One-step reverse transcription-polymerase chain reaction (RT-PCR) was employed to amplify a cDNA fragment including L region by synthetic oligonucleotides as follows: (LF) 5-ACAAACACGGTCTAAGCAGG-3; (LR) 5-GTTCTGGTATTGCTGCAT-3. that both the relative usage of the two initiation sites and overall translation efficiency were changed by Lb AUG modifications. In addition, the in vitro translation activity was also negatively regulated by Lb AUG mutations. Collectively, these findings suggested that this restricted replications of Lb AUG-modified FMDVs were related to the delayed eIF4GI cleavage and decreased ability to block IFN expression but were mainly determined by the inefficient translation initiation. FMDVs precisely with modifications of Lb AUG initiation codon may represent safer seed viruses for Leuprolide Acetate vaccine production. Key points I and II and constructed back between I and II sites of the full-length cDNA pOFS for FMDV O/HN/CHN/93, producing recombinant full-length plasmids pLab3m, pLab4m, pLab5m, and pLab9m. The resultant plasmids were verified by sequencing. Table 1 Primers used for the mutagenesis of Lb AUG initiation codon I and I sites of psiCHECK?-2 vector to produce psicIRESWT, psicIRESLab3m, psicIRESLab4m, psicIRESLab5m, and psicIRESLab9m, respectively. The detailed construction processes were described previously (Han et al. 2020; Huang et al. 2012). The introduced fragments were analyzed by sequencing as well. Rescue of mutant viruses The four recombinant plasmids in addition to the control Vav1 plasmid pOFS (2 g), which were linearized by I, were transfected into BSR/T7 cells using LipofectamineTM 2000 (Invitrogen). The transfected cells were incubated at 37 C for 48 h, and the supernatants were harvested following freezing. The viruses recovered from transfections were amplified by 6 rounds of contamination of BHK-21 cells. Virus titers were decided in BHK cells by calculating the 50% tissue culture infectious dose (TCID50) as described previously (Li et al. Leuprolide Acetate 2012b). The viral RNA was extracted from the passage 3 and 6 of each recovered virus using RNeasy mini kit (Qiagen) according to the manufacturers manual, and the remaining DNA was eliminated by digestion with RNase-free DNase I (Qiagen). One-step reverse transcription-polymerase chain reaction (RT-PCR) was employed to amplify a cDNA fragment including Leuprolide Acetate L region by synthetic oligonucleotides as follows: (LF) 5-ACAAACACGGTCTAAGCAGG-3; (LR) 5-GTTCTGGTATTGCTGCAT-3. The resulted fragments were sequenced using the following primer: (LCF) 5-AGGTAACACGAGACACTC-3. These results were confirmed by at least two impartial sequencing reactions. Immunofluorescence assay BHK-21 cell monolayers were produced on 20-mm glass-bottomed culture dish (NEST) and infected with modified FMDVs at a multiplicity of contamination (MOI) of 10 for 4 h. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS), and then blocked with 1% bovine serum albumin (BSA) overnight at 4 C. FMDV 3A protein was detected with primary antibody MAb 3A24 and Cy3-conjugated secondary antibody, and nuclei were detected with 4,6-diamidino-2-phenylindole (DAPI) staining (Beyotime Institute of Biotechnology, Shanghai, China). Eventually, Alexa Fluor 405 and Alexa Fluor 561 were examined using a Leuprolide Acetate laser-scanning confocal microscope (LSCM; Leica SP8, Leica Camera AG, Wetzlar, Germany). The growth of FMDV mutants in culture cells To analyze the effects of Lb AUG mutations on FMDV replication, plaque morphology of modified FMDVs was evaluated in BHK-21 or PK-15 cells as described previously (Bai et al. 2019; Li et al. 2012a; Vazquez-Calvo et al. 2014). The virus-infected cells were.