PMC:7537531 / 2848-7815 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7537531","sourcedb":"PMC","sourceid":"7537531","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7537531","text":"2 MATERIALS AND METHODS\n\n2.1 Viruses and cells\nThe SARS‐CoV‐2 München isolate (BetaCoV/Germany/BavPat1/2020) (Wölfel et al., 2020) was used for virus challenge. Stocks were propagated and titrated on Vero E6 cells (ATCC CRL‐1586) in minimal essential medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% foetal bovine serum (PAN Biotech), 100 IU/ml penicillin G and 100 µg/ml streptomycin (Carl Roth, Karlsruhe, Germany) and stored at −80°C prior to experimental infections.\n\n2.2 Experimental infection of Chinese hamsters\n24 five‐ to seven‐week‐old female and male Chinese hamsters (Cricetulus griseus) were obtained via the German pet trade and kept in groups of six animals in enriched, individually ventilated cages. The hamsters had ad libidum access to food and water, and were allowed acclimatization to these conditions for seven days prior to SARS‐CoV‐2 infection. Cage temperatures and relative humidities were recorded daily and ranged from 23 to 24°C and from 50% to 65%, respectively. The animals were randomly distributed into two groups: SARS‐CoV‐2‐infected (n = 12; 1 × 105 plaque‐forming units (pfu) in a volume of 30 µl) and mock‐infected (n = 12; 30 µl cell culture supernatant from uninfected Vero E6 cells). The infection was performed exactly as previously described (Osterrieder et al., 2020). Baseline body weights of all hamsters and body temperatures of at least three hamsters per group were measured before infection and then recorded daily throughout the 14‐day experiment. Moreover, clinical signs of all animals were monitored twice daily throughout the experiment. On days 2, 3, 5 and 14 post‐infection (dpi), three randomly assigned hamsters of each group were euthanized by exsanguination under general anaesthesia as described (Nakamura et al., 2017). For virus titrations and RT‐qPCR and/or histopathological examinations, blood, bucco‐laryngeal swabs and lungs (left and right) were collected.\n\n2.3 Histopathology and in situ hybridization\nFor histopathology, lungs were processed as previously described (Osterrieder et al., 2020). In brief, the left lung lobe was carefully removed and immersion fixed in buffered 4% formalin, pH 7.0, for 48 hr, embedded in paraffin and cut at 2 μm thickness. Sections were stained with haematoxylin and eosin (HE) followed by blinded microscopic evaluation by board certified veterinary pathologists (K.D., A.D.G.). For in situ hybridization (ISH), the ViewRNA™ ISH Tissue Assay Kit (Invitrogen by Thermo Fisher Scientific, Darmstadt, Germany) was used following the manufacturer's instructions with minor adjustments. A probe for the detection of the N gene RNA of SARS‐CoV‐2 (NCBI database NC_045512.2, nucleotides 28,274–29,533, assay ID: VPNKRHM) was employed. Next, lung sections of 2 μm thickness were mounted on adhesive glass slides, dewaxed in xylol and dehydrated in graded ethanols. Tissues were incubated at 95°C for 10 min, and subsequently, protease digested for 20 min. Sections were fixed with 4% paraformaldehyde dissolved in phosphate‐buffered saline (PBS) and hybridized with the probes. Amplifier and label probe hybridizations were performed according to the manufacturer's instructions using fast red as chromogen. Sections were counterstained with haematoxylin for 45 s, washed in tap water for 5 min and mounted with Roti®‐Mount Fluor‐Care DAPI (4, 6‐diaminidino‐2‐phenylindole; Carl Roth). For negative and morphologically intact controls, lungs from uninfected hamsters were included. An irrelevant probe for the detection of streptococcal pneumolysin was used as a control for sequence‐specific binding. HE‐stained and ISH slides were analysed and photographs were taken using an Olympus BX41 microscope with a DP80 Microscope Digital Camera and the cellSens™ Imaging Software, Version 1.18 (Olympus Corporation, Münster, Germany). For overviews, slides were automatically digitized using the Aperio CS2 slide scanner (Leica Biosystems Imaging Inc., Vista, CA, USA) followed by image file generation using the Image Scope Software (Leica Biosystems Imaging Inc.).\n\n2.4 Virus titrations, RNA extractions and RT‐qPCR\nVirus titres from 50 mg lung tissue were assessed by serial dilution of tissue homogenates and subsequent titrations on Vero E6 cells. 3 dpi, cells were formalin‐fixed, stained with crystal violet, and plaques were counted. RNA was extracted from tissue samples, blood and tracheal swabs using the innuPREP Virus RNA kit (Analytic Jena, Jena, Germany) and quantified with a one‐step RT‐qPCR reaction (NEB Luna Universal Probe One‐Step RT‐qPCR kit; New England Biolabs, Ipswitch, MA, USA) and previously published TaqMan primers and probe (Corman et al., 2020).\n\n2.5 Statistical analyses\nData were analysed with GraphPad PRISM. We used Mann–Whitney U tests to compare body weights and body temperatures in mock‐infected versus SARS‐CoV‐2 infected Chinese hamsters. Data were considered significant if p \u003c .05 (*p \u003c .05; 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