Contemporary Zika Virus Isolates Induce More dsRNA and Produce More Negative-Strand Intermediate in Human Astrocytoma Cells

Abstract

The recent emergence and rapid geographic expansion of Zika virus (ZIKV) poses a significant challenge for public health. Although historically causing only mild febrile illness, recent ZIKV outbreaks have been associated with more severe neurological complications, such as Guillain-Barré syndrome and fetal microcephaly. Here we demonstrate that two contemporary (2015) ZIKV isolates from Puerto Rico and Brazil may have increased replicative fitness in human astrocytoma cells. Over a single infectious cycle, the Brazilian isolate replicates to higher titers and induces more severe cytopathic effects in human astrocytoma cells than the historical African reference strain or an early Asian lineage isolate. In addition, both contemporary isolates induce significantly more double-stranded RNA in infected astrocytoma cells, despite similar numbers of infected cells across isolates. Moreover, when we quantified positive- and negative-strand viral RNA, we found that the Asian lineage isolates displayed substantially more negative-strand replicative intermediates than the African lineage isolate in human astrocytoma cells. However, over multiple rounds of infection, the contemporary ZIKV isolates appear to be impaired in cell spread, infecting a lower proportion of cells at a low MOI despite replicating to similar or higher titers. Taken together, our data suggests that contemporary ZIKV isolates may have evolved mechanisms that allow them to replicate with increased efficiency in certain cell types, thereby highlighting the importance of cell-intrinsic factors in studies of viral replicative fitness.

Keywords: Zika virus; astrocytomas; dsRNA; flavivirus; viral fitness.

Figure 1

ZIKV isolates demonstrate unique plaque morphology and different growth kinetics in A549 and U-251 MG cell lines. (A) Representative images of Vero cell plaque assays of the indicated ZIKV isolates. (B–E) Cell culture supernatants were collected at the indicated time points and viral titer was determined by plaque assay. (B) A549 and (C) U-251 MG cells were infected with ZIKV at MOI = 10. (D) A549, and (E) U-251 MG cells were infected with ZIKV at MOI = 0.01. Values represent mean ± SD of at least three independent experiments. Asterisks indicate significant differences in viral titer relative to ZIKV^AF: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

ZIKV isolates elicit different cytopathic effects in A549 and U-251 MG cell lines. (A) A549 and (B) U-251 MG cells were infected with ZIKV at MOI = 10 and cell viability was determined by MTT assay at 24 h post-infection. (C) A549 and (D) U-251 MG cells were infected with ZIKV at MOI = 0.01 and cell viability was determined by MTT assay 72 h post-infection. % Cytopathicity = 100% − ((Uninfected Absorbance − Infected Absorbance)/(Uninfected Absorbance) × 100%). Values represent the mean ± SEM of three independent experiments. Asterisks indicate significant differences in % cytopathicity: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Contemporary ZIKV isolates induce more dsRNA than pre-epidemic isolates, despite similar numbers of infected cells. (A) A549 and (B) U-251 MG cells were infected with ZIKV at MOI = 10 and at 24 h post-infection cells were stained with the pan-flavivirus (4G2) antibody and the percentage of infected cells was determined by flow cytometry. (C) A549 and (D) U-251 MG cells were infected with ZIKV at MOI = 10 and 24 h post-infection the percentage of dsRNA-positive cells was determined by flow cytometry. The percentage of positive cells was determined by comparison to mock-infected cells. Values represent mean ± SEM of at least three independent experiments. Asterisks indicate significant differences in % infected cells: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Isolate-specific differences are observed in number and fluorescence intensity of dsRNA foci in infected cell. (A) A549 and (B) U-251 MG cells were infected with ZIKV at MOI = 10 and 24 h post-infection dsRNA expression was analyzed by immunofluorescence microscopy. Scale bar, 20 μm. The number of dsRNA foci per cell in (C) A549 and (D) U-251 MG cells was quantified using Imaris software (>100 cells/condition). (E) A549 and (F) U-251 MG cells were infected with ZIKV at MOI = 10 and 24 h post-infection the mean fluorescence intensity (MFI) of dsRNA-positive cells was determined by flow cytometry. Values represent mean ± SEM of at least three independent experiments. Asterisks indicate significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Asian lineage ZIKV isolates induce a higher ratio of negative:positive strand RNA. (A) A549 and (B) U-251 MG cells were infected with ZIKV at MOI = 10 and 24 h post-infection intracellular positive strand viral RNA was quantified by qRT-PCR. Data are normalized to GAPDH and expressed relative to a standard curve of PFU equivalents per ng input RNA. (C) A549 cells and (D) U-251 MG cells were infected with ZIKV at MOI = 10 and 24 h post-infection the relative amounts of positive and negative strand ZIKV genomes was quantified by qRT-PCR. Data are expressed as a ratio of negative:positive strand RNA. Values represent mean ± SEM of two or three independent experiments. Asterisks indicate significant differences: * p < 0.05, ** p < 0.01.