Fig 1.
Production of infectious Zika viral particles.
In target cell lines over time as measured by plaque assay. PFU/mL = plaque forming units/milliliter. Error rate for plaque assay is +/- 0.5 log. Asterisks denote data with statistically significant change from baseline.
Fig 2.
Determined by qRT-PCR in target cell lines: Vero, Huh7.5, and HepG2, in a) cell culture supernatants, and b) cell lysates. Results are the percent inverse of the PCR cycles over time of infection. Data are means with bars representing standard error of the mean. Asterisks denote data with statistically significant change from baseline.
Fig 3.
Measurement of Zika NS1 protein in target cell line supernatant over time, by ELISA. Results are expressed as the log ng/mL of the mean of 2–4 replicates in two experiments. Bars represent standard error of the mean. Asterisks denote data with statistically significant change from baseline.
Fig 4.
Agarose gel visualization of RT-PCR 113 bp products.
Amplification of Zika RNA extracted from cell lysates from the target cell lines, at days 1,2,3,6, and 8 of infection. Positive strand products were amplified using the negative primer and negative strand products using the positive primer in the initial reverse transcription (RT) step. Product size is indicated by a 100 base pair ladder. To verify that no products were present without the initial RT step, representative HepG2 cell lysates were amplified with and without the reverse transcription enzyme. No bands appear in the lanes which had no RT enzyme. Positive strand virus with RT is more abundant than negative strand virus.
Fig 5.
Cell lines with either uninfected or Zika-infected cells were visualized by electron microscopy to observe ultrastructure, after three days of infection. Black arrows point to ZIKV. a) EM image of uninfected Vero cells. Magnification = 80000x. Scale bar = 100 nm. b) Uninfected Huh7.5 cells. Magnification = 80000x. Scale bar = 100 nm. c) Uninfected HepG2 cells. Magnification = 80000x. Scale bar = 100 nm. d) EM image using negative staining of Zika virions in Vero cell culture supernatant. Particle sizes in nm. Magnification = 300000x. Scale bar = 100 nm. e) EM image of Zika infection in Huh7.5 cells. Magnification = 80000x. Scale bar = 100 nm. f) EM image of HepG2 cells infected with Zika virus. Magnification = 60000x. Scale bar = 100 nm.
Fig 6.
Apoptosis as measured by M30 ELISA.
Determination of apoptosis in undiluted cell supernatants over time by M30 CytoDeath ELISA (enzyme-linked immunosorbent assay). Results are the mean of 2–4 replicates, bars representing standard error of the mean. Assay detection limit is 60–3000 U/L; LLOQ is 250 U/L. Asterisk denotes data with statistically significant changes from baseline.