86 research outputs found

    Small Interfering RNA Inhibition of Andes Virus Replication

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    <div><p>Andes virus (ANDV) is the most common causative agent of hantavirus pulmonary syndrome (HPS) in the Americas, and is the only hantavirus associated with human-to-human transmission. Case fatality rates of ANDV-induced HPS are approximately 40%. There are currently no effective vaccines or antivirals against ANDV. Since HPS severity correlates with viral load, we tested small interfering RNA (siRNA) directed against ANDV genes as a potential antiviral strategy. We designed pools of 4 siRNAs targeting each of the ANDV genome segments (S, M, and L), and tested their efficacy in reducing viral replication <i>in vitro</i>. The siRNA pool targeting the S segment reduced viral transcription and replication in Vero-E6 cells more efficiently than those targeting the M and L segments. In contrast, siRNAs targeting the S, M, or L segment were similar in their ability to reduce viral replication in human lung microvascular endothelial cells. Importantly, these siRNAs inhibit ANDV replication even if given after infection. Taken together, our findings indicate that siRNAs targeting the ANDV genome efficiently inhibit ANDV replication, and show promise as a strategy for developing therapeutics against ANDV infection.</p></div

    siRNA inhibits production of infectious ANDV.

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    <p>(A) Viral protein levels determined by Western blot analysis. Vero-E6 cells were transfected with 33 nM of each siRNA pool (S, L, or M) alone or in combination (S+L, S+M, L+M, or S+L+M) for 6 h. The total concentration of each transfection was brought up to 100 nM with scrambled siRNA. Cells treated with only scrambled siRNA were used as control. The cells were then infected with ANDV at MOI  = 0.5. ANDV Gc and N protein levels were determined by densitometry. (B) ANDV production and release were determined by immunofocus assays. All experiments were performed in quadruplicate.</p

    siRNA inhibits ANDV replication and release when administered post-infection in Vero-E6 cells.

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    <p>Vero-E6 cells were infected with ANDV at MOI  = 0.5. After virus adsorption for 2 h, the virus inoculum was removed and replaced with fresh media. Cells were then transfected with 100 nM siRNA 2, 6, 12, or 24 h post-infection. Time shown in parentheses indicates the total h post-infection. (A) N protein levels as determined by immunolabeling assays. (B) Infectious virus release as measured by immunofocus assays. After virus adsorption for 2 h, virus was removed and fresh media replaced. The cells were transfected with 100 nM siS for 6, 12, 24, or 48 h post-infection. Cell supernatants were harvested after 2 days, and infectious virus production was determined by immunofocus assays. All experiments were performed in triplicate. Data indicated above each bar represent percentage reduction in comparison to scrambled siRNA control.</p

    siRNA inhibits ANDV replication in human primary lung endothelial cells (HMVEC-L).

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    <p>HMVEC-L were transfected with siRNAs for 6 h, and then infected with ANDV at MOI  = 0.5. After 2 h of virus adsorption, the virus inoculum was removed and replaced with fresh media. (A) Viral protein levels as determined by Western blotting. Percent downregulation (%↓) of N or Gc protein represents percent decrease compared to non-targeting siRNA control. (B) Viral production as measured by immunofocus assays 48 h post-infection.</p

    Survival data for C57BL/6 mice following infection with KFDV or AHFV sc.

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    <p>Values indicate the number of mice (out of 5) that survived infection with KFDV or AHFV as of 30 dpi.</p

    More severe histopathologic lesions were apparent in the brains and gastrointestinal tracts of KFDV-infected mice than AHFV-infected mice.

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    <p>(A) Brain, hippocampus, KFDV-infected mouse, 6 dpi; acute necrosis and loss of hippocampal neurons (segment delineated by arrowheads), with adjacent, relatively unaffected neurons (arrow). (B) Brainstem, KFDV-infected mouse, 6 dpi; severe meningoencephalitis with perivascular cuffing (arrow) and widespread gliosis in the adjacent neuropil (asterisk). (C) Brainstem, AHFV-infected mouse, 7 dpi; mild meningoencephalitis characterized by perivascular cuffing (arrows). (D) Small intestine, mock-inoculated mouse; normal tissue section demonstrating anatomic location of submucosal (arrow) and myenteric (arrowhead) nerve plexi and normal intestinal villi; inset shows higher magnification of neuron cell bodies in plexus. (E) Small intestine, KFDV-infected mouse, 7 dpi; moderate, predominantly histiocytic, inflammatory infiltrate in submucosa and muscularis, involving and disrupting myenteric plexi (arrowheads). Inset shows higher magnification of infiltrate and cell debris in plexus. (F) Small intestine, KFDV-infected mouse, 7 dpi; intestinal crypts are dilated and filled with necrotic cells (asterisks), intestinal lumen contains abundant cellular debris (top of image), and villi are blunted and fused. All H&E images, original magnification 200x.</p

    KFDV-infected mice have higher viral RNA loads than AHFV-infected mice early in infection.

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    <p>Viral RNA loads in the (A) blood, (B) spleen, (C) gastrointestinal tract and (D) brain were compared between KFDV- and AHFV-infected mice on 1, 4, 6 and 7 dpi. Asterisks indicate significant differences between KFDV- and AHFV-infected mice (n = 5; p<0.05). The gray dotted line denotes limit of detection of the assay.</p

    KFDV and AHFV-infected mice developed significant lymphopenia relative to control mice.

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    <p>Complete blood counts were run on KFDV-, AHFV- and mock-infected mice on 1, 4, 6, and 7 dpi. (A) Leukocyte, (B) lymphocyte, (C) monocyte and (D) granulocyte counts are displayed. Asterisks indicate significant differences between KFDV-infected mice (blue) or AHFV-infected mice (green) relative to mock-infected animals (p<0.05). On each day, data was collected from a minimum of 3 and a maximum of 5 mice per group.</p

    KFDV- and AHFV-infected mice mount an early innate response to infection in the CNS.

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    <p>In the brains of mice infected with KFDV or AHFV, there was significant upregulation of (A) interferon-stimulated genes and (B) pathogen recognition receptors (PRRs). There were not significant differences in gene expression between mice infected with AHFV and those infected with KFDV (n = 5; p>0.05).</p

    KFDV is more virulent than AHFV in 3 immunocompetent mouse strains.

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    <p>C3H, A/J and C57BL/6J mice were inoculated subcutaneously with either 1×10<sup>5</sup> TCID<sub>50</sub> AHFV or 1×10<sup>5</sup> TCID<sub>50</sub> KFDV (n = 10/group) and monitored for 28 days. Regardless of strain, KFDV infection resulted in higher mortality and more severe disease than AHFV.</p
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