Penicillin streptomycin

The Jordan MERS-CoV strain (GenBank accession no. KC776174.1, MERS-CoV-Hu/Jordan-N3/2012) was kindly provided by Kanta Subbarao (National Institutes of Health, Bethesda, MD), Gabriel Defang (Naval Medical Research Unit 3 [NAMRU-3], Cairo, Egypt), Michael Cooper (Armed Forces Health Surveillance Center [AFHSC]) and Emad Mohereb (NAMRU-3). All experiments with live MERS-CoV (Jordan) were performed under biosafety level 3 conditions at the University of Maryland School of Medicine. Vero E6 (monkey kidney epithelial cells, ATCC CRL-1586) for plaque assays were grown in minimal essential medium (Corning) supplemented with 10% (vol/vol) FBS (Sigma Aldrich), 1% l-glutamine (Gibco) and 1% penicillin-streptomycin (Gemini Bio-Products) at 37°C in a 5% CO₂ atmosphere.

All serum samples were heat-inactivated at 56 °C for 30 min to deactivate complement and allowed to equilibrate to RT prior to processing for neutralization titer. Samples were diluted in duplicate to an initial dilution of 1:20 followed by 1:2 serial dilutions (vaccinated samples), resulting in a 12-dilution series with each well containing 60 µl. All dilutions employed DMEM (Quality Biological), supplemented with 10% (v/v) fetal bovine serum (heat-inactivated, Gibco), 1% (v/v) penicillin/streptomycin (Gemini Bio-products), and 1% (v/v) L-glutamine (2 mM final concentration, Gibco). Dilution plates were then transported into the BSL-3 laboratory, and 60 µl of diluted SARS-CoV-2 (WA-1, courtesy of Dr. Natalie Thornburg/CDC) inoculum was added to each well to result in a multiplicity of infection (MOI) of 0.01 upon transfer to titering plates. A non-treated, virus-only control and mock infection control were included on every plate. The sample/virus mixture was then incubated at 37 °C (5.0% CO₂) for 1 h before transferring 100 µl to 96-well titer plates with 5e3 VeroE6 cells. Titer plates were incubated at 37 °C (5.0% CO₂) for 72 h, followed by cytopathic effect (CPE) determination for each well in the plate. The first sample dilution to show CPE was reported as the minimum sample dilution required to neutralize >99% of the concentration of SARS-CoV-2 tested (NT99).

Conditionally immortalized wild-type STHdh^Q7 (female, Coriell CH00097, RRID: CVCL_M590) and mutant huntingtin homozygous knock-in STHdh^Q111 (female, Coriell CH00095, RRID: CVCL_M591) murine striatal progenitor cell lines were purchased from Coriell. Cells were maintained at 33 °C with 5% CO₂ and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning 10-013) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products 100–106), and 1% penicillin/streptomycin (Gemini Bio-Products 400–109).