Skip to main content
. Author manuscript; available in PMC: 2019 Oct 10.
Published in final edited form as: Cell Host Microbe. 2018 Oct 10;24(4):487–499.e5. doi: 10.1016/j.chom.2018.09.008

Figure 3.

Figure 3.

Characterization of the C7 mutant with adaptive mutations (C7a). (a) Comparison of specific infectivity between C7 and C7a (with adaptive prM E21K and NS2B E27G mutations). Upper-left panel outlines the experimental procedures. Infectious cDNA clone-derived C7 and C7a RNAs were transfected into BHK-HA-C cells. Transfected cells were serially diluted and seeded onto BHK-HA-C cell monolayers. Focus-forming units were detected by immunostaining on day 4 post-transfection. PFU/μg RNA values were calculated and presented in the upperright panel. The lower panel shows the morphology of the foci after immunostaining. Statistically significant differences were determined using a t-test. (b) IFA of RNA-transfected Vero, BHK-21, and BHK-HA-C cells. C7a RNAs (containing the C7 deletion plus prM E21K and NS2B E27G mutations) were electroporated into BHK-21 or BHK-HA-C cells. The viral E protein expression was monitored by IFA. (c) Virus titers in supernatants on day 4 p.t. determined by focus-forming assay. (d) Focus morphology. C7a/t viruses were harvested from the C7a RNA-transfected BHK-HA-C cells on day 4 post-transfection. Focus morphology of C7a/t virus was determined on Vero, BHK-21, and BHK-HA-C cells. (e) Viral replication kinetics. BHK-21 or Vero cells were infected with C7a/t or WT ZIKV at an MOI of 0.5. Virus titers were measured by focus-forming assay on BHK-HA-C cells. Statistical significance was determined using multiple t-test. (f) IFA for monitoring the spread of WT ZIKV infection. (g) IFA for monitoring the spread of C7a/t virus infection. Cells were infected with WT or C7a/t virus (MOI 0.25). Viral E protein expressing cells were quantified by IFA using 4G2 antibody. Percentages of E-positive cells are presented. CPE, viral infection-mediated cytopathic effect; +, mild; ++, moderate; +++, severe. (h) Western blot analysis of C7a/t virus. Supernatants from replicon RNA-transfected BHK-HA-C cells (day 4 p.t.), WT RNA-transfected Vero cells (day 4 p.t.), and C7a RNA-transfected BHK-HA-C cells (day 4 p.t.) were analyzed for viral proteins (HA-C, C, M, E, and NS1) using Western blotting.