All animal experiments were approved by the Institutional Animal Care and Use Committee of the Rocky Mountain Laboratories (ASP #2009-43), and performed following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) by certified staff in an AAALAC approved facility.
Biosafety and containment
All work with infected hamsters and potentially infectious materials derived from hamsters was conducted in the Biosafety Level 4 facility at the Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Sample inactivation and removal was performed according to standard operating protocols approved by the local Institutional Biosafety Committee.
Inoculation of hamsters
ANDV, strain Chile 9717869 
, was propagated and titered as previously described 
. Syrian hamsters (female, aged 4–6 weeks) were inoculated with 200 focus forming units (FFU) of ANDV (100×LD50
), a dose previously determined to be uniformly lethal in this model, diluted in sterile DMEM and administered through the intranasal route with 50 µl of virus suspension delivered per nare using a sterile p200 pipette. Control animals received an equivalent volume of sterile DMEM alone.
At 12 hours and 1, 3, 5, 7, 8, 9, 10, 11 and 12 days p.i., one control and six infected hamsters were anesthetized via inhalational isoflurane and chest radiographs (ventral dorsal, right and left lateral) were taken using a portable digital radiography unit with a flat panel digital detector (TruDR, Sound-Eklin, Carlsbad, CA) and veterinary specific software (VET-PACS, London, United Kingdom). Hamsters were weighed, bled (EDTA and heparin treated 1.8 mL vacutainer tubes) and exsanguinated via cardiac puncture. Necropsies were performed to collect trachea, lung, liver, spleen, heart, kidney, digestive tract (stomach and intestines) and cervical lymph nodes. Lungs were removed, photographed, weighed, and a representative sample was taken from each lobe for histopathology (see below). In addition, a piece of the left lung was processed for RT-PCR as outlined below. Organ samples were processed for molecular analysis, histopathology and IHC. For molecular analysis, pieces of tissue (approximately 100 mg) were immersed in 1 mL of RNAlater (Qiagen, Valencia, CA) for overnight at 4°C, removed and stored at −80°C until RNA extraction. The remainders of each organ were fixed in 10% neutral buffered formalin for seven days at 4°C and processed as outlined below for histopathology and IHC. The head was skinned and fixed in 10% neutral buffered formalin for histological analysis of the upper respiratory tract. Lung weight to body weight ratios were calculated and pairwise comparisons of data from control and infected hamsters performed at each interval using t-tests.
Hamsters were assigned a clinical score based on signs of infection/disease as determined by clinical presentation and gross pathology of lungs as follows: no signs
indistinguishable from controls; mild
hamsters appeared lethargic, lesions on single lung lobes; moderate
onset of breathing abnormalities (rapid shallow breathes), lesions apparent on multiple lung lobes; severe
breathing distress, multi-focal lesions of increased size on all lung lobes; terminal
moribund, epistasis, lungs hemorrhagic in appearance.
Disease progression was monitored in a subset of five ANDV infected hamsters with chest radiographs taken at one to two day intervals until day 7 p.i. and twice daily (approximately 12 hours apart) from day 7 p.i. until the point of euthanasia.
Hematology and blood chemistry
Blood chemistry was monitored in heparinized blood using a portable iSTAT instrument (Abbott Point of Care, Princeton, NJ) with EC8+ cartridges measuring Na, K, Cl, TCO2, anion gap, glucose, urea nitrogen, hematocrit, hemoglobin, pH, pCO2, pO2, TCO2, HCO3, and base excess. Hematology, including white blood cell count, lymphocyte, platelet, reticulocyte and red blood cell counts, hematocrit values, mean cell volume, mean corpuscular volume, and mean corpuscular hemoglobin concentrations, was performed on EDTA blood using a Hemavet 950FS (Drew Scientific, Waterbury, CT).
Due to the volume of plasma required for the coagulation examination, a second group of one control and six infected hamsters were exsanguinated via cardiac puncture at days 1, 3, 5, 7, 8, 9, 10, 11 and 12 p.i. with blood collected into 1.8 mL citrate vacutainers. Plasma was tested for coagulation parameters, including activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), fibrinogen concentration, and Protein S and Protein C activity, on a STart4 instrument using the PTT Automate, STA Neoplastine CI plus, STA Thrombin, Fibri-Prest automate, STA Staclot Protein S and Protein C kits, respectively (all from Diagnostica Stago, Parsippany, NJ). Data was analyzed using a one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparison post-test comparing values from infected hamsters to uninfected controls.
Histopathology and IHC
Formalin fixed tissues were processed and embedded in paraffin according to standard procedures. For sectioning of the nasal tract, the mandible and any extraneous muscle was removed from formalin fixed skulls to expose the cranium. Skulls were rinsed in running tap water, placed in a decalcifying solution consisting of 20% EDTA in sucrose (Newcomer Supply, Middleton WI) and allowed to sit at room temperature for 3–5 weeks, with 2–3 changes of decalcification solution over that period. Following decalcification, skulls were processed and embedded in paraffin.
Thin (5 µm) sections were cut and stained with hematoxylin and eosin or tested for the presence of viral antigen by IHC using a monoclonal antibody generated against the ANDV nucleoprotein (1:500 dilution, clone 1A8F6, Austral, San Ramon, CA). IHC was accomplished on a Discovery XT instrument (Ventana Medical Systems, Tucson, AZ) using a biotinylated goat anti-mouse (1:250 dilution, BioGenex, San Ramon, CA) secondary antibody and DAB Map kit. Following immunological staining, slides were counterstained with hematoxylin, dehydrated, cleared in xylene, and coverslipped. In addition, lung sections were stained for vascular endothelial growth factor (VEGF, 1:100 dilution, sc-507, Santa Cruz Biotechnology, Santa Cruz, CA) using a cross-reactive polyclonal antibody generated in rabbits essentially as outlined above with an alkaline phosphatase conjugated secondary antibody (Ventana Medical Systems).
Slides were evaluated by a Veterinary Pathologist. The scoring for H and E stained lung specimens were as follows: 1
Minimally increased numbers of inflammatory cells within alveolar septae without widening of the septae; 2
Mildly increased numbers of inflammatory cells within septae and mild expansion, or thickening, of septal walls and occasional extension of inflammation into the lumen of alveoli and bronchioles; 3
Interstitial inflammation (alveolar septae and perivascular, peribronchiolar and peribronchial) and inflammation within alveoli and larger airways (inflammatory cells, fibrin, hemorrhage). Inflammation may be extensive and involve large areas of the tissue section; 4
Changes are as described for #3; however, extent of lesions is greater and involves most of the tissue section.
Transcriptional profiling of host response and virus replication
RNA was extracted from solid tissues (30 mg pieces) and blood samples using RNeasy or QIAamp viral RNA kits (Qiagen), respectively, according to manufacturers' instructions. Immediately after extraction, RNA was quantified on a nanodrop 8000 spectrophotometer (Thermo Scientific, Wilmington, DE). Sample concentration was adjusted to 40 ng/µl and aliquots frozen at -80°C. Real-time quantitative (q) RT-PCR was performed on a rotor-gene 6000 instrument (Corbett Life Science, Sydney Australia) using QuantiFast probe reagents (Qiagen) and 200 ng of template RNA. The presence of ANDV RNA was quantified using a previously described nucleoprotein specific assay 
. Host responses, including interleukin (IL) 1β, 2, 4, 6, 10, 12p35, and 21, tumor necrosis factor alpha (TNF α), interferon gamma (IFN γ), transforming growth factor beta (TGF β), forkhead box P3 (FoxP3), myxovirus resistance protein 2 (Mx2), interferon regulatory factor 1 (IRF1) and 2 (IRF2), and signal transducer and activator of transcription 1 (STAT1) and 2 (STAT2), were monitored in lung, spleen, heart, cervical lymph node and blood samples using recently developed Syrian hamster specific, real-time qRT-PCR assays using ribosomal protein L18 as an internal control 
. The small group sizes used in these studies prevented a thorough statistical analysis of temporal transcriptional data; however, patterns of up-regulation of pro-inflammatory markers were analyzed using Chi square tests.
Multiplex cytokine, chemokine, and plasma factor analysis
Plasma samples from a subset of infected (n
22) and control (n
6) hamsters were sent to Rules-Based Medicine, Inc (Austin, TX) for analysis of cytokine and chemokine concentrations using a 58-biomarker Multi-Analyte Profile (MAP) approach (RodentMAP version 2.0). Samples from infected animals were divided into three categories; early (n
6, collected on day 1 p.i.), middle (n
8, collected on days 7–8 p.i.), and late (n
8, collected on days 11–12 p.i.) stages of disease. Although this platform has only been validated against mouse and rat samples, recently it was suggested to work for selected hamster biomarkers, especially chemokines 
. Data was analyzed using a one-way ANOVA with Tukey-Kramer multiple comparison post-test.
Infectious viral loads were measured in a subset of lung homogenates (10% [w/v]) from infected hamsters using a focus forming unit assay essentially as previously described 
GenBank accession numbers are as follows: IL-1β AB028497; IL-2 EU729351; IL-4 AF046213; IL-6 AB028635; IL-10 AF046210; IL-12p35 AB085791; IL-21 FJ664142; TNF α AF315292; IFN γ AF034482; TGF β AF046214; FoxP3 FJ 664148; Mx2 EU616539; IRF1 DQ092344; IRF2 AY714581; STAT1 DQ092343; STAT2 AB177399; ribosomal protein L18 DQ403027.