Figure 1.
PMOplus targeting of LCMV in vivo inhibits viral replication.
C57BL/6 mice were treated daily with 4 mg/kg antisense targeting the terminal sequences of LCMV starting either 2 days prior to infection (pre-infection) or 2 days post-infection or with a scrambled antisense sequence (SCR) 2 days prior to infection with 1–2×106 p.f.u. LCMV clone-13. Viral load was quantified by qRT-PCR in tissue isolated from kidney, spleen, brain and liver 8 days post-infection. LCMV antisense PMOplus led to a significant reduction in viral RNA compared to PBS and scramble control in all tissues (Mann Whitney; p<0.05) except for pre-treatment compared to scramble in spleen and post-treatment compared to PBS in brain. Dotted line indicates lower level of assay detection. Solid lines indicate the mean LCMV copies/mg for each tissue type.
Table 1.
Sequence of AVI-7012 and alignment to Arenavirus L and S genome and anti-genome segments.
Figure 2.
Overt signs of tissue morbidity and hemorrhage in the LCMV infected FVB mouse.
Images were taken of FVB mice 6 to 8 days post-infection with LCMV -13. A) and B) Subcutaneous tissue in infected and uninfected FVB mice, respectively. C) and D) Gastro intestinal track of infected and normal FVB mice, respectively. E) General sites of blood pooling in the liver, stomach and GI track. F) and G) Cutaneous bleeding in the hindquarters and pooling of blood in the extremities, respectively. Arrows indicate sites of petechie and tissue hemorrhage.
Figure 3.
FVB mice develop an acute lethal disease after LCMV-13 infection independent of reduced viral titers in tissues.
FVB or C57BL/6 mice were infected with 1–2×106 p.f.u. and monitored for up to 11-days post-infection for development of disease signs or death. Animals were humanely killed upon development of severe disease. A) Survival curves of FVB and C57BL/6 mice (n = 11). B) Survival curves of FVB mice infected with LCMV-13 and LCMV-Arm (n = 3 mice) C) Weight change of FVB (n = 8) and C57BL/6 (n = 10) mice starting from pre-infection levels. Results are presented as a mean percentage of initial weight +/− standard deviation. D) Viral RNA in tissues of FVB (n = 13) and C57BL/6 (n = 15) mice infected with LCMV-13. Levels of viral RNA in indicated tissues at time of death were quantified by qRT-PCR for LCMV RNA. Results are presented as mean +/− standard deviation (*, p<0.05. One-way Anova). E) Viral burden as assessed by plaque assay. Infectious virus was quantified day 7 post-infection for all mice (n = 5). Results are presented as mean +/− standard deviation (*, p<0.05. t-test). F) FVB mice treated with anti-arenavirus antisense did not exhibit increased survival to LCMV infection compared to PBS treated animals (n = 5).
Figure 4.
Effects of LCMV infection on blood and splenic cellularity in LCMV infected mice.
A) Complete blood count with differential of LCMV-13 infected mice for platelet, neutrophil, lymphocyte, eosinphil and basophil counts. FVB (n = 4) or C57BL/6 (n = 1) mice were infected with 1–2×106 p.f.u. LCMV-13 and blood was taken at day 7 post-infection. Additionally, naïve FVB (n = 2) blood was collected as a reference. Counts are presented as mean +/− standard deviation. Results for FVB mice are combined from two independent experiments. B) Spleen size in FVB mice exhibits an inverse correlation with infections dose of LCMV compared to C57BL/6. C) FVB mice have reduced T cell numbers as assessed by staining with anti-CD3 antibody in spleens after LCMV-13 infection. FVB (n = 10) or C57BL/6 (n = 8) mice were infected with 1–2×106 p.f.u. LCMV-13 and spleens were harvested between day 7–11 post-infection. Results are presented as mean +/− standard deviation (*, p<0.05. t-test). Similar results were found in two additional independent experiments.
Figure 5.
Indications of tissues damage in FVB mice following LCMV infection.
FVB or C57BL/6 mice were infected with 1–2×106 p.f.u. LCMV-13 and blood or tissue samples were taken at day 7 post-infection. A) Histological comparison of infected diseased and normal tissue in FVB mice. Signs of splenic, hepatic and pulmonary necrosis in FVB diseased mice in upper, middle and lower panels, respectively. B) Clinical chemistry profiles of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and blood urea nitrogen (BUN) are shown. LCMV infected C57BL/6 data are presented as the values from combined serum collected from 4–5 mice. LCMV infected FVB data is presented as the mean +/− standard deviation of two groups of 3–5 mice. Results are representative of three independent experiments.
Figure 6.
FVB mice infected with LCMV produce increased systemic cytokine and chemokine profiles.
FVB or C57BL/6 mice were infected with 1–2×106 p.f.u. LCMV-13 and serum harvested from 4–5 mice was combined and assayed for cytokine and chemokine levels. A) Serum levels of IL-6, IFN-γ, MCP-1, MCP-3 and CXCL1/KC measured 3 days post infection in FVB and C57BL/6 mice and IL-17 was measured day 1 post-infection. B) Serum levels of TNF-α were measured in FVB and C57BL/6 mice at indicated times post-infection. Results are representative of three independent experiments.
Figure 7.
CD4 or CD8 depletion provides FVB mice protection from LCMV-13 induced death.
FVB mice were treated with anti-CD8 antibody (n = 3), anti-CD4 antibody (n = 3) or PBS (n = 3) at days 0 and 1 post-infection with 1×106 p.f.u. LCMV-13. Survival (A) and weight (B) were monitored daily until day 17 post-infection. Animals were humanely killed upon development of severe disease. C) Serum from 3 mice was combined and analyzed for AST and ALT levels. D) Viral burden in tissues measured after sacrificing animals. Levels of infectious virus in indicated tissue at time of death were quantitated by qRT-PCR for LCMV RNA. Results are presented as mean +/− standard deviation. Results are representative of two independent experiments.
Figure 8.
Blockade of the IL17 signaling pathway increases survival and reduces disease signs in LCMV infected FVB mice.
FVB mice were infected with LCMV-13 and either treated with 7.5 mg/kg IL17RC SD12 antisense or PBS at days 0, 1, and 2 post-infection. A) Survival curves of PBS (n = 8) and IL17RC SD12 (n = 3) treated mice. B) Viral burden in tissues of FVB mice treated with PBS (n = 8) or IL17RC SD12 (n = 3). C) Clinical chemistry profiles of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALK), total bilirubin (TBIL) are presented. Untreated infected FVB data are presented as the mean +/− standard deviation of combined serum collected from three groups of 1–3 mice. Naïve FVB and IL17RC SD12 treated data are presented as the values from combined serum collected from 3–5 mice.
Figure 9.
FVB×C57BL/6 F1 hybrids succumb to hemorrhagic disease while retaining H-2Db restricted T cell response to LCMV infection.
A) FVB mice were infected with 2×106 PFU LCMV-13 and F1 hybrids were infected with the indicated doses of LCMV-13 and monitored for survival (n = 3). B) C57BL/6 or F1 hybrid mice were infected with the indicated virus and body temperature was assessed day 7 post-infection (n = 5). C–D) Splenic T cell numbers were assessed day 7 post-infection. E) Virus specific CD8 T cell numbers in the spleen as assessed by MHC pentamer stain (top) and IFN-γ production (bottom) after stimulation with the indicated peptides. Data are presented as the mean +/− standard deviation (*, p<0.05. t-test).
Table 2.
Clinical presentation of arenaviral hemorrhagic diseases in comparison to LCMV-13 infected FVB mice.