Human respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI),and no vaccine against LRTI has proven to be safe and effective in infants.Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice.The constructed recombinant plasmids harbored (5\'to 3\') a T7 promoter,hammerhead ribozyme,RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion(A2cpts△SH),HDV ribozyme (δ),and a T7 terminator.These vectors were subsequently co-transfected with four helper plasmids encoding N,P,L,and M2-1 viral proteins into BHK/T7-9 cells,and the recovered viruses were then passaged in Vero cells.The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp,rRSV-Long/A2cpts,and rRSV-Long/A2cpts△SH,respectively,and stably passaged in vitro,without reversion to wild type (wt) at sites containing introduced mutations or deletion.Although rRSV-Long/A2cpts and rRSV-Long/A2cpts△SH displayed temperature-sensitive (ts)phenotype in vitro and in vivo,all rRSVs were significantly attenuated in vivo.Furthermore,BALB/c mice immunized with rRSVs produced Thl-biased immune response,resisted wtRSV infection,and were free from enhanced respiratory disease.We showed that the combination of △SH with attenuation (att) mutations of cpts contributed to improving att phenotype,efficacy,and gene stability of rRSV.By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains,we have laid an important foundation for the development of RSV live attenuated vaccines.