Subsequently values from the predefined timepoints

were analyzed with the pre inoculation (P.I.) values using paired t-test (Y to Z). Ethics statement To reduce the numbers of experimental animals used, we combined the earlier published influenza pathogenesis study [21] with the current study addressing questions related to activation of coagulation and tissue fibrin deposition during influenza virus infection. Animal housing and experiments were all in compliance with European guidelines (EU directive on animal testing 86/609/EEC) and Dutch legislation (Experiments on Animals Act, 1997) as documented previously [21]. The study protocol was approved by the independent animal experimentation ethical review committee of the Netherlands Vaccine Institute (permit number 200900201). Animal welfare was observed on a daily basis, and

animal handling was performed under light anesthesia C59 wnt molecular weight using a mixture of ketamine and medetomidine. After handling, atipamezole was administered to antagonize the effect of medetomidine. Coagulation assays Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured CT99021 using a BCS-XP coagulation analyzer (Siemens Healthcare Diagnostics) according to the instructions of the manufacturer. Clotting was initiated with Thromborel S (PT) and Pathrombin SL (APTT). VWF ristocetin cofactor activity was also determined on the BCS-XP with reagents of the manufacturer, and was expressed as percentage of normal pooled human plasma. Thrombin-antithrombin complexes (TAT, Siemens Healthcare Diagnostics) and D-dimer levels (Asserachrom, Roche, The Netherlands) were measured using enzyme-linked immunosorbent assay. All these assays were carried out within the BSL-3 setting after careful calibration and validation. Pathology and fibrin staining Gross pathology

and histopathology were evaluated as previously described [21]. Relative lung weight was used as a validated measure of gross pathology and lung inflammation [47]. Phosphatidylinositol diacylglycerol-lyase For detection of fibrin, tissues were stained with the Lendrum staining according manufacturers’ protocol (MSB RRSK2-100 stain kit, Atom scientific). On each slide a small piece of human placenta was added as a positive control. Semi-quantitative assessment of fibrin expression in the lungs was performed as follows: for the alveoli, 25 arbitrarily chosen, 20x objective, fields of lung parenchyma of one lung section were examined by light microscopy for the presence of fibrin, without the knowledge of the identity of the animals. The scores (+ or -) were multiplied by 4 and presented as percentage. Virology The presence of virus and virus replication in the respiratory tract were measured by determining infectious virus titers at different sites of the upper respiratory tract (URT) and lower respiratory tract (LRT).