Journal: PLoS ONE

Article Title: African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast

doi: 10.1371/journal.pone.0200086

Figure Lengend Snippet: (A) Published amino acid sequences (NCBI GenBank) of the six ZIKV strains were used to construct a phylogenetic tree using the neighbor-joining method. The AS (red) and AF strains (blue) show a clear separation into two distinct clusters based on their amino acid sequences. (B) Comparison of amino acid sequence identities between the six viruses when clustered into the AF and AS lineages.

Article Snippet: After fixing the cells in 4% v/v paraformaldehyde in PBS for 12 min and permeabilizing in 1% Triton X-100/PBS, coverslips were placed in 5% w/v bovine serum albumin in PBS for 1 h. Cells were then incubated overnight at 4°C with polyclonal primary anti-ZIKV antibodies (SAB-153 Biotherapeutics), diluted 1:2000 in 5% w/v bovine serum albumin in PBS.

Techniques: Construct, Comparison, Sequencing

Journal: PLoS ONE

Article Title: African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast

doi: 10.1371/journal.pone.0200086

Figure Lengend Snippet: Colonies of ESC were grown in 6-well matrigel-coated cell culture dishes and differentiated for 4 days by BAP treatment. After 4 days, cells were infected with each ZIKV strain at 1, 0.1 or 0 (mock) MOI. (A) At 48 h PI, cells were fixed and stained with crystal violet to observe CPE. Cell lysis was evident in cells infected with all three of the AF strains, even at 0.1 MOI, whereas minimal cell lysis was evident in AS ZIKV infected (1 MOI) colonies. (B) Cell viability was measured at 48 h and 60 h PI to demonstrate the extent of lysis induced by each ZIKV strain at 1 MOI. At 48 h post- infection, only the three AF strains of ZIKV (shades of blue) showed a significant reduction in cell viability when compared to the mock infected control (one-way ANOVA, p < 0.001). Conversely, none of the AS strains of ZIKV (shades of red) induced a significant reduction in cell viability. All six ZIKV strains showed a significant reduction in cell viability when compared to the mock infected control at 60 h PI ( p < 0.01 (Mexico), p < 0.001 (all other strains)). Data are presented as mean ± SEM (n = 5). (C) The extent of cell lysis at 72 h PI is accentuated by images taken at a higher magnification. At 72 h PI, severe cell lysis was evident in cells infected with all three AF strains and became apparent in cells infected with the AS strains. In the images, areas of deep purple staining highlight the areas of multinucleated syncytia. There were no syncytial areas present in the cultures infected with the AF strains of ZIKV, while small areas remained visible after infection with the AS strains. At 72 h PI, mock infected controls showed non-virus induced cell lysis, as the cells naturally started to die off at this time point post-differentiation. These areas are indicated by black arrows. Scale bars are 3 mm (A) and 1 mm (C).

Article Snippet: After fixing the cells in 4% v/v paraformaldehyde in PBS for 12 min and permeabilizing in 1% Triton X-100/PBS, coverslips were placed in 5% w/v bovine serum albumin in PBS for 1 h. Cells were then incubated overnight at 4°C with polyclonal primary anti-ZIKV antibodies (SAB-153 Biotherapeutics), diluted 1:2000 in 5% w/v bovine serum albumin in PBS.

Techniques: Cell Culture, Infection, Staining, Lysis, Control, Virus

Journal: PLoS ONE

Article Title: African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast

doi: 10.1371/journal.pone.0200086

Figure Lengend Snippet: (A) Two separately cultured groups of JAr cells were exposed continuously to either enzymatic dispersal (>10 passages, trypsin) or non-enzymatic dispersal (gentle disassociation reagent, STEMCELL Technologies). Prior to infection, each group was dispersed by a non-enzymatic agent, to allow uniform cell dispersal. Two days after plating, the groups were infected with the AF Uganda strain of ZIKV at an MOI of 0.27. At 48 hours PI, live cell images were captured to demonstrate CPE (bottom panel). Cells were fixed and stained with crystal violet (top panel). Areas showing clear CPE are highlighted by red rectangles. (B) JAr cells were infected with each ZIKV strain at 1 MOI and fixed either at 48 h or 60 h PI. Respective mock infected controls are shown below. Lysis is only evident after infection with the three AF strains of ZIKV. Scale bars are 100 μm for live cell images in (A) and 1 mm in (A) and (B) for crystal violet stained images.

Article Snippet: After fixing the cells in 4% v/v paraformaldehyde in PBS for 12 min and permeabilizing in 1% Triton X-100/PBS, coverslips were placed in 5% w/v bovine serum albumin in PBS for 1 h. Cells were then incubated overnight at 4°C with polyclonal primary anti-ZIKV antibodies (SAB-153 Biotherapeutics), diluted 1:2000 in 5% w/v bovine serum albumin in PBS.

Techniques: Cell Culture, Gentle, Infection, Staining, Lysis

Journal: PLoS ONE

Article Title: African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast

doi: 10.1371/journal.pone.0200086

Figure Lengend Snippet: Vero cells were infected with each ZIKV strain at 1 MOI and fixed either at 48 h or 60 h PI. Respective mock infected controls are shown below. Similar CPE are evident after infection with all AF and AS ZIKV strains, although the AF Nigeria and AS Panama strains elicited weaker CPE when compared to the other strains. Scale bar is 1 mm.

Article Snippet: After fixing the cells in 4% v/v paraformaldehyde in PBS for 12 min and permeabilizing in 1% Triton X-100/PBS, coverslips were placed in 5% w/v bovine serum albumin in PBS for 1 h. Cells were then incubated overnight at 4°C with polyclonal primary anti-ZIKV antibodies (SAB-153 Biotherapeutics), diluted 1:2000 in 5% w/v bovine serum albumin in PBS.

Techniques: Infection

Journal: PLoS ONE

Article Title: African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast

doi: 10.1371/journal.pone.0200086

Figure Lengend Snippet: Cells were infected with the six ZIKV strains at a MOI of 0.1. Cell supernatants were harvested at the indicated time points for titration by plaque assay in Vero cells. Growth curve analyses were performed in triplicate and plotted as SEM. Growth curves representing the AF strains are highlighted in shades of blue and those representing the AS strains are shown in shades of red. (A) In ESCd, significantly higher virus concentrations were observed by 48 h PI for the AF Uganda and the AF Senegal strains when compared to all other strains ( p < 0.05). No significant differences were observed between the Nigeria, Cambodia, Mexico, and Panama strains. (B) In JAr cells, significantly higher concentrations were observed by 48 h PI for the AF Uganda strain when compared to all other strains ( p < 0.001). No significant differences were observed between the Senegal, Nigeria, Cambodia, Mexico, and Panama strains. (C) In Vero cells, significantly higher virus concentrations became obvious by 48 h PI for the AF Uganda, the AF Senegal, and the AS Mexico strains when compared to the other three viruses ( p < 0.01). No significant differences were observed between the Nigeria, Cambodia and Panama strains.

Article Snippet: After fixing the cells in 4% v/v paraformaldehyde in PBS for 12 min and permeabilizing in 1% Triton X-100/PBS, coverslips were placed in 5% w/v bovine serum albumin in PBS for 1 h. Cells were then incubated overnight at 4°C with polyclonal primary anti-ZIKV antibodies (SAB-153 Biotherapeutics), diluted 1:2000 in 5% w/v bovine serum albumin in PBS.

Techniques: Infection, Titration, Plaque Assay, Virus