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Figure 5
Creation and in vitro replication of the mutant virus (vPIP mI). (a) A schematic diagram of the ORF49 gene locus in the MHV-68 genome. Open reading frames are shown as boxes. The arrowheads of the boxes indicate the direction of transcription. The numbers indicate the positions of each part within the viral genome. The recombinant viruses vPIP mI and vPIP mI-MR were constructed as indicated and the mutated positions in the recombinant viruses were confirmed by sequencing. (b) The genome integrity of the recombinant virus BAC clones was verified by digestion with the EcoRI or NotI restriction enzyme. (c) Multiple-step replication curves of the WT, vPIP-S, vPIP mI and vPIP mI-MR viruses. BHK21 cells were infected with the WT, vPIP-S, vPIP mI or vPIP mI-MR virus in triplicate at a multiplicity of infection (MOI) of 0.05 and were harvested at the indicated time points. The virus titers in the cells and the supernatants were analyzed by plaque assays. (d, e) Plaque sizes of the WT, vPIP-S, vPIP mI and vPIP mI-MR viruses. Plaque assays were performed on Vero cells and the diameters of the plaques were determined for at least 100 plaques per virus. The average plaque sizes are shown with the standard error in (d). Statistical analysis was performed by a two-sided Student's t-test (*** denotes P < 0.005). Representative pictures of actual plaques are shown in (e). The scale bar is 200 µm in length.

IUCrJ
Volume 5| Part 6| November 2018| Pages 866-879
ISSN: 2052-2525
https://doi.org/10.1107/S2052252518013854