Figure 1.
Radiographic imaging and gross lung pathology associated with HPS in hamsters.
Hamsters were infected with 200 FFU of Andes virus as outlined in the materials and methods section. Beginning at 12 hours post-infection and continuing every 1–2 days, infected hamsters were euthanized and necropsied. Macroscopic evidence of pulmonary damage was first noted at day 7 post-infection (see arrows) with the frequency and severity of lesions increasing until the terminal phase of disease. Radiographic imaging (X-ray) demonstrated a progression of lung infiltrations beginning between days 7 and 9 post-infection. Shown are ventral dorsal chest radiographs and gross pathology from a representative hamster collected at the indicated time points.
Figure 2.
Lung weight to body weight ratios.
Whole lungs were removed from Andes virus infected hamsters at indicated time points post-infection and lung to body weight ratios were determined. Individual dots represent data from a single hamster (expressed as a %) and are color coded to reflect the signs of infection/disease (as determined by clinical presentation and gross pathology of lungs, see inset for legend) of the specific animal. The blue dashed line represents the average lung to body weight ratio of control (uninfected) hamsters (n = 13) and the red dashed line represents the average ratios of infected hamsters at the indicated time point. Error bars represent the standard error of the mean. * Time point p<0.05; ** Time point p<0.01 (infected hamsters compared to controls).
Figure 3.
ANDV RNA was quantified in organs collected from infected hamsters at the indicated time points using primers and probe designed against the nucleoprotein coding region (A). Heart samples were not available for analysis at the day 10 time point due to insufficient levels of RNA. Error bars represent the standard error of the mean. LN, lymph nodes. Infectious ANDV titers were determined in lung samples from infected hamsters at the indicated time points using a focus-forming unit assay (B). Error bars represent the standard error of the mean.
Figure 4.
Histological examination of lungs from Andes infected hamsters.
Hamsters were infected with 200 FFU of ANDV as outlined in the materials and methods section. Beginning at 12 hours post-infection and continuing every 1–2 days, infected hamsters were euthanized and necropsied. Shown are representative sections of lungs stained with hematoxylin and eosin (H&E, 200X magnification) or a monoclonal antibody targeting the ANDV nucleoprotein (IHC, 400X magnification) from a representative hamster collected at the indicated time points. Histological changes were first apparent at day 7 post-infection and included increased immune cell (predominantly macrophages, neutrophils and lymphocytes) infiltration (circled areas), hemorrhage (closed arrow heads), fibrin deposition (F) and edema (e). Diffuse staining of lung endothelium and alveolar septal cells (arrows) was also apparent beginning at 7 days post-infection. Note, samples shown correspond to the same hamsters in Figure 1.
Figure 5.
Histological analysis of peripheral organs from Andes virus infected hamsters.
Representative samples of heart, kidney, liver, lymph nodes and spleen sections collected at day 12 post-infection from a terminally ill, ANDV infected hamster are shown (H&E 200X, anti-Andes virus nucleoprotein IHC 400X). Extensive viral antigen was noted in endothelial cells of all tissues analyzed (black arrow), including endocardial endothelium in heart sections (open arrow) and glomerular tuft endothelium in kidney sections (circled areas) as well as hepatocytes and Kupffer cells in liver sections (denoted with an H and K, respectively) and dendritic cells and macrophages in lymph nodes (circled area). Despite the high frequency of antigen positivity in peripheral tissue samples, histological abnormalities were infrequent and largely unremarkable in these organs.
Figure 6.
Histological analysis of the upper respiratory tract of Andes virus infected hamsters.
At the conclusion of each necropsy, hamsters were decapitated and the head was skinned, fixed in 10% neutral buffered formalin, decalcified and sectioned for histological analysis of the upper respiratory tract. Throughout the course of the study no discernable virus induced pathology was noted in the upper respiratory tract of infected hamsters. (A, 40X magnification of upper respiratory tract of infected hamsters collected at day 10 post-infection). Immunohistochemistry demonstrates the presence of ANDV infected olfactory epithelial cells (black arrow) and endothelium (open arrow) in the upper respiratory tract of hamsters at days 10 (B, 200X magnification) and 12 (C, 400X magnification) p.i.
Table 1.
Summary of coagulation parameters monitored in control and Andes infected hamsters.
Figure 7.
Host responses to Andes virus infection.
Host responses to ANDV infection were monitored using recently developed, hamster specific, real-time RT-PCR assays. The average fold changes in lung, heart, spleen, cervical lymph nodes and blood samples from a minimum of 6 infected animals per time point are shown. Data collected is normalized uninfected animals. Day 10 heart samples were not tested due to insufficient RNA concentrations following extraction. Spleen samples were not tested for STAT-2 or IRF-2 for the same reason.
Figure 8.
Analysis of plasma concentrations of inflammatory mediators.
Plasma samples from control (n = 6) and Andes virus infected (n = 22) hamsters were tested for analytes using the RodentMAP version 2.0 platform. Samples from infected hamsters were divided into early (n = 6, collected on day 1 post-infection, p.i.), middle (n = 8, collected on days 7–8 p.i.), or late (n = 8, collected on days 11–12 p.i.) stages of disease. Shown are data from biomarkers that sufficiently cross-reactive with the mouse/rat specific assay. IP, inducible protein; M-CSF, macrophage-colony stimulating factor; MCP, monocyte chemoattractant protein; VCAM, vascular cell adhesion molecule; vWF, von Willebrand factor; VEGF, vascular endothelial cell growth factor; MDC, macrophage-derived chemokine; SCF, stem cell factor. * p<0.01, ** p<0.001; *** p<0.0001 (indicated time point to uninfected controls).