PMC:6354430 / 7624-15246 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/6354430","sourcedb":"PMC","sourceid":"6354430","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6354430","text":"Methods\n\nEthics statement\nAll experiments were performed in strict compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and according to the international Guiding Principles for Biomedical Research Involving Animals. The protocol was approved by the Washington University School of Medicine in St Louis Animal Safety Committee (#20170064).\n\nViruses\nWNV-NY is strain 3000.0259, isolated in New York in 2000 [42] and passaged twice in C6/36 Aedes albopictus cells to generate an insect cell-derived stock for f.p. inoculations. For i.c. inoculations of WNV-NY, virus strain 3000.0259 was passaged once in Vero cells to generate mammalian cell-derived stock. Attenuated WNV-NS5-E218A was constructed from WNV 3356 strain as described previously [43, 44] and was passaged once in Vero cells, as described previously [45]. Stock titers for all viruses were determined using BHK21 cells for viral plaque assay as previously described [46].\n\nMouse experiments\nAll mice used in these experiments were male C57BL/6 inbred mice obtained commercially (The Jackson Laboratory). All mice were housed in pathogen-free facilities at the Washington University School of Medicine. PLX5622 was provided by Plexxikon Inc. and formulated in AIN-76A rodent diet at a dose of 1200 mg/kg by Research Diets. Mice were provided PLX5622 or control AIN-76A chow starting at 6 weeks of age for 2 weeks. For viral inoculations, mice were anesthesthetized with a cocktail of ketamine/xylazine/acepromazine, then inoculated subcutaneously via footpad injection (f.p., 50 μl) or intracranially (i.c., 10 μl) with a guided 29G needle into the brain’s third ventricle.\n\nViral tissue burden and viremia quantification\nFor in vivo virus replication experiments, mice were infected with WNV and euthanized at specific days post-infection, as indicated. For tissue collection, mice were deeply anesthetized with ketamine/xylazine/acepromazine, blood collected in serum separator tubes, then transcardially perfused with sterile Dulbecco’s phosphate-buffered saline (dPBS; Gibco). The spleen and kidneys were collected, then the brain and spinal cord removed and microdissected. All organs were snap frozen, weighed, and then homogenized in dPBS. Virus was titered by standard plaque assay with BHK21 cells, as described previously [46]. Serum viremia was measured using TaqMan quantitative RT-PCR (qRT-PCR) primers and probe listed in Table 1, as described previously [47].\nTable 1 Primer and probe sequences used for qRT-PCR\n\nLeukocyte isolation and flow cytometry\nFor flow cytometry experiments, mice were deeply anesthetized with ketamine/xylazine/acepromazine, then blood was collected in 5 μM EDTA in dPBS (Gibco). Mice were transcardially perfused with dPBS, and then, the spleen, brain, popliteal LNs, and femur were removed. Bone marrow was collected from the femur by centrifuging the bone in a 0.65-ml microtube punctured with an 18G needle nested inside a 1.7-ml tube. Brain tissue was minced and digested in a HBSS (Gibco) containing 0.05% collagenase D (Sigma), 0.1 μg/ml TLCK trypsin inhibitor (Sigma), 10 μg/ml DNase I (Sigma), and 10 mM Hepes pH 7.4 (Gibco) for 1 h at 22 °C with shaking. Brain, spleen, and LN tissues were pushed through a 70-μm strainer and centrifuged at 500g for 10 min. Brain cell pellets were resuspended in 37% isotonic Percoll (GE healthcare) and centrifuged at 1200g for 30 min to remove myelin debris, and pellet was resuspended in dPBS. Red blood cells were lysed in blood, spleen, and bone marrow samples with ACK lysing buffer (Gibco) for 5 min, then centrifuged at 500g for 10 min and resuspended in dPBS. Prior to immunostaining, all cells were blocked with 1:50 TruStain FcX anti-mouse CD16/32 (Clone 93, Biolegend, Cat 101320) for 5 min. Cells were stained with antibodies, as indicated for 15 min at 22 °C, then washed thrice with dPBS, and fixed with 2% paraformaldehyde (PFA). Data were collected with a BD LSR Fortessa X-20 flow cytometer and analyzed with FlowJo software.\n\nFlow cytometry antibodies\nAll used at 1:200: CD11b (Clone M1/70, Biolegend, Cat 10137), CD45 (Clone 30-F11, eBioscience, Cat 56-0451-82), MHCII (Clone M5/114.15.2, Biolegend, Cat 107626), CD86 (Clone GL1, BD Biosciences, 553691), CD80 (Clone 16-10A1, Biolegend, 104706), CD11c (Clone N418, Biolegend, Cat 117335), CD103 (Clone 2E7, eBioscience, Cat 48-1031-80), CD8a (Clone 53-6.7, Biolegend, Cat 100712), CD4 (Clone RM4-5, BD Biosciences, Cat 550954), CD69 (Clone H1.2F3, eBiosciene, Cat 12-0691-81), Ly6C (Clone HK1.4, Biolegend, Cat 128003), Ly6G (Clone 1A8, Biolegend, Cat 127616), CD160 (Clone 7H1, Biolegend, Cat 143007), Rat-anti-P2RY12 (Clone S16007D, Biolegend, Cat 848002), and Goat-anti-Rat-AlexaFluor 350 (Invitrogen, Cat A21093). WNV-specific CD8+ T cells were identified with fluorescent-labeled immunodominant Db-restricted NS4B peptide.\n\nQuantitative RT-PCR\nRNA was isolated from tissue homogenates using Qiagen RNeasy kit according to the manufacturer’s instructions. RNA was treated with DNAse, then cDNA was synthesized using random primers and MultiScribe reverse transcriptase (Applied Biosystems). A single reverse-transcriptase master mix was used to reverse-transcribe all samples in order to minimize differences in reverse-transcription efficiency using the following conditions: 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. For all primers except WNV, qRT-PCR was performed using Power SYBR Green PCR mastermix, and data are reported as ΔCt (Cttarget − CtGAPDH). Viral RNA was isolated from serum using Qiagen Viral RNA kit. A standard curve of viral genome with known PFU and serum samples were quantified using TaqMan reagents and the primers and probe listed in Table 1.\n\nTunel stain, immunohistochemistry, and confocal microscopy\nFollowing perfusion with ice cold PBS and 4% paraformaldehyde (PFA), brains were immersion-fixed overnight in 4% PFA, followed by cryoprotection in two exchanges of 30% sucrose for 48 h, then frozen in OCT (Fisher). Ten-micrometer sagittal sections were cut, then washed with PBS, and permeabilized with 0.1% Triton X-100/0.1% sodium citrate. Tunel reaction was prepared according to the manufacturer’s directions (Roche in situ cell death kit, TMR Red, Cat 12-156-792-910). Sections were immunostained for NeuN or Iba1 by blocking sections with 5% goat serum/0.1% Tween 20/PBS, then incubating overnight at 4 °C with rabbit-anti-NeuN (1:1000, Clone D3S3I, Cell Signaling Technology Cat 12943S) or rabbit-anti-Iba1 (1:1000, Wako Cat 019-19741). Sections were washed with 0.1% Tween 20/PBS, then incubated with goat-anti-rabbit-AF488 (4 μg/ml, Invitrogen, Cat A11008), counterstained with 1 μg/ml DAPI, and coverslipped with Prolong Gold Antifade Mountant (ThermoFisher, Cat P36930). Z-stack images (seven images separated by 1 μm) were captured using a Zeiss LSM 510 laser-scanning confocal microscope at × 40 objective magnification then compressed into a maximum intensity projection with accompanying software. For each region of each mouse, three to six images were taken from two different sagittal sections spaced at least 50 μm apart. The number of DAPI-positive nuclei also positive for NeuN and Tunel were counted by an individual blinded to the conditions.\n\nStatistical analysis\nStatistical analyses were performed using Prism 7.0 (GraphPad Software). All data were analyzed using an unpaired t test or two-way ANOVA with Sidak’s post-test to correct for multiple comparisons, as indicated in the corresponding figure legends. A P value \u003c 0.05 was considered significant.\n","divisions":[{"label":"Title","span":{"begin":0,"end":7}},{"label":"Section","span":{"begin":9,"end":415}},{"label":"Title","span":{"begin":9,"end":25}},{"label":"Section","span":{"begin":417,"end":1009}},{"label":"Title","span":{"begin":417,"end":424}},{"label":"Section","span":{"begin":1011,"end":1710}},{"label":"Title","span":{"begin":1011,"end":1028}},{"label":"Section","span":{"begin":1712,"end":2563}},{"label":"Title","span":{"begin":1712,"end":1758}},{"label":"Table caption","span":{"begin":2512,"end":2563}},{"label":"Section","span":{"begin":2565,"end":4065}},{"label":"Title","span":{"begin":2565,"end":2603}},{"label":"Section","span":{"begin":4067,"end":4919}},{"label":"Title","span":{"begin":4067,"end":4092}},{"label":"Section","span":{"begin":4921,"end":5779}},{"label":"Title","span":{"begin":4921,"end":4940}},{"label":"Section","span":{"begin":5781,"end":7306}},{"label":"Title","span":{"begin":5781,"end":5839}},{"label":"Section","span":{"begin":7308,"end":7621}},{"label":"Title","span":{"begin":7308,"end":7328}}],"tracks":[{"project":"2_test","denotations":[{"id":"30704498-11585527-63046397","span":{"begin":483,"end":485},"obj":"11585527"},{"id":"30704498-12021317-63046398","span":{"begin":819,"end":821},"obj":"12021317"},{"id":"30704498-17267492-63046399","span":{"begin":823,"end":825},"obj":"17267492"},{"id":"30704498-22589727-63046400","span":{"begin":889,"end":891},"obj":"22589727"},{"id":"30704498-12551996-63046401","span":{"begin":1005,"end":1007},"obj":"12551996"},{"id":"30704498-12551996-63046402","span":{"begin":2370,"end":2372},"obj":"12551996"},{"id":"30704498-16809306-63046403","span":{"begin":2507,"end":2509},"obj":"16809306"}],"attributes":[{"subj":"30704498-11585527-63046397","pred":"source","obj":"2_test"},{"subj":"30704498-12021317-63046398","pred":"source","obj":"2_test"},{"subj":"30704498-17267492-63046399","pred":"source","obj":"2_test"},{"subj":"30704498-22589727-63046400","pred":"source","obj":"2_test"},{"subj":"30704498-12551996-63046401","pred":"source","obj":"2_test"},{"subj":"30704498-12551996-63046402","pred":"source","obj":"2_test"},{"subj":"30704498-16809306-63046403","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ecbc93","default":true}]}]}}