Journal: Viruses

Article Title: Generation of Recombinant Authentic Live Attenuated Human Rotavirus Vaccine Strain RIX4414 (Rotarix ® ) from Cloned cDNAs Using Reverse Genetics

doi: 10.3390/v16081198

Figure Lengend Snippet: Generation of a panel of recombinant SA11-L2-based single-segment reassortants having one gene segment from RIX4414. ( A ) Schematic presentation of an 11-plasmid reverse genetics system to generate SA11-L2-based single-segment reassortants. The 11 rescue T7 plasmids include the full-length segment of cDNA of each dsRNA segment of RVA, flanked by the T7 RNA polymerase promoter (PT7) and the HDV ribozyme (Rib). To generate SA11-L2-based single-segment reassortants having one gene segment from RIX4414, BHK/T7-9 cells were cotransfected with the 11 rescue T7 plasmids (10 for SA11-L2 plus one for RIX4414) with 3-fold increased amounts of the two plasmids carrying the NSP2 and NSP5 genes. A panel of recombinant SA11-L2-based single-segment reassortants having one segment from RIX4414 were rescued from the cultures of the transfected BHK/T7-9 cells. ( B ) PAGE analysis of recombinant SA11-L2-based single-segment reassortants with each of the 11 segments from RIX4414. Lanes 1 and 13, dsRNAs from rSA11-L2 (lane 1) and RIX4414 (lane 13); lanes 2–12, dsRNAs from rescued rSA11-VP1RIX (lane 2), rSA11-VP2RIX (lane 3), rSA11-VP3RIX (lane 4), rSA11-VP4RIX (lane 5), rSA11-NSP1RIX (lane 6), rSA11-VP6RIX (lane 7), rSA11-NSP3RIX (lane 8), rSA11-NSP2RIX (lane 9), rSA11-VP7RIX (lane 10), rSA11-NSP4RIX (lane 11), and rSA11-NSP5RIX (lane 12). Red asterisks indicate the positions of the cDNA-derived RIX4414 segments. The numbers on the left and right indicate the orders of the genomic dsRNA segments of rSA11-L2 and RIX4414, respectively. ( C ) Infectivity of recombinant SA11-L2-based single-segment reassortants with each of the 11 segments from RIX4414. MA104 cells were infected with RVAs at an MOI of 0.01 and then incubated for 36 h. The viral titers in the cultures were determined by plaque assay. The data shown are the mean viral titers and standard deviations (SDs) for three independent cell cultures. Asterisks indicate significant differences between rSA11-L2 and recombinant single-segment reassortants; *, p < 0.05 (as calculated by two-way ANOVA with Sidak’s post-test).

Article Snippet: A baby hamster kidney cell line stably expressing the T7 RNA polymerase (BHK/T7-9) [ ] was cultured in Dulbecco’s modified Eagle medium (DMEM; Nacalai, Kyoto, Japan) supplemented with 5% fetal calf serum (FCS; Gibco, Tokyo, Japan) (complete medium) in the presence of 600 ng/mL hygromycin (Invitrogen, Tokyo, Japan).

Techniques: Recombinant, Plasmid Preparation, Transfection, Derivative Assay, Infection, Incubation, Plaque Assay

Journal:

Article Title: Cell-to-Cell Contact as an Efficient Mode of Epstein-Barr Virus Infection of Diverse Human Epithelial Cells

doi:

Figure Lengend Snippet: Cells used in this study

Article Snippet: BHK , Baby hamster kidney cells , JCRB , 37.

Techniques: