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

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

ZIKV infection induces a Th1 CD4⁺ T cell response.

<p>Representative plots of IFN-γ <b>(A)</b>, TNF-α <b>(B)</b>, and IL-2 <b>(C)</b> production from splenic CD11a⁺CD49d⁺ CD4⁺ T cells from ZIKV-infected mice 7 dpi. Total splenocytes were incubated in media alone in the presence of Brefeldin A (top) or stimulated with PMA and ionomycin in the presence of Brefeldin A for 3 h at 37°C (bottom). Percentage <b>(D)</b>, number <b>(E)</b>, and gMFI <b>(F)</b> of cytokine-producing CD11a⁺CD49d⁺ CD4⁺ T cells from the spleens of ZIKV-infected mice 7 dpi. <b>(G)</b> Representative histograms of T-bet expression in splenic CD11a⁺CD49d⁺ CD4⁺ T cells from mock- and ZIKV-infected mice 7 dpi. Shaded histograms represent isotype control. Percentage <b>(H)</b> and gMFI <b>(I)</b> of T-bet expression in splenic CD11a⁺CD49d⁺ CD4⁺ T cells from mock- and ZIKV-infected mice 7 dpi. Error bars represent mean ± SEM. Data are pooled from two independent experiments, n = 3–5 mice per group per experiment. Data in <b>(H</b> and <b>I)</b> were analyzed with a two-tailed, unpaired Student’s t test. ****p<0.0001.</p

Analysis of the T Cell Response to Zika Virus and Identification of a Novel CD8⁺ T Cell Epitope in Immunocompetent Mice

<div><p>Zika virus (ZIKV) is an emerging arbovirus of the <i>Flaviviridae</i> family. Although ZIKV infection is typically mild and self-limiting in healthy adults, infection has been associated with neurological symptoms such as Guillain-Barré syndrome, and a causal link has been established between fetal microcephaly and ZIKV infection during pregnancy. These risks, and the magnitude of the ongoing ZIKV pandemic, have created an urgent need for the development of animal models to study the immune response to ZIKV infection. Previous animal models have primarily focused on pathogenesis in immunocompromised mice. In this study, we provide a model of ZIKV infection in wild-type immunocompetent C57BL/6 mice, and have provided an analysis of the immune response to infection. We evaluated the activation of several innate immune cell types, and studied the kinetics, phenotype, and functionality of T cell responses to ZIKV infection. Our results demonstrate that ZIKV infection is mild in wild-type immunocompetent C57BL/6 mice, resulting in minimal morbidity. Our data establish that at the peak of the adaptive response, antigen-experienced CD4⁺ T cells polarize to a Th1 phenotype, and antigen-experienced CD8⁺ T cells exhibit an activated effector phenotype, producing both effector cytokines and cytolytic molecules. Furthermore, we have identified a novel ZIKV CD8⁺ T cell epitope in the envelope protein that is recognized by the majority of responding cells. Our model provides an important reference point that will help dissect the impact of polymorphisms in the circulating ZIKV strains on the immune response and ZIKV pathogenesis. In addition, the identification of a ZIKV epitope will allow for the design of tetramers to study epitope-specific T cell responses, and will have important implications for the design and development of ZIKV vaccine strategies.</p></div

ZIKV induces innate immune cell activation.

<p>Representative histograms and geometric mean fluorescence intensity (gMFI) of CD40 <b>(A</b>-<b>B)</b>, CD80 <b>(C</b>-<b>D)</b>, and CD86 <b>(E-F)</b> on splenic dendritic cells (DCs) at 2 dpi with 10⁶ PFU of ZIKV i.v. (open histograms with solid line), an equivalent dose of UV-inactivated ZIKV (open histograms with dashed line) or mock infection (shaded histograms). Histograms represent DCs gated as CD3^- CD19^- NK1.1^- MHC-II⁺ CD11c⁺ cells. Representative histogram <b>(G)</b>, percentage <b>(H)</b>, and number <b>(I)</b> of CD69⁺ NK1.1⁺CD3^- natural killer cells from the spleens of ZIKV- (open histogram with solid line), UV-inactivated ZIKV- (open histogram with dashed line) and mock-infected (shaded histogram) mice 2 dpi. Number on histogram indicates percentage of CD69⁺ cells from ZIKV-infected mouse. <b>(J)</b> Mice were infected with 10⁶ PFU of ZIKV or an equivalent dose of UV-inactivated virus, spleens were harvested 12 h post-infection and total RNA was extracted. IFN-α and IFN-β mRNA expression was assessed by qRT-PCR, normalized to TATA-binding protein mRNA expression, and expressed as fold change over mRNA expression in mock-infected mice. Error bars represent mean ± SEM. Data in (<b>A</b>-<b>I</b>) are pooled from two independent experiments, n = 3 mice per group per experiment. Data in (<b>J</b>) are pooled from three independent experiments, n = 3 mice per group per experiment. Data in <b>(B</b>, <b>D</b>, <b>F</b>, <b>H</b> and <b>I)</b> were analyzed by one-way ANOVA with Tukey’s post-test of multiple comparisons. ****p<0.0001.</p

ZIKV infection kinetics.

<p><b>(A)</b> Viral RNA was quantified in the spleens of C57BL/6 mice infected i.v. with 10⁶ PFU of ZIKV at 6, 12, 24, 48 and 72 h post-infection using qRT-PCR analysis. <b>(B)</b> Viral RNA was quantified in the spleens of C57BL/6 mice infected i.v. with 10⁶ PFU of ZIKV or an equivalent dose of UV-inactivated virus at 12 h post-infection using qRT-PCR analysis. Data are presented as plaque forming unit (PFU) equivalents per gram of tissue after normalization to a standard curve (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006184#ppat.1006184.s002" target="_blank">S2 Fig</a>). The dotted line indicates limit of detection based on the average number of PFU equivalents per gram of tissue from the spleens of mock-infected mice at 48 h post-infection <b>(A)</b> or 12 h post-infection <b>(B)</b>. Error bars represent mean ± SEM. Data are pooled from two independent experiments, n = 3 mice per group per experiment.</p

ZIKV infection induces a prototypical effector CD8⁺ T cell response in immunocompetent mice.

<p>Representative plots of IFN-γ <b>(A)</b>, TNF-α <b>(B)</b>, and IL-2 <b>(C)</b> production from splenic CD8α^loCD11a^hi CD8⁺ T cells from ZIKV-infected mice 7 dpi. Total splenocytes were incubated in media alone in the presence of Brefeldin A (top) or stimulated with PMA and ionomycin in the presence of Brefeldin A for 3 h at 37°C (bottom). Percentage <b>(D)</b>, number <b>(E)</b>, and gMFI <b>(F)</b> of cytokine-producing CD8α^loCD11a^hi CD8⁺ T cells from the spleens of ZIKV-infected mice 7 dpi. <b>(G)</b> Representative histograms of T-bet expression in splenic CD8α^loCD11a^hi CD8⁺ T cells from mock- and ZIKV-infected mice 7 dpi. Shaded histograms represent isotype control. Percentage <b>(H)</b> and gMFI <b>(I)</b> of T-bet expression in splenic CD8α^loCD11a^hi CD8⁺ T cells from mock- and ZIKV-infected mice 7 dpi. Error bars represent mean ± SEM. Data are pooled from two independent experiments, n = 3–5 mice per group per experiment. Data in <b>(H</b> and <b>I)</b> were analyzed with a two-tailed, unpaired Student’s t test. ****p<0.0001.</p

CD8⁺ T cells respond to an epitope within the ZIKV Envelope protein.

<p><b>(A)</b> Representative plots of IFN-γ production from antigen-experienced CD8α^loCD11a^hi CD8⁺ T cells (top row) and naïve CD8α^hiCD11a^lo CD8⁺ T cells (bottom row) from the spleens of ZIKV-infected mice 7 dpi. Total splenocytes were incubated for 5.5 h at 37°C with media alone or 200nM of the indicated peptide in the presence of Brefeldin A. <b>(B)</b> Representative plots of IFN-γ production from antigen-experienced CD8α^loCD11a^hi CD8⁺ T cells (top row) and naïve CD8α^hiCD11a^lo CD8⁺ T cells (bottom row) from the spleens LCMV-infected mice 7 dpi. Total splenocytes were incubated for 5.5 h at 37°C with media alone or 200nM of the indicated peptide in the presence of Brefeldin A. <b>(C-D)</b> Percentage of IFN-γ⁺ antigen-experienced CD8α^loCD11a^hi or naïve CD8α^hiCD11a^lo CD8⁺ T cells from mock-, UV-inactivated ZIKV-, ZIKV- or LCMV-infected mice 7 dpi after restimulation with media alone or 200nM of GP_33-41 (LCMV peptide) <b>(C)</b> or Env_294-302 (ZIKV peptide) <b>(D)</b> for 5.5 h at 37°C in the presence of Brefeldin A. Error bars represent mean ± SEM. Data are pooled from two independent experiments, n = 3 mice per group per experiment. Data in <b>(C</b> and <b>D)</b> were analyzed with a two-tailed, paired Student’s t test. ****p<0.0001.</p

ZIKV induces CD4⁺ and CD8⁺ T cell activation at the peak of the T cell response.

<p>Representative plots of CD11a⁺CD49d⁺ antigen-experienced CD4⁺ T cells from spleens of mock- <b>(A)</b>, UV-inactivated ZIKV- <b>(B)</b> and ZIKV-infected <b>(C)</b> mice 7 dpi. Percentage <b>(D)</b> and number <b>(E)</b> of CD11a⁺CD49d⁺ antigen-experienced CD4⁺ T cells from spleens of mock-, UV-inactivated ZIKV- and ZIKV-infected mice 7 dpi. Representative plots of CD8α^loCD11a^hi antigen-experienced CD8⁺ T cells from the spleens of mock- <b>(F)</b>, UV-inactivated ZIKV- <b>(G)</b> and ZIKV-infected <b>(H)</b> mice 7 dpi. Percentage <b>(I)</b> and number <b>(J)</b> of CD8α^loCD11a^hi antigen-experienced CD8⁺ T cells from the spleens of mock-, UV-inactivated ZIKV- and ZIKV-infected mice 7 dpi. Error bars represent mean ± SEM. Data are pooled from two independent experiments, n = 3 mice per group per experiment. Data in <b>(D</b>, <b>E</b>, <b>I</b> and <b>J)</b> were analyzed by one-way ANOVA with Tukey’s post-test of multiple comparisons. ****p<0.0001.</p