Penicillin streptomycin amphotericin b psa

All chemicals used in buffer preparations were from Gerbu, except imidazole, which was from Merck. LB broth for small-scale bacterial cultures was from Conda. Mammalian cell culture media, DMEM and PEM, were from BioConcept and Thermo Fischer Scientific, respectively. Fetal Calf Serum was purchased from Seraglob. Penicillin-streptomycin-amphotericin B (PSA) and Glutamax were from Gibco. Detergents, including dodecylmaltoside (DDM), decyl maltoside (DM), lauryldimethylamine oxide (LDAO), C₁₂E₈, octyl glucoside (OG), nonyl glucoside (NG), octyl maltoside (OM), nonyl maltoside (NM) and octyl thioglucopyranoside (OTG) were purchased from Anatrace (all detergents were ana-grade; DDM used for solubilisation was sol-grade). HisPur Cobalt resin and StrepTactinXT Superflow resin were purchased from Thermo Fischer Scientific and iba, respectively. Cholesteryl hemisuccinate, 25 kDa branched PEI, sodium butyrate and tetracycline were from Sigma. Biotin was purchased from Fluorochem. Radioligand [³H]PK11195 was from PerkinElmer.

Monolayers of baby hamster kidney (BHK21) cells cultured in Dulbecco’s Modified Essential Medium (DMEM) (Gibco; Thermo Fisher Scientific, Waltham, USA) supplemented with 5% (v/v) foetal bovine serum (FBS), L-glutamine (200 mM) and 25 µg/mL Penicillin-Streptomycin-Amphotericin B (PSA) (Gibco) were infected with freeze-dried individual AHSV isolates (Table S1). Total dsRNA was extracted and cDNA synthesized and individual genome segments amplified using the loop-mediated approach, previously described by Potgieter et al. [29 (link)]. Purified PCR products were sequenced using Nextera DNA Sample Preparation Kit (Illumina, Inc., San Diego, CA, USA) with an Illumina MiSeq instrument (Illumina, Inc.) at Inqaba Biotec (Pretoria, South Africa). Between 250 and 500 Mb of data was obtained for each isolate and this was imported into CLC Genomics Workbench v7.0 (CLC bio, Aarhus, Denmark). The reads were trimmed and low-quality score data was removed. Contigs were generated by either de novo assembly or mapping against known references. These contigs were used to assemble ten complete genome segments per sample. The final consensus sequence of each African horse sickness virus genome segment was submitted to GenBank and accession numbers assigned (KP939368–KP940236).

MDCK was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). MDCK cells were maintained in Dulbecco's Modified Eagle medium (DMEM) (Invitrogen) with 10% of fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and 1% of penicillin/streptomycin/amphotericin B (PSA) (Invitrogen), and cultured in 37 °C and 5% CO2. When influenza virus infection in MDCK cells, the infection medium (DMEM containing 0.075% BSA, 1% non‐essential amino acid, 1% sodium pyruvate, 1% HEPES, 1% PSA, and 2 µg mL⁻¹ TPCK‐treated trypsin) was used. Influenza A virus strain A/Puerto Rico/8/1934 (H1N1) was kindly provided by Professor Shin‐Ru Shih at Chang Gung University. Influenza A virus strain A/HKx31 (H3N2) was kindly provided by Professor Hung‐Chih Yang at the National Taiwan University College of Medicine, and A/California/7/2009 (pdmH1N1) was kindly provided by Professor Li‐Min Huang at National Taiwan University Hospital. All viruses were propagated in the allantoic cavity of 10‐day‐old specific pathogen‐free (SPF) chicken embryos (JD‐SPF Biotech, Miaoli, Taiwan). Virus titer was determined by plaque assays as previously described.^[41]