Smai restriction enzyme

Pulsed-field gel electrophoresis (PFGE) was performed using the SmaI restriction enzyme (New England Biolabs, UK) [18] (link). Banding patterns were analysed using BioNumerics software version 6.01 (Applied Maths, Sint-Martens-Latem, Belgium) as described [19] (link). A similarity coefficient of 80% was used to define PFGE types [18] (link). DNA was extracted and MLST was performed as described elsewhere [2] (link), [6] (link), [20] (link). Sequence types (ST) were assigned using the MLST database (www.mlst.net) [20] (link). Phylogenetic analysis of concatenated MSLT data was performed using ClustalW for the alignment and the maximum likelihood method for construction of the phylogenetic tree in MEGA version 6. Two multiplex PCR assays were used to detect the presence of virulence and other genes (assay 1 for tst, eta, etb, sea, seb, sec, sed, see[21] (link), assay 2 for mecA and pvl) [22] (link).

Chromosomal DNA was digested with SmaI restriction enzyme (New England BioLabs) as described previously (46 (link)). The samples were loaded on pulsed-field certified agarose (1%) (Bio-Rad) using a CHEF mapper system (Bio-Rad, Munich, Germany). A Salmonella serotype Braenderup strain (H9812) was used as the molecular size standard. Running parameters were as follows: initial switch, 5 s; final switch, 40 s; time, 20 h; voltage, 6 V/cm; and temperature, 14°C. Gels were stained with 500 mL ethidium bromide (EB) for 25 min, destained in water for 40 min, and viewed using the Gel doc XR imaging system to obtain electrophoretic images. The similarity coefficients of DNA banding patterns were analyzed using BioNumerics software (version 4.0, Applied Maths, Ghent, Belgium). Similarity coefficients of 100% were determined as the same PFGE type, and those less than 100% were determined as different PFGE type. A similarity cutoff value of 80% was set to define the PFGE cluster. Isolates exhibiting identical DNA patterns were regarded as genotypically indistinguishable.

Chromosomal DNA was digested with SmaI restriction enzyme (New England BioLabs) as described previously (46 (link)). The samples were loaded on pulsed-field certified agarose (1%) (Bio-Rad) using a CHEF mapper system (Bio-Rad, Munich, Germany). A Salmonella serotype Braenderup strain (H9812) was used as the molecular size standard. Running parameters were as follows: initial switch, 5 s; final switch, 40 s; time, 20 h; voltage, 6 V/cm; and temperature, 14°C. Gels were stained with 500 mL ethidium bromide (EB) for 25 min, destained in water for 40 min, and viewed using the Gel doc XR imaging system to obtain electrophoretic images. The similarity coefficients of DNA banding patterns were analyzed using BioNumerics software (version 4.0, Applied Maths, Ghent, Belgium). Similarity coefficients of 100% were determined as the same PFGE type, and those less than 100% were determined as different PFGE type. A similarity cutoff value of 80% was set to define the PFGE cluster. Isolates exhibiting identical DNA patterns were regarded as genotypically indistinguishable.

All infectious clone plasmids were purified using a HiPure Plasmid Micro Kit (Magen, Guangzhou, China). The RNA transcription procedure was performed as previously reported^{25 (link)}. Briefly, the plasmid was linearized using SmaI restriction enzyme (NEB, Beijing, China) and was then purified as a template for RNA transcription. An mMESSAGE mMACHINE T7 Transcription kit (Ambion, USA) was used for the transcription of RNA in vitro in a 20 μL reaction with an additional 1.5 μL of GTP solution, which was incubated at 37 °C for 3 h. The DNA template was then removed using DNase I. Afterward, the RNA was purified using the lithium chloride precipitation method, quantitated by spectrophotometry, and stored at –80 °C in aliquots.
For virus rescue, BHK-21 cells were seeded in 12-well plates for 16 h, and the cells (70–90% confluence) were then transfected with 1 μg of RNA per well using Lipofectamine MessengerMAX reagent (Invitrogen, CA, USA). After transfection, the cell plates were incubated at 37 °C with 5% CO₂. The harvested supernatants from transfected cells (F0 virus) were used to prepare F1 virus stock, and F1 viruses were confirmed by Sanger sequencing.