NextSeq 500 sequencing; RNA-seq of Golden Hamster lung tissues according to sex, SARS-CoV-2 infection and treatment
At 4 days post-infection, animals were euthanized with an excess of anesthetics (ketamine and xylazine) and exsanguination (AVMA, 2020), and samples of nasal turbinates and lungs were collected and immediately frozen at -80°C. All animal experiments were performed according to the French legislation and in compliance with the European Communities Council Directives (2010/63/UE, French Law 2013–118, February 6, 2013) and according to the regulations of Pasteur Institute Animal Care Committees. The Animal Experimentation Ethics Committee (CETEA 89) of the Institut Pasteur approved this study (200023; APAFIS#25326-2020050617114340 v2) before experiments were initiated. Hamsters were housed by groups of 4 animals in isolators and manipulated in class III safety cabinets in the Pasteur Institute animal facilities accredited by the French Ministry of Agriculture for performing experiments on live rodents. Production and titration of SARS-CoV-2 virus. The isolate BetaCoV/France/IDF00372/2020 (EVAg collection, Ref-SKU: 014V-03890) was kindly provided by Sylvie Van der Werf. Viral stocks were produced on Vero-E6 cells infected at a multiplicity of infection of 1x10-4 PFU (plaque-forming units). The virus was harvested 3 days post-infection, clarified and then aliquoted before storage at -80°C. Viral stocks were titrated on Vero-E6 cells by classical plaque assays using semisolid overlays (Avicel, RC581-NFDR080I, DuPont) (Baer & Kehn-Hall, 2014). Infected and mock-infected animals were housed in separate isolators and all hamsters were followed-up daily during four days at which the body weight and the clinical score were noted. The clinical score was based on a cumulative 0-4 scale: ruffled fur, slow movements, apathy, absence of exploration activity. Male and female Syrian hamsters (Mesocricetus auratus; RjHan:AURA) of 5-6 weeks of age (average weight 60-80 grams) were purchased from Janvier Laboratories and handled under specific pathogen-free conditions. The animals were housed and manipulated in isolators in a Biosafety level-3 facility, with ad libitum access to water and food. Before manipulation, animals underwent an acclimation period of one week.  Animals were anesthetized with an intraperitoneal injection of 200 mg/kg ketamine (Imalgène 1000, Merial) and 10 mg/kg xylazine (Rompun, Bayer), and received one single subcutaneous injection of 200 µL of freshly-diluted ivermectin (I8898, Sigma-Aldrich) at the classical anti-parasitic dose of 400 µg/kg (Beco et al., 2001) (or at 100-200 µg/kg for the dose-response experiment). Non-treated animals received one single subcutaneous injection of 200 µL of physiological solution. 100 µL of physiological solution containing 6x104 PFU of SARS-CoV-2 was then administered intranasally to each animal (50 µL/nostril). Mock-infected animals received the physiological solution only.  Frozen tissues were homogenized with Trizol (15596026, Invitrogen) in Lysing Matrix D 2 mL tubes (116913100, MP Biomedicals) using the FastPrep-24™ system (MP Biomedicals) at the speed of 6.5 m/s during 1 min. Total RNA was extracted using the Direct-zol RNA MicroPrep Kit (R2062, Zymo Research: nasal turbinates) or MiniPrep Kit (R2052, Zymo Research: lung) RNA preparation was used to construct strand specific single end cDNA libraries according to manufacturers' instructions (Truseq Stranded mRNA sample prep kit, Illumina)