumptive target of the <i>S. purpurea</i> extract.</p

The specificity of <i>S. purpurea</i> extracts on <i>Orthopoxvirus.</i>

<p>A) HeLa cells were mock-infected or infected with monkeypox virus (MPXV) at an MOI = 10 followed by the addition of ethanol/glycerol carrier or 25 microL <i>S. purpurea</i> extract/ml media, either 0 or 15 min after infection. At 4 HPI, cell lysates were prepared and the MPXV F3L protein detected by Western blot. B) HeLa cells were mock-infected or infected with variola virus (VARV) at an MOI = 10 followed by the addition of the indicated concentrations of ethanol/glycerol carrier or <i>S. purpurea</i> extract to the media at 0 min after infection. At 4 HPI, cell lysates were prepared and the VARV E3L protein detected by Western blot. C) HeLa cells were infected with adenovirus (Adeno), vesicular stomatitis virus (VSV), mouse hepatitis virus (MHV) or reovirus (reo) followed by the addition of ethanol/glycerol carrier or 25 microL <i>S. purpurea</i> extract/ml media. At 15 min and 8 hours post-infection, cell lysates were prepared and representative viral proteins detected by Western blot (E1A, G, E, and core proteins, respectively). D) HeLa cells were infected with VACV at an MOI = 10 followed by the addition of ethanol/glycerol carrier (none) or <i>S. purpurea</i> extract, <i>Echinacea</i> extract, <i>Astragalus</i> extract, <i>Coriolus</i> extract, or <i>Glycyrrhiza</i> extract (25 microL/ml media). At 4 HPI, cell lysates were prepared and the VACV E3L protein detected by Western blot.</p

The effect of <i>S. purpurea</i> extracts on VACV transcription <i>in vivo</i> and <i>in vitro.</i>

<p>A) HeLa cells were infected with VACV at an MOI = 10 followed by the addition of 25 microL <i>S. purpurea</i> extract/ml media. At 4 HPI, total RNA was isolated and VACV-E3L RNA levels determined by real-time PCR. Reactions contained total RNA from mock-infected cells, ethanol/glycerol carrier-treated VACV-infected cells, or <i>S. purpurea</i>-treated VACV-infected cells. C(t) values were calculated using manufacturer's software. Graph illustrates data from a representative experiment. C(t) values and fold change from two separate experiments are shown. B) Purified VACV virion cores were incubated in the presence of the indicated concentrations of <i>S. purpurea</i> or ethanol/glycerol carrier and [³⁵S]-UTP. Newly synthesized RNA products were spotted onto glass-fiber filters, washed in TCA, and quantified by scintillation counting (counts per minute). A no template reaction was performed by excluding the addition of the virion cores.</p

The effect of <i>S. purpurea</i> extracts on VACV replication.

<p>A) RK-13 cells were infected with 150 pfu of VACV followed by the addition of the indicated concentration of <i>S. purpurea</i> extract to the cell culture media. Cells were treated one-time only with the extract (black bars) or every 6 hours with fresh extract (gray bars). After 48 hours, plaques were visualized and quantified. Error bars represent standard deviation (n = 3). B) RK-13 cells were infected with VACV at an multiplicity of infection (MOI) = 10 followed by addition of ethanol/glycerol carrier (closed diamonds) or the addition of 25 microL <i>S.purpurea</i> extract/ml media (open squares and closed triangles). Cells were treated one-time only with the extract (open squares) or every 6 hours with fresh extract (closed diamonds). Cells were harvested at the indicated times and viral titers determined. Error bars represent deviation between assays (n = 2). C) For viral translation levels (closed squares), HeLa cells were infected with VACV at an MOI = 10 followed by the addition of the indicated concentrations of <i>S. purpurea</i> extract/ml media. At 6 HPI, cell lysates were prepared, the VACV E3L protein detected by Western blot, and quantified. For cell viability, HeLa cells were treated with the indicated concentrations of <i>S. purpurea</i> extract (closed diamonds) or ethanol/glycerol carrier (closed triangles) for 6 hours and the number of viable cells determined by a trypan blue exclusion assay. D) For viral plaque formation (closed squares), HeLa cells were infected with VACV (approx. 200 pfu) followed by the addition of the indicated concentrations of cidofovir to the media. After 48 hours, plaques were visualized and quantified. Error bars represent standard deviation (n = 3). For cell viability, HeLa cells were treated with the indicated concentrations of cidofovir (closed diamonds) for 48 hours and the number of viable cells determined by a trypan blue exclusion assay. E) HeLa cells were mock-infected or infected with VACV at an MOI = 10 followed by the immediate addition of media, 25 microL <i>S. purpurea</i> extract/ml media, or 320 microg cidofovir/ml media. At 4 HPI, the cell monolayers were photographed.</p