Interaction of silver nanoparticles with Tacaribe virus

Abstract

Background: Silver nanoparticles possess many unique properties that make them attractive for use in biological applications. Recently they received attention when it was shown that 10 nm silver nanoparticles were bactericidal, which is promising in light of the growing number of antibiotic resistant bacteria. An area that has been largely unexplored is the interaction of nanomaterials with viruses and the possible use of silver nanoparticles as an antiviral agent.

Results: This research focuses on evaluating the interaction of silver nanoparticles with a New World arenavirus, Tacaribe virus, to determine if they influence viral replication. Surprisingly exposing the virus to silver nanoparticles prior to infection actually facilitated virus uptake into the host cells, but the silver-treated virus had a significant reduction in viral RNA production and progeny virus release, which indicates that silver nanoparticles are capable of inhibiting arenavirus infection in vitro. The inhibition of viral replication must occur during early replication since although pre-infection treatment with silver nanoparticles is very effective, the post-infection addition of silver nanoparticles is only effective if administered within the first 2-4 hours of virus replication.

Conclusions: Silver nanoparticles are capable of inhibiting a prototype arenavirus at non-toxic concentrations and effectively inhibit arenavirus replication when administered prior to viral infection or early after initial virus exposure. This suggests that the mode of action of viral neutralization by silver nanoparticles occurs during the early phases of viral replication.

Figure 1

Biocompatibility of Ag-NPs in Vero cells. Vero cells were exposed to Ag-NPs for 24 hours (A), 48 hours (B), or 8 days (C) and the cell viability was determined using a standard MTS assay. The effects of the Ag-NPs on cellular viability are expressed as percent of control (untreated Vero cells) with error bars representing standard error of the mean (S.E.M.).

Figure 2

TCRV replication following exposure to Ag-NPs. TCRV was treated with uncoated and PS-coated 10 and 25 nm Ag-NPs for 1 hour. Treated or control virus suspensions were used to infect Vero cells for 8 days. Progeny virus was recovered from the cell culture supernatant and was quantified using a standard tissue culture infectious dose assay. Progeny virus titers are expressed as the mean log₁₀ TCID₅₀/mL +/- S.E.M. (T = concentration of nanoparticles was toxic at this dose; *p < 0.05; student's t test; n = 6).

Figure 3

Confocal imaging of untreated and Ag-NP-treated TCRV in Vero cells. TCRV was labeled with a fluorophore that excites at a wavelength of 488 nm and was treated with Ag-NPs for 1 hour. Treated or control virus was used to infect Vero cells for 4 hours. The supernatant was removed and the cells were washed 2 times with PBS to remove non-adherent virus and were fixed in 3% paraformaldehyde. The nuclei were stained with Hoechst (blue) and the images were taken using spinning disc Confocal microscopy with the pictures representative of collapsed z-stacks of sections through the cell (15 sections). (a) Vero cells alone (b) TCRV in Vero cells (c) TCRV + 10 nm Ag 50 μg/ml (d) TCRV + 10 nm Ag 10 μg/ml (e) TCRV + 10 nm PS-Ag 50 μg/ml (f) TCRV + 10 nm PS-Ag 10 μg/ml (g) TCRV + 25 nm Ag 50 μg/ml (h) TCRV + 25 nm Ag 10 μg/ml (i) TCRV + 25 nm PS-Ag 50 μg/ml (j) TCRV + 25 nm PS-Ag 10 μg/ml (representative picture of 3 separate trials).

Figure 4

TEM of TCRV internalization into Vero cells. TEM micrographs depict Vero cells infected with untreated TCRV (a, b), 10 nm (uncoated, 50 μg/ml) treated TCRV (c, d), or 25 nm (uncoated, 50 μg/ml) treated TCRV (e, f) with b, d, and f being zoomed-in images of the white squares of a, b, and c. Images g and h depict virus and the Ag-NPs localizing within the same cell, and i and j depict the interaction of TCRV with the Ag-NPs outside of the cell. White arrows are pointing towards the virus and black arrows show the Ag-NPs.

Figure 5

S segment quantitative real time PCR analysis. TCRV was treated with uncoated and PS-coated 10 and 25 nm Ag-NPs for 1 hour. Treated or control virus was used to infect Vero cells for 4 days. The supernatant was removed and the cells were washed 2 times with PBS to remove non-adherent virus. RNA was isolated from the Vero cells and S segment gene expression was determined using qRT-PCR with Sybr green. Changes in S segment gene expression are expressed as fold change over untreated TCRV infection with error bars representing the S.E.M. and statistical analysis comparing the untreated control to each of the treatment groups. (*p < 0.05; student's t test; n = 6).

Figure 6

Vero cells were infected with TCRV and were exposed to 10 nm Ag-NPs at 25 μg/ml at 1, 2, 4, 8, and 24 hours post-infection or the Ag-NPs were added concurrently with the TCRV (0 hour). The -1 hour reflected the pre-exposure scenario of figure 1 and was used as a control. The progeny virus produced was harvested 8 days post-infection and is expressed as the mean log₁₀ TCID₅₀/mL. All treatments were significantly lower than the "No NP" control as determined by the student's t test with n = 6.