Mouse experiments

For VEEV ZPC738 infections, 7 to 11-week-old male and female Ldlrad3^Δ14/Δ14 and wild-type C57BL/6J mice were anesthetized as described above and inoculated with 10² focus-forming units (FFU) of virus diluted in sterile PBS (Gibco #14190–136) via a subcutaneous, intranasal, or intracranial routes. For subcutaneous inoculations, 50 μL of diluted virus was injected into the left rear footpad. For intranasal inoculations, 20 μL of diluted virus was delivered dropwise into the nares (10 μL per nare) using a micropipettor. For intracranial inoculations, 10 μL of diluted virus was injected into the left hind cortex using a pre-measured needle guide and a 0.3 mL 29G × ½” insulin syringe (Exel Int #26018). For MADV infections, six to seven-week-old male and female Ldlrad3^Δ14/Δ14 and wild-type C57BL/6J mice were inoculated via a subcutaneous route with 10³ FFU of virus diluted in sterile PBS as described above. Survival and body weight were measured on the day of inoculation and daily thereafter for 14 days.

For sample collection, peripheral blood was collected via cardiac puncture and added to serum-separating tubes (BD Microtainer #365967) or tubes with K₂EDTA (BD Microtainer #365974) supplemented with 30 μL of 0.5 M EDTA (Corning #46–034-CI) for peripheral blood leukocyte (PBL) collection. After obtaining blood samples, mice were perfused with 20 mL of PBS. Depending on the experiment, tissues in the periphery (foot skin, underlying tissue of the foot, popliteal or inguinal DLN, spleen, liver, heart, lung, thymus) and CNS (olfactory bulb, cortex, cerebellum, hippocampus, brainstem, other brain, and spinal cord) were collected at 6 h, 12 h, day 1, 3, 5, 7, 10, or 14 after inoculation. Samples were immediately placed on dry ice. PBLs were further processed to remove red blood cells with ACK lysing buffer (Gibco #A10492–01). PBLs were then washed twice with 15 mL of iced FACS buffer (2% FBS, 2 mM EDTA in PBS) and resuspended in 5X MagMax-96 Viral RNA Isolation Kit (Applied Biosystems #{"type":"entrez-protein","attrs":{"text":"AMB18365","term_id":"982818187","term_text":"AMB18365"}}AMB18365) lysis buffer. All samples were stored at −80°C before viral RNA and infectious virus quantification. During the kinetic analysis of viral burden after intracranial inoculation, two Ldlrad3^Δ14/Δ14 mice died before the timepoint of tissue collection and were necessarily excluded from analysis.