Optimization Of Titer Detection Method For Respiratory Syncytial Virus(RSV)and Modification Of RSV Genome

Here,we analyzed the plaque effect produced by RSV infection in different cells and compared the effect of different covering media on the plaque effect caused by RSV infection.It was observed that the virus produced different lesions on different cells:typical cell fusion(syncytosis)was observed in 3 dpi after Hep-2 cell infection;syncytosis was not observed until 7 dpi after Vero cell infection,but it was atypical and not obvious,and 14 dpi cells showed obvious cell shedding;after infection with Hela cells,the cells appeared round shrinkage at 3-4 dpi,and the cells shed rapidly without syncytosis.Further,we analyzed the effects of different covering media on the size and number of plaques generated by RSV infected Hep-2 cells.We have tested four medium,including classic commonly used high viscosity medium(a kind of solid medium,agarose),classic commonly used low viscosity medium(the semisolid medium,methyl cellulose(MC)and carboxymethyl cellulose(CMC),the new type of low viscosity medium(has not yet been used in the study of RSV microcrystalline cellulose,Avicel).However,after comparison,we found that there was no significant difference in the diameter of plaque between the agarose group and the Avicel group,however,the number of agarose group plaques in the same proportion of infection was less,about 20%of that in the Avicel group,and the operation of the medium required technicians to master the optimal temperature after melting;all the cells in the MC coverage media group were exfoliated,and no number of plaques were formed;The diameter of the macrophage plaques in the CMC coverage media group was too small for observation and counting,which was almost the same as the spot signals in the classical immunochemical staining method.However,in the Avicel overlying mediators group,the diameter of the plaques was larger and the number was larger for observation and counting.In summary,we found that Hep-2 cells are a cell line suitable for the analysis of RSV infected phagocytosis,and we successfully tried for the first time to apply the new covering medium,Avicel,to RSV phagocytosis,which does not rely on immunocytochemical staining but is a convenient and rapid crystal violet staining method.This study compared three low-copy,high-capacity vectors,including pBeloBAC(which is about 2),pACNR1180(which is strictly low-copy if the copy number is below 5),and p15A(which is about 15).Firstly,segmented RSV cDNA was cloned into different vectors by Gibson recombinant enzyme,and it was found that the viral genome was only intact in pACNR1180 and pBeloBAC vectors,while the 2 KB gene(G and SH gene sequences)were lost in p15A vectors for many times.Further,we selected the pACNR1180-RSV cDNA recombinant plasmid for the study of fast and seamless site-directed cloning transformation.Taking the deletion of the NS1 gene as an example,we used three different homologous recombination techniques to realize the connection between the 1.5kb PCR product and the 15kb recombinant plasmid enzyme digestion product,including SLIC,Gibson assembly technology,Red/ET recombination technology.In the attempt of this study,we found that the positive rate of successful ligation in the SLIC group was 0,about 25%in the Gibson assembly,and about 65%in the Red/ET group.Therefore,we successfully established an efficient and rapid RSV cDNA modification method based on the Red/ET system,and further,we successfully constructed the M2-2 gene deletion and the deletion of NS1 and M2-2 gene deletion cDNA with this method,for the next step to study the effect of the deletion gene on viral virulence.In summary,this paper successfully established a convenient and stable plaque method to detect RSV titer,the rapid construction of RSV cDNA based on Gibson assembly and the rapid site-directed seamless cloning of RSV cDNA based on Red/ET homologous recombination.It provides a more convenient method for the laboratory to detect the titer of RSV virus by the porphyritic method,provides a favorable molecular method for the basic laboratory research,such as the study of gene function,and further provides an alternative method for the virulence assessment of strains in the vaccine development and the artificial manufacture of weak strains.