key: cord-014938-7evmiuv5 authors: Wei-ming, Yan; Jia-quan, Huang; Xiao-ping, Luo; Qin, Ning title: Expression of prothrombinase/fibroleukin gene fg12 in lung impairment in a murine severe acute respiratory syndrome model date: 2008-01-13 journal: Virol Sin DOI: 10.1007/s12250-007-0020-5 sha: doc_id: 14938 cord_uid: 7evmiuv5 To evaluate the role of murine fibrinogen like protein 2 (mfgl2) /fibroleukin in lung impairment in Severe acute respiratory syndrome (SARS), a murine SARS model induced by Murine hepatitis virus strain 3 (MHV-3) through trachea was established. Impressively, all the animals developed interstitial pneumonia with extensive hyaline membranes formation within alveoli, and presence of micro-vascular thrombosis in the pulmonary vessels. MHV-3 nucleocapsid gene transcripts were identified in multiple organs including lungs, spleen etc. As a representative proinflammatory gene, mfgl2 prothrombinase expression was evident in terminal and respiratory bronchioles, alveolar epithelia and infiltrated cells in the lungs associated with fibrin deposition and micro-vascular thrombosis. In summary, the established murine SARS model could mimic the pathologic characteristics of lungs in patients with SARS. Besides the physical damages due to virus replication in organs, the up-regulation of novel gene mfgl2 in lungs may play a vital role in the development of SARS associated lung damage. . All animal studies were carried out according to the Animals were acclimatized to laboratory conditions for 1 week before experiments and all procedures that involved animals were conducted according to the hospital guidelines. Culture Collection (ATCC) and plaque purified on monolayers of DBT cells and titred on L2 cells using a standard plaque assay (12, 16) . To setup the murine SARS model twenty Balb/cJ mice were inoculated with 100PFU of MHV-3 through trachea. Another twenty Balb/cJ mice injected with sterilized saline through trachea in the same volume were set up as a control. Animal survival rate was then observed. Additionally, 10 Balb/cJ mice were injected with sterilized saline as a control. In a separate experiment, tissues including lungs, spleen, liver, kidneys, intestine, heart, brain were collected at a series of time points from Balb/cJ mice infected with MHV-3 through trachea. The samples were snap freezed and kept at a -80 freezer or fixed with 10% formalin for further experiments. The tissues that had been fixed with 10% formalin were dehydrated in graded alcohol solutions and xylene and embedded in paraffin. 4-m sections were cut and tissues were then stained with hematoxylin and eosin (HE). Approximate 40mg of each frozen organ was homogenized in 0.1 mol/L PBS (pH7.4), and the supernatants were subjected to titer analysis on L2 cells by a standard plaque assay as described elsewhere (12, 16) . The titration examination in each organ was repeated for three times in duplicate. Quantitative data were reported as the means standard deviation for at least three separate experiments. Differences of virus titers between organs at 48h following MHV-3 infection were analyzed by using one-way analysis of variance, and a P value of less than 0.05 was considered statistically significant. Balb/cJ mice inoculated with MHV-3 through trac- In a separate set of experiment, tissues including lungs, spleen, liver, kidneys, intestine, heart and brain from Balb/cJ mice infected with MHV-3 through trachea were examined as described above. Various histopathological changes were found in these organs (shown in Fig.2 Intestine, heart and brain: No significant pathological changes were found in these organs ( Fig.3 . D4, E4, F4). To determine viral titers, tissues were homogenized and the supernatants were subjected to titer determination on L2 cells as described previously (12, 16) . Virus plaques were found in all collected organs and increased with the progress of the infection Widespread fibrin deposition in association with the mfgl2 expression, especially in microvasculars (Fig.7 . A) and hyaline-membranes ( Fig.7 . B) was detected in the lungs. 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