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4 pages, 183 KiB

Open AccessEditorial

New Advances on Zika Virus Research

by Luis Martinez-Sobrido and Fernando Almazán

Viruses 2019, 11(3), 258; https://doi.org/10.3390/v11030258 - 14 Mar 2019

Cited by 4 | Viewed by 3716

Abstract

Zika virus (ZIKV) is an emerging mosquito-borne member of the Flaviviridae family that has historically been known to cause sporadic outbreaks, associated with a mild febrile illness, in Africa and Southeast Asia [...] Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

Research

Jump to: Editorial, Review

11 pages, 438 KiB

Open AccessArticle

The Application and Interpretation of IgG Avidity and IgA ELISA Tests to Characterize Zika Virus Infections

by Fátima Amaro, María P. Sánchez-Seco, Ana Vázquez, Maria J. Alves, Líbia Zé-Zé, Maria T. Luz, Teodora Minguito, Jesús De La Fuente and Fernando De Ory

Viruses 2019, 11(2), 179; https://doi.org/10.3390/v11020179 - 20 Feb 2019

Cited by 16 | Viewed by 4308

Abstract

In the absence of viremia, the diagnostics of Zika virus (ZIKV) infections must rely on serological techniques. In order to improve the serological diagnosis of ZIKV, ZIKV-IgA and ZIKV-IgG avidity assays were evaluated. Forty patients returning from ZIKV endemic areas, with confirmed or [...] Read more.

In the absence of viremia, the diagnostics of Zika virus (ZIKV) infections must rely on serological techniques. In order to improve the serological diagnosis of ZIKV, ZIKV-IgA and ZIKV-IgG avidity assays were evaluated. Forty patients returning from ZIKV endemic areas, with confirmed or suspected ZIKV infections were studied. Samples were classified as early acute, acute and late acute according to the number of days post illness onset. Low avidity IgG was only detected at acute and late acute stages and IgA mostly at the early acute and acute stages. The date of sampling provides useful information and can help to choose the best technique to use at a determined moment in time and to interpret low avidity IgG and IgA results, improving the serological diagnosis of ZIKV. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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22 pages, 3157 KiB

Open AccessArticle

Integrated MicroRNA and mRNA Profiling in Zika Virus-Infected Neurons

by Francine Azouz, Komal Arora, Keeton Krause, Vivek R. Nerurkar and Mukesh Kumar

Viruses 2019, 11(2), 162; https://doi.org/10.3390/v11020162 - 16 Feb 2019

Cited by 43 | Viewed by 5841

Abstract

Zika virus (ZIKV) infections have caused a wide spectrum of neurological diseases, such as Guillain-Barré syndrome, myelitis, meningoencephalitis, and congenital microcephaly. No effective therapies currently exist for treating patients infected with ZIKV. MicroRNAs (miRNAs) are a group of small RNAs involved in the [...] Read more.

Zika virus (ZIKV) infections have caused a wide spectrum of neurological diseases, such as Guillain-Barré syndrome, myelitis, meningoencephalitis, and congenital microcephaly. No effective therapies currently exist for treating patients infected with ZIKV. MicroRNAs (miRNAs) are a group of small RNAs involved in the regulation of a wide variety of cellular and physiological processes. In this study, we analyzed digital miRNA and mRNA profiles in ZIKV-infected primary mouse neurons using the nCounter technology. A total of 599 miRNAs and 770 mRNAs were examined. We demonstrate that ZIKV infection causes global downregulation of miRNAs with only few upregulated miRNAs. ZIKV-modulated miRNAs including miR-155, miR-203, miR-29a, and miR-124-3p are known to play critical role in flavivirus infection, anti-viral immunity and brain injury. ZIKV infection also results in downregulation of miRNA processing enzymes. In contrast, ZIKV infection induces dramatic upregulation of anti-viral, inflammatory and apoptotic genes. Furthermore, our data demonstrate an inverse correlation between ZIKV-modulated miRNAs and target host mRNAs induced by ZIKV. Biofunctional analysis revealed that ZIKV-modulated miRNAs and mRNAs regulate the pathways related to neurological development and neuroinflammatory responses. Functional studies targeting specific miRNA are warranted to develop therapeutics for the management of ZIKV neurological disease. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Figure 1
Zika virus (ZIKV) infection of the primary mouse neurons. Mouse cortical neuron cultures were prepared from one-day old pups. Neurons were infected with ZIKV (PRVABC59 strain) or PBS (Mock) at multiplicity of infection (MOI)-1. (A) ZIKV titers in culture supernatant were determined by plaque assay. Viral titers are expressed as plaque forming units (PFU)/mL of supernatant. Data represents the mean ± SEM. Neurons grown and fixed on coverslips at 48 h after infection were stained with anti-dsRNA antibody (red) and counterstained with DAPI (blue). (B) Mock-infected cells. 20× magnification. (C) ZIKV-infected cells demonstrate robust virus staining in the cytoplasm. 20× magnification. Full article ">Figure 2
ZIKV infection of the primary mouse neurons causes changes in cellular miRNA expression. Neurons were infected with ZIKV (PRVABC59 strain) or PBS (Mock) at MOI-1. (A) Venn diagram showing the number of differentially expressed miRNAs at 24 and 48 h after infection. Sets of upregulated miRNAs are represented by upward red arrows and sets of downregulated miRNAs are represented by downward green arrows. Pairs of arrows in the intersection refer to the number of miRNAs upregulated (double red arrows) or down regulated (double green arrows) at both 24 and 48 h after infection. (B) qRT-PCR was conducted on RNA extracted from mock and ZIKV-infected neurons to determine fold-change in miR-155, miR-203, miR-29a, and miR-124-3p expression. Changes in the levels of each miRNA were first normalized to the snoRNA and then the fold-change in ZIKV-infected cells was calculated in comparison to corresponding mock-infected cells. Data represents the mean ± SEM. (C) qRT-PCR was conducted on RNA extracted from mock and ZIKV-infected neurons to determine fold-change in Dicer-1, Drosha, DGCR8, AGO1, and AGO2 expression. Changes in the levels of each mRNA were first normalized to the β-actin and then the fold-change in ZIKV-infected cells was calculated in comparison to corresponding mock-infected cells. Data represents the mean ± SEM. Full article ">Figure 3
ZIKV infection of the primary mouse neurons causes changes in cellular mRNA expression. Neurons were infected with ZIKV (PRVABC59 strain) or PBS (Mock) at MOI-1. (A) Venn diagram showing the number of differentially expressed mRNAs at 24 and 48 h after infection. Sets of upregulated mRNAs are represented by upward red arrows and sets of downregulated mRNAs are represented by downward green arrows. Pairs of arrows in the intersection refer to the number of mRNAs upregulated (double red arrows) or down regulated (double green arrows) at both 24 and 48 h after infection. (B) qRT-PCR was conducted on RNA extracted from mock and ZIKV-infected neurons to determine fold-change in IFIT1, IFIT3, IL6, and Caspase1 expression. Changes in the levels of each mRNA were first normalized to the β-actin and then the fold-change in ZIKV-infected cells was calculated in comparison to corresponding mock-infected cells. Data represents the mean ± SEM. Full article ">Figure 4
Enhanced production of cytokines and chemokines in ZIKV-infected neurons. Mouse cortical neuron cultures were infected with ZIKV (PRVABC59 strain) or PBS (Mock) at MOI-1 and supernatants were collected at 24 and 48 h after infection. Levels of chemokines and cytokines as noted in the figure were measured in cell supernatants using multiplex immunoassay and are expressed as the mean concentration (pg/mL) ± SEM. *p < 0.05, **p < 0.001. Full article ">Figure 5
Core functional pathway analysis of ZIKV-modulated mRNAs using IPA. Top canonical signaling pathways regulated by significantly modulated mRNAs. Threshold bar indicates cut-off point of significance p < 0.05, using Fisher’s exact test. Range of activation z-score is also depicted in the figure. The color of the bars indicates predicted pathway activation based on z-score (orange = activation; blue = inhibition; gray = no prediction can be made; white = z-score close to 0). Orange line represents the ratio = number of genes in dataset/total number of genes that compose that pathway. Full article ">Figure 6
Pathway analysis for PRR and IFN signaling. Genes associated with (A) PRR and (B) IFN signaling activated by ZIKV infection are shown. Differentially expressed mRNAs are highlighted in color. Color intensity indicates the degree of upregulation (red) relative to the mock-infected neurons. Solid lines represent direct interactions and dashed lines indirect interactions. Shading intensity indicates the degree that each mRNA was upregulated. Full article ">Figure 7
Networks of the interactions of the miRNA target genes. IPA tool was used to generate the miRNA-mRNA interaction network of (A) miR-124-3p, (B) miR-654-3p, (C) miR-331-5p, (D) miR-509-5p, and (E) miR-335-3p and mRNAs significantly modulated in neurons after ZIKV infection. Red (increased expression) and green (decreased expression). Full article ">

21 pages, 3474 KiB

Open AccessArticle

The Roles of prM-E Proteins in Historical and Epidemic Zika Virus-mediated Infection and Neurocytotoxicity

by Ge Li, Sandra Bos, Konstantin A. Tsetsarkin, Alexander G. Pletnev, Philippe Desprès, Gilles Gadea and Richard Y. Zhao

Viruses 2019, 11(2), 157; https://doi.org/10.3390/v11020157 - 14 Feb 2019

Cited by 28 | Viewed by 5695

Abstract

The Zika virus (ZIKV) was first isolated in Africa in 1947. It was shown to be a mild virus that had limited threat to humans. However, the resurgence of the ZIKV in the most recent Brazil outbreak surprised us because it causes severe [...] Read more.

The Zika virus (ZIKV) was first isolated in Africa in 1947. It was shown to be a mild virus that had limited threat to humans. However, the resurgence of the ZIKV in the most recent Brazil outbreak surprised us because it causes severe human congenital and neurologic disorders including microcephaly in newborns and Guillain-Barré syndrome in adults. Studies showed that the epidemic ZIKV strains are phenotypically different from the historic strains, suggesting that the epidemic ZIKV has acquired mutations associated with the altered viral pathogenicity. However, what genetic changes are responsible for the changed viral pathogenicity remains largely unknown. One of our early studies suggested that the ZIKV structural proteins contribute in part to the observed virologic differences. The objectives of this study were to compare the historic African MR766 ZIKV strain with two epidemic Brazilian strains (BR15 and ICD) for their abilities to initiate viral infection and to confer neurocytopathic effects in the human brain’s SNB-19 glial cells, and further to determine which part of the ZIKV structural proteins are responsible for the observed differences. Our results show that the historic African (MR766) and epidemic Brazilian (BR15 and ICD) ZIKV strains are different in viral attachment to host neuronal cells, viral permissiveness and replication, as well as in the induction of cytopathic effects. The analysis of chimeric viruses, generated between the MR766 and BR15 molecular clones, suggests that the ZIKV E protein correlates with the viral attachment, and the C-prM region contributes to the permissiveness and ZIKV-induced cytopathic effects. The expression of adenoviruses, expressing prM and its processed protein products, shows that the prM protein and its cleaved Pr product, but not the mature M protein, induces apoptotic cell death in the SNB-19 cells. We found that the Pr region, which resides on the N-terminal side of prM protein, is responsible for prM-induced apoptotic cell death. Mutational analysis further identified four amino-acid residues that have an impact on the ability of prM to induce apoptosis. Together, the results of this study show that the difference of ZIKV-mediated viral pathogenicity, between the historic and epidemic strains, contributed in part the functions of the structural prM-E proteins. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Different infectivity between the epidemic Brazilian Zika virus (BR15 and ICD) molecular clones and the historical African MR766 molecular clone in human brain glial SNB-19 cells. (A) Viral binding to SNB-19 cells was measured by presence of cell-associated vRNA one-hour post-infection (p.i.). A housekeeping gene GAPDH was used as an endogenous control for the measurement of viral bindings. (B) Zika virus (ZIKV) replication was measured by quantitative RT-PCR with timeframe as indicated. SNB-19 cells were infected by Zika viruses with multiplicity of infection (MOI) 1.0. Results represent average and standard deviation (X ± SD) of four independent experiments. (C) Viral infectivity measured by anti-E mAb 4G2 at 48 h p.i. (D) Viral infectivity is shown as an average of three different experiments, each carried out in triplicates. Average cell number counted was about 100–200. Results represent average and standard deviation (X ± SD). Levels of statistical significance were calculated by two-tailed and paired t-test for (A), and Two-way ANOVA was used for (B). **, p < 0.05; ***, p < 0.01. Full article ">Figure 2
Different neuro-cytopathic effects of epidemic and historical molecular clones of Zika viruses on human brain glial SNB-19 cells. (A) Cellular survival was measured by the MTT assay. The graph is plotted as the relative growth in relevance to mock infected SNB19 cells. Statistic t-test shows that the differences among three viruses are not statistically significant. (B) ZIKV-induced cell death as measured by the Trypan blue assay. **, p < 0.05 for BR15 and ***, p < 0.01 for ICD. Three different experiments were carried out. Average cell number counted was about 100–200. (C) ZIKV-induced apoptosis was measured by caspase-3 cleavages using immunostaining as reported previously [<a href="#B32-viruses-11-00157" class="html-bibr">32</a>]. Cells were collected at 72 h p.i. Two experiments were conducted and cells showing caspase-3 cleavages were counted at 10 different areas with an average number of cells counted at 25–75. All results represent average and standard deviation (X+SD). Levels of statistical significance were calculated by Two-way ANOVA for (A). The difference between MR766 and BR15 is highly significant with p < 0.01 (***). Full article ">Figure 3
Correlation of the ZIKV C-prM with viral attachment and viral infection. (A) Generation of chimeric ZIKV molecular clones are shown along with their parental clones. The chimeric viruses were made between the MR766 and the BR15 ZIKV molecular clones. The viral genome exchange is at the junction of prM and E protein. (B) Viral binding was measured by presence of cell-associated vRNA 1 h p.i. A housekeeping gene GAPDH was used as an endogenous control for the measurement of viral bindings. Results represent average and standard deviation (X ± SD) of four independent experiments. (C) ZIKV viral replication was measured by RT-qPCR with timeframe as indicated. (D) Viral infection was measured by anti-E mAb 4G2 at 48 h p.i. (E) Quantification of the results shown in (D). SNB-19 cells were infected with Zika viruses with MOI of 1.0. Three different experiments were carried out in triplicates. Average cell number counted was about 100–200. All quantitative results represent average and standard deviation (X ± SD). Levels of statistical significance were calculated by two-tailed and paired t-test for (B), and Two-way ANOVA was used for (C). **, p < 0.05; ***, p < 0.01. Full article ">Figure 4
Correlation of the C-prM with ZIKV-induced growth restriction and apoptotic cell death. (A) Cell proliferation was measured by the MTT assay. Statistic t-test shows the differences shown among three viruses are not significant. (B) ZIKV-induced cell death was measured by the Trypan blue assay 72 h p.i. Two different experiments were carried out. Average number of cells counted was about 100-200. Differences between MR766 vs. BR15, and MR766 vs. B/M were both high significant with p ≤ 0.05 (**) for both comparisons. The difference between MR766 and M/B was not significant with p = 0.11. (C) ZIKV-induced apoptosis was measured by cleavage of caspase-3. Cells were collected at 72 h p.i. All results represent average and standard deviation (X ± SD). Levels of statistical significance were calculated by Two-way ANOVA for (A). The difference between MR766 and BR15 is highly significant with p < 0.01 (***). Full article ">Figure 5
Effect of prM protein processing on ZIKV-induced neurocytotoxicity. (A) Cell proliferation and viability was measured overtime by the MTT assay. The graph is plotted as the relative growth in relevance to mock infected SNB-19 cells. The differences shown were highly significant with p < 0.05 (**). (B) Measurement of apoptosis by Annexin V over time. The difference between Adv-prM and Adv-M was highly significant with p < 0.01 (***), but the difference between Adv-prM and Adv-Pr was not significant with p = 0.84. (C) Measurement of cellular necrosis over time. Two-way ANOVA was used to calculate the difference between Adv-prM and Adv-M for (B) and (C). The differences between Adv-prM and Adv-M were highly significant with p < 0.01 (***). More than two different experiments were conducted to evaluate apoptosis and necrosis. All results represent average and standard deviation (X ± SD). Full article ">Figure 6
Mutational analysis of the Pr region of the prM protein. (A) Mutagenesis of the Pr protein. Four amino acids mutations (A148P, V153M, H157Y and V158I) were generated as shown by “*” on the top of the prM sequence alignments between MR766 (top) and BR15 (bottom). The resulting mutant adenoviral construct is labeled as Adv-Pr*MR766. The site of the reverse N139S mutation is shown by “†” on the top of the prM sequence. The corresponding mutant adenoviral construct is labeled as Adv-Pr†BR15. (B) Cell proliferation and viability by the MTT assay. Only Adv-prM showed significant differences overtime with p < 0.05 (**). Adv-Pr-induced cell death was measured by (C) apoptosis, and (D) necrosis over time. The underlined RSRR sequence indicates the putative furin cleavage site on the prM protein, based on its consensus target site Arg-X-Lys/Arg-Arg↓, where the arrow indicates the location of furin cleavage site. Two-way ANOVA was used to calculate the differences between Adv-prM and Adv-M for (C) and (D). The differences between Adv-Pr MR766 and Adv-Pr* MR766 were highly significant with p < 0.01 (***). However, the difference between Adv-Pr BR15 and Adv-Pr† BR15 was not significant with p > 0.99 for both apoptosis and necrosis. All quantitative results represent average and standard deviation (X ± SD). Full article ">

11 pages, 4383 KiB

Open AccessCommunication

Differential Zika Virus Infection of Testicular Cell Lines

by Luwanika Mlera and Marshall E. Bloom

Viruses 2019, 11(1), 42; https://doi.org/10.3390/v11010042 - 9 Jan 2019

Cited by 21 | Viewed by 5206

Abstract

Background: Zika virus is a mosquito-borne flavivirus responsible for recent outbreaks of epidemic proportions in Latin America. Sexual transmission of the virus has been reported in 13 countries and may be an important route of infection. Sexual transmission of ZIKV has mostly been [...] Read more.

Background: Zika virus is a mosquito-borne flavivirus responsible for recent outbreaks of epidemic proportions in Latin America. Sexual transmission of the virus has been reported in 13 countries and may be an important route of infection. Sexual transmission of ZIKV has mostly been male-to-female, and persistence of viral RNA in semen for up to 370 days has been recorded. The susceptibility to ZIKV of different testicular cell types merits investigation. Methods: We infected primary Sertoli cells, a primary testicular fibroblast Hs1.Tes, and 2 seminoma cell lines SEM-1 and TCam-2 cells with ZIKV Paraiba and the prototype ZIKV MR766 to evaluate their susceptibility and to look for viral persistence. A human neuroblastoma cell line SK-N-SH served as a control cell type. Results: Both virus strains were able to replicate in all cell lines tested, but ZIKV MR766 attained higher titers. Initiation of viral persistence by ZIKV Paraiba was observed in Sertoli, Hs1.Tes, SEM-1 and TCam-2 cells, but was of limited duration due to delayed cell death. ZIKV MR766 persisted only in Hs1.Tes and Sertoli cells, and persistence was also limited. In contrast, SK-N-SH cells were killed by both ZIKV MR766 and ZIKV Paraiba and persistence could not be established in these cells. Conclusions: ZIKV prototype strain MR766 and the clinically relevant Paraiba strain replicated in several testicular cell types. Persistence of ZIKV MR766 was only observed in Hs1.Tes and Sertoli cells, but the persistence did not last more than 3 or 4 passages, respectively. ZIKV Paraiba persisted in TCam-2, Hs1.Tes, Sertoli and SEM-1 cells for up to 5 passages, depending on cell type. TCam-2 cells appeared to clear persistent infection by ZIKV Paraiba. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Figure 1
Replication kinetics of ZIKV MR766 and Paraiba over the course of 7 days. Error bars represent standard deviation (SD) from the mean for 3 independent replicates. Full article ">Figure 2
Microscopic evaluation of testicular cell lines infected with ZIKV Paraiba. No obvious cytopathic effect was observed in all testicular cell lines infected with ZIKV Paraiba. Images were captured at a magnification of 400×. Full article ">Figure 3
Cytopathic effect of ZIKV MR766 on testicular cell lines at 7 dpi. We noted that ZIKV MR766-infected Sertoli and SEM-1 cells appeared smaller than uninfected controls. TCam-2 and Hs1.Tes cells did show any CPE at 7 dpi. Images were obtained at a magnification of 400×. Full article ">Figure 4
ZIKV titers in persistently infected testicular cell lines. The clinical isolate ZIKV Paraiba was able to persist in all cell lines tested for up to 5 passages (P1 through to P5), depending on cell line. ZIKV MR766 was only able to persist in Sertoli and Hs1.Tes cells. Virus titration was performed using supernatants collected at the end of each 7-day period and each data point represents an average of 3 biological replicates. Error bars represent standard deviation from the mean. Each passage was done after 7 days by washing the monolayer twice with PBS, trypsinizing the cells and seeding into new flasks with fresh culture medium at 1:10. Full article ">Figure 5
Analysis of continued cell death in ZIKV-infected testicular cells. (a) Cell count graph showing that there were 2-times more cells in the uninfected Hs1.Tes control, compared to ZIKV Paraiba-infected Hs1.Tes cells at P1. For both control and ZIKV-infected cells, the count was done after 7 days of cell passage. ****, p < 0.0001 (unpaired t-test). (b) Microscopic images showing loss of the Hs1.Tes monolayer in ZIKV-infected cells at P3. The morphology of infected cells at this time point appeared grossly aberrant and pleomorphic when compared to that of uninfected control cells. Cells were imaged at a magnification of 400×. (c) Confocal microscopy images showing cleaved caspase 3 in some ZIKV-infected Sertoli cells at P1. Not all ZIKV E protein-expressing cells stained positive for cleaved caspase 3, supporting the notion that cell death in persistently infected cells was progressive. Cells were imaged at a magnification of 400×. Full article ">Figure 6
ZIKV infection in a human neuroblastoma SK-N-SH cell line. (a) Replication kinetics of ZIKV MR766 and Paraiba strains in SK-N-SH cells. Error bars represent SD from the mean for 3 independent experiments. (b) Cell death in SK-N-SH cell monolayers infected with ZIKV Paraiba or ZIKV MR766. ZIKV MR766 was more aggressive at killing SK-N-SH cells. Images of the SK-N-SH monolayer were acquired at a magnification of 400×. Full article ">

17 pages, 11363 KiB

Open AccessArticle

The Oxysterol 7-Ketocholesterol Reduces Zika Virus Titers in Vero Cells and Human Neurons

by Katherine A. Willard, Christina L. Elling, Steven L. Stice and Melinda A. Brindley

Viruses 2019, 11(1), 20; https://doi.org/10.3390/v11010020 - 30 Dec 2018

Cited by 21 | Viewed by 4881

Abstract

Zika virus (ZIKV) is an emerging flavivirus responsible for a major epidemic in the Americas beginning in 2015. ZIKV associated with maternal infection can lead to neurological disorders in newborns, including microcephaly. Although there is an abundance of research examining the neurotropism of [...] Read more.

Zika virus (ZIKV) is an emerging flavivirus responsible for a major epidemic in the Americas beginning in 2015. ZIKV associated with maternal infection can lead to neurological disorders in newborns, including microcephaly. Although there is an abundance of research examining the neurotropism of ZIKV, we still do not completely understand the mechanism by which ZIKV targets neural cells or how to limit neural cell infection. Recent research suggests that flaviviruses, including ZIKV, may hijack the cellular autophagy pathway to benefit their replication. Therefore, we hypothesized that ZIKV replication would be impacted when infected cells were treated with compounds that target the autophagy pathway. We screened a library of 94 compounds known to affect autophagy in both mammalian and insect cell lines. A subset of compounds that inhibited ZIKV replication without affecting cellular viability were tested for their ability to limit ZIKV replication in human neurons. From this second screen, we identified one compound, 7-ketocholesterol (7-KC), which inhibited ZIKV replication in neurons without significantly affecting neuron viability. Interestingly, 7-KC induces autophagy, which would be hypothesized to increase ZIKV replication, yet it decreased virus production. Time-of-addition experiments suggest 7-KC inhibits ZIKV replication late in the replication cycle. While 7-KC did not inhibit RNA replication, it decreased the number of particles in the supernatant and the relative infectivity of the released particles, suggesting it interferes with particle budding, release from the host cell, and particle integrity. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Figure 1

Figure 1
Autophagy compound screen in Vero cells. (A) ZIKV SPH growth in compound-treated Vero cells as a percent of ZIKV titer in untreated Vero cells. Red symbols represent compounds that increased ZIKV titer compared to the untreated control, yellow symbols represent compounds that reduced titer between 10 and 99% of control, and green symbols represent compounds that reduced ZIKV titer below 10% of control (at least 1 log of viral growth). Error bars represent the standard error of the mean (SEM) from three independent trials. The compounds are numbered according to <a href="#viruses-11-00020-t001" class="html-table">Table 1</a>. (B) ZIKV growth in Vero cells versus viability of compound-treated Vero cells. The shaded box represents the zone in which ZIKV titer is reduced to 10% or lower than the untreated control and cell viability is at least 80% of control. Compounds that meet these criteria are in green and labeled with their names. Full article ">Figure 1 Cont.
Autophagy compound screen in Vero cells. (A) ZIKV SPH growth in compound-treated Vero cells as a percent of ZIKV titer in untreated Vero cells. Red symbols represent compounds that increased ZIKV titer compared to the untreated control, yellow symbols represent compounds that reduced titer between 10 and 99% of control, and green symbols represent compounds that reduced ZIKV titer below 10% of control (at least 1 log of viral growth). Error bars represent the standard error of the mean (SEM) from three independent trials. The compounds are numbered according to <a href="#viruses-11-00020-t001" class="html-table">Table 1</a>. (B) ZIKV growth in Vero cells versus viability of compound-treated Vero cells. The shaded box represents the zone in which ZIKV titer is reduced to 10% or lower than the untreated control and cell viability is at least 80% of control. Compounds that meet these criteria are in green and labeled with their names. Full article ">Figure 2
African- lineage ZIKV IbH growth in compound-treated Vero cells as a percent of ZIKV titer in untreated Vero cells (A). The color-codes are the same as in <a href="#viruses-11-00020-f001" class="html-fig">Figure 1</a>A. (B) ZIKV IbH growth in Vero cells versus viability of compound-treated Vero cells. The shaded box represents the zone in which ZIKV titer is reduced to 10% or lower than the untreated control and cell viability is at least 80% of control. Full article ">Figure 3
Autophagy compound screen in C6/36 cells. (A) ZIKV growth in compound-treated C6/36 cells as a percent of ZIKV titer in untreated C6/36 cells. The labels and color-codes are the same as in <a href="#viruses-11-00020-f001" class="html-fig">Figure 1</a>A. (B) ZIKV growth in C6/36 cells versus viability of compound-treated C6/36 cells. As in <a href="#viruses-11-00020-f001" class="html-fig">Figure 1</a>, the shaded box represents the zone in which ZIKV titer is reduced to 10% or lower than the untreated control and cell viability is at least 80% of control. No compounds fit these criteria in C6/36 cells. Full article ">Figure 4
Autophagy compound screen in human neurons. (A) Impact of autophagy compounds on ZIKV replication in neurons and (B) neuron viability. (C) Chemical structure of cholesterol (PubChem CID 5997) versus 7-ketocholesterol (PubChem CID 91474). Carbon atoms are shaded in purple, hydrogen atoms are shaded in grey, and oxygen atoms are shaded in red. Full article ">Figure 5
Dose-response curve and viability of Vero cells treated with varying doses (0–27 µM) of 7-ketocholesterol. The curves were generated by GraphPad Prism version 7.04 using the log (inhibitor) vs. normalized response variable slope regression. The regression curve estimates the IC50 at 4.064 μM (R2 = 0.9078). The cell viability did not drop below 80% preventing us from determining the EC50. Full article ">Figure 6
Analysis of 7-ketocholesterol inhibition of ZIKV SPH replication. (A) ZIKV SPH preincubation with 200 µM of 7-KC before Vero cell infection. (B) 7-KC versus ammonium chloride time-of-addition assay in Vero cells. Error bars represent the SEM. *, p < 0.05; **, p < 0.01. Full article ">Figure 7
7-KC reduced ZIKV budding efficiency and infectious virion production. (A) ZIKV infection was inhibited by adding 7-KC at the indicated time points following infection. Twenty-four hours post infection (hpi), total cellular RNA was harvested and ZIKV genomes were quantified using q-RT-PCR. 7-KC did not significantly alter the level of ZIKV genomes in the cells. (B) ZIKV genome copies present in the supernatants were also quantified. To determine the budding/release efficiency we compared the number of genomes present in the supernatant to cell associated genomes and determined the budding efficiency based on DMSO control. (C) ZIKV produced in the presence of 7-KC is less infectious than virus grown in DMSO control cells. The specific infectivity of the virions was determined by comparing the titer to the number of genome copies found in the supernatant (×1000). Each RNA sample was run in triplicate during each trial for a total of 3 trials. *, p < 0.05; **, p < 0.01. Full article ">

13 pages, 1092 KiB

Open AccessArticle

Subversion of the Heme Oxygenase-1 Antiviral Activity by Zika Virus

by Chaker El Kalamouni, Etienne Frumence, Sandra Bos, Jonathan Turpin, Brice Nativel, Wissal Harrabi, David A. Wilkinson, Olivier Meilhac, Gilles Gadea, Philippe Desprès, Pascale Krejbich-Trotot and Wildriss Viranaïcken

Viruses 2019, 11(1), 2; https://doi.org/10.3390/v11010002 - 20 Dec 2018

Cited by 39 | Viewed by 5463

Abstract

Heme oxygenase-1 (HO-1), a rate-limiting enzyme involved in the degradation of heme, is induced in response to a wide range of stress conditions. HO-1 exerts antiviral activity against a broad range of viruses, including the Hepatitis C virus, the human immunodeficiency virus, and [...] Read more.

Heme oxygenase-1 (HO-1), a rate-limiting enzyme involved in the degradation of heme, is induced in response to a wide range of stress conditions. HO-1 exerts antiviral activity against a broad range of viruses, including the Hepatitis C virus, the human immunodeficiency virus, and the dengue virus by inhibiting viral growth. It has been reported that HO-1 displays antiviral activity against the Zika virus (ZIKV) but the mechanisms of viral inhibition remain largely unknown. Using a ZIKV RNA replicon with the Green Fluorescent Protein (GFP) as a reporter protein, we were able to show that HO-1 expression resulted in the inhibition of viral RNA replication. Conversely, we observed a decrease in HO-1 expression in cells replicating the ZIKV RNA replicon. The study of human cells infected with ZIKV showed that the HO-1 expression level was significantly lower once viral replication was established, thereby limiting the antiviral effect of HO-1. Our work highlights the capacity of ZIKV to thwart the anti-replicative activity of HO-1 in human cells. Therefore, the modulation of HO-1 as a novel therapeutic strategy against ZIKV infection may display limited effect. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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17 pages, 2031 KiB

Open AccessArticle

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

by Trisha R. Barnard, Maaran M. Rajah and Selena M. Sagan

Viruses 2018, 10(12), 728; https://doi.org/10.3390/v10120728 - 19 Dec 2018

Cited by 17 | Viewed by 5169

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. [...] Read more.

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. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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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 ZIKVAF: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">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. Full article ">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. Full article ">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. Full article ">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. Full article ">

22 pages, 4515 KiB

Open AccessArticle

Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay

by Sanchita Bhadra, Miguel A. Saldaña, Hannah Grace Han, Grant L. Hughes and Andrew D. Ellington

Viruses 2018, 10(12), 714; https://doi.org/10.3390/v10120714 - 14 Dec 2018

Cited by 9 | Viewed by 5943

Abstract

We have developed a generalizable “smart molecular diagnostic” capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth [...] Read more.

We have developed a generalizable “smart molecular diagnostic” capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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16 pages, 3809 KiB

Open AccessArticle

Tet-Inducible Production of Infectious Zika Virus from the Full-Length cDNA Clones of African- and Asian-Lineage Strains

by Lizhou Zhang, Wei Ji, Shuang Lyu, Luhua Qiao and Guangxiang Luo

Viruses 2018, 10(12), 700; https://doi.org/10.3390/v10120700 - 9 Dec 2018

Cited by 5 | Viewed by 4147

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as an important human viral pathogen, causing congenital malformation including microcephaly among infants born to mothers infected with the virus during pregnancy. Phylogenetic analysis suggested that ZIKV can be classified into African and [...] Read more.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as an important human viral pathogen, causing congenital malformation including microcephaly among infants born to mothers infected with the virus during pregnancy. Phylogenetic analysis suggested that ZIKV can be classified into African and Asian lineages. In this study, we have developed a stable plasmid-based reverse genetic system for robust production of both ZIKV prototype African-lineage MR766 and clinical Asian-lineage FSS13025 strains using a tetracycline (Tet)-controlled gene expression vector. Transcription of the full-length ZIKV RNA is under the control of the Tet-responsive P_tight promoter at the 5′ end and an antigenomic ribozyme of hepatitis delta virus at the 3′ end. The transcription of infectious ZIKV RNA genome was efficiently induced by doxycycline. This novel ZIKV reverse genetics system will be valuable for the study of molecular viral pathogenesis of ZIKV and the development of new vaccines against ZIKV infection. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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11 pages, 3246 KiB

Open AccessArticle

An Evolutionary Insight into Zika Virus Strains Isolated in the Latin American Region

by Diego Simón, Alvaro Fajardo, Pilar Moreno, Gonzalo Moratorio and Juan Cristina

Viruses 2018, 10(12), 698; https://doi.org/10.3390/v10120698 - 8 Dec 2018

Cited by 9 | Viewed by 3716

Abstract

Zika virus (ZIKV) is an emerging pathogen member of the Flaviviridae family. ZIKV has spread rapidly in the Latin American region, causing hundreds of thousands of cases of ZIKV disease, as well as microcephaly in congenital infections. Detailed studies on the pattern of [...] Read more.

Zika virus (ZIKV) is an emerging pathogen member of the Flaviviridae family. ZIKV has spread rapidly in the Latin American region, causing hundreds of thousands of cases of ZIKV disease, as well as microcephaly in congenital infections. Detailed studies on the pattern of evolution of ZIKV strains have been extremely important to our understanding of viral survival, fitness, and evasion of the host’s immune system. For these reasons, we performed a comprehensive phylogenetic analysis of ZIKV strains recently isolated in the Americas. The results of these studies revealed evidence of diversification of ZIKV strains circulating in the Latin American region into at least five different genetic clusters. This diversification was also reflected in the different trends in dinucleotide bias and codon usage variation. Amino acid substitutions were found in E and prM proteins of the ZIKV strains isolated in this region, revealing the presence of novel genetic variants circulating in Latin America. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Figure 1
Bayesian maximum clade credibility tree representing the time-scale of ZIKV, obtained by the analysis of 61 complete coding sequences using the Tamura-Ney (TN93) + Γ model, the Bayesian Skyline model, and a relaxed exponential clock. The tree is rooted to the Most Recent Common Ancestor (MRCA) of strains included. The scale at the bottom is in units of evolutionary time and represents the years before the last sampling date. Strains in the tree are shown by their accession number, geographical location, and year of isolation expressed in decimal format. Clades are indicated in blue, red, green, violet, and black. Full article ">Figure 2
Positions of the ZIKV strains for the first two major axes of principal component analysis of the (a) dinucleotide observed/expected ratios, (b) relative synonymous codon usage, and (c) amino acid frequencies. The proportion of variance explained by each axis is displayed, placed between parentheses. Strains in the plot are colored according to their clade assignment depicted in <a href="#viruses-10-00698-f001" class="html-fig">Figure 1</a> (i.e., in blue, red, green, violet, and black). Full article ">Figure 3
An amino acid sequence alignment of the DIII domain of ZIKV E proteins. Strains are shown by accession number, geographic location, and year of isolation. Identity of the strain H/PF/2013 from the Asian genotype (accession number KJ776791) is shown by a dash. Sequence position relative to the E protein of that strain is shown on the top of the figure. Predicted coiled regions of the protein are indicated by a blue arrow on top of the alignment. Predicted exposed residues are indicated by an asterisk on the upper part of the alignment. Previously described conformational epitopes ABDE, C-C’, and LR [<a href="#B30-viruses-10-00698" class="html-bibr">30</a>] are shown in green, blue, and magenta. Full article ">Figure 4
An amino acid sequence alignment of ZIKV prM proteins. Strains are shown by accession number, geographic location, and year of isolation. Identity of the strain H/PF/2013 from the Asian genotype (accession number KJ776791) is shown by a dash. Sequence position relative to the prM protein of that strain is shown on the top of the figure. Predicted coiled regions of the protein are indicated by a blue arrow on top of the alignment. Predicted exposed residues are indicated by an asterisk on the upper part of the alignment. Positions where amino acid substitutions were found between pre-epidemic and epidemic strains are highlighted in green. Position 139 is shown in red. Full article ">

16 pages, 2933 KiB

Open AccessArticle

Characterizing the Different Effects of Zika Virus Infection in Placenta and Microglia Cells

by Maria del Pilar Martinez Viedma and Brett E. Pickett

Viruses 2018, 10(11), 649; https://doi.org/10.3390/v10110649 - 18 Nov 2018

Cited by 20 | Viewed by 5431

Abstract

Zika virus (ZIKV) is a neuropathic virus that causes serious neurological abnormalities such as Guillain-Barre syndrome in adults and congenital Zika syndrome (CZS) in fetuses, which makes it an important concern for global human health. A catalogue of cells that support ZIKV replication, [...] Read more.

Zika virus (ZIKV) is a neuropathic virus that causes serious neurological abnormalities such as Guillain-Barre syndrome in adults and congenital Zika syndrome (CZS) in fetuses, which makes it an important concern for global human health. A catalogue of cells that support ZIKV replication, pathogenesis, and/or the persistence of the virus still remains unknown. Here, we studied the behavior of the virus in human placenta (JEG-3) and human microglia (HMC3) cell lines in order to better understand how different host tissues respond during infection. We quantified the host transcriptional response to ZIKV infection in both types of cells at 24 and 72 h post-infection. A panel of 84 genes that are involved in the innate or adaptive immune responses was used to quantify differential expression in both cell lines. HMC3 cells showed a unique set of significant differentially expressed genes (DEGs) compared with JEG-3 cells at both time points. Subsequent analysis of these data using modern pathway analysis methods revealed that the TLR7/8 pathway was strongly inhibited in HMC3 cells, while it was activated in JEG-3 cells during virus infection. The disruption of these pathways was subsequently confirmed with specific small interfering RNA (siRNA) experiments that characterize their role in the viral life cycle, and may partially explain why ZIKV infection in placental tissue contributes to extreme neurological problems in a developing fetus. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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12 pages, 2243 KiB

Open AccessArticle

Development and Characterization of Double-Antibody Sandwich ELISA for Detection of Zika Virus Infection

by Liding Zhang, Xuewei Du, Congjie Chen, Zhixin Chen, Li Zhang, Qinqin Han, Xueshan Xia, Yuzhu Song and Jinyang Zhang

Viruses 2018, 10(11), 634; https://doi.org/10.3390/v10110634 - 15 Nov 2018

Cited by 29 | Viewed by 6185

Abstract

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defect and Guillain−Barré syndrome during pregnancy. Although, several molecular diagnostic methods have been developed to detect the ZIKV, these methods pose challenges as they cannot detect early [...] Read more.

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defect and Guillain−Barré syndrome during pregnancy. Although, several molecular diagnostic methods have been developed to detect the ZIKV, these methods pose challenges as they cannot detect early viral infection. Furthermore, these methods require the extraction of RNA, which is easy to contaminate. Nonstructural protein 1 (NS1) is an important biomarker for early diagnosis of the virus, and the detection methods associated with the NS1 protein have recently been reported. The aim of this study was to develop a rapid and sensitive detection method for the detection of the ZIKV based on the NS1 protein. The sensitivity of this method is 120 ng mL⁻¹ and it detected the ZIKV in the supernatant and lysates of Vero and BHK cells, as well as the sera of tree shrews infected with the ZIKV. Without the isolation of the virus and the extraction of the RNA, our method can be used as a primary screening test as opposed to other diagnosis methods that detect the ZIKV. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Analysis of the recombinant vector construction and expression of recombinant ZIKV-NS1 protein. (A) Identification of the positive clone of pET-32a-NS1 by bacteria polymerase chain reaction, lane 1: positive clone; lane 2: blank control. (B) Lane 1: E. coli was induced with IPTG; lane 2: E. coli before induced; lane 3: the purified ZIKV-NS1 after dialysis. Full article ">Figure 2
Evaluation of the pAb R1 and mAbs against ZIKV-NS1 protein. (A) Indirect immunofluorescent assay of the mAbs and pAb R1, , Scale bar = 100 μm. (B) ELISA test of the mAbs and pAb R1. (C) Western blot analysis of the pAb R1; lane 1: E. coli Rosetta (DE3) without IPTG; lane 2: E. coli Rosetta (DE3) induced with IPTG; lane 3: the purified ZIKV-NS1. (D) ZIKV infected (lane 1) and mock-infected Vero cells (lane 2) were used for Western blot analysis using pAb R1. (E) Mock-infected (lane 1) and ZIKV infected Vero cells (lane 2) were used for Western blot analysis using mAb 1F12. Full article ">Figure 3
Characteristics of the key components of the DAS-ELISA. (A) The reactivity of nine mAbs conjugated with HRP, 5% skimmed milk as the blank control. (B) Nine different groups for DAS-ELISA, the blank control was 5% skimmed milk. The titer of pAb R1 (C) and mAb 1F12 (D) at different dilution ratios were determined by ELISA, 5% skimmed milk as the blank control. (E) The saturation curves for determination of the dissociation constants of mAb 1F12 and pAb R1. (F) SDS-PAGE analysis of purified mAb 1F12 (lane 1) and pAb R1 (lane 2). Full article ">Figure 4
Optimization operations of DAS-ELISA. Optimum coating solution (A), coating temperatures and times (B) of pAb R1. Optimum working concentration of pAb R1 (C) and HRP-labeled mAb 1F12 (D). (E) Optimum incubating temperatures and times of antigen. (F) Optimum incubating times and temperatures of mAb 1F12-HRP probe. Full article ">Figure 5
Specificity and sensitivity of the DAS-ELISA. (A) Specificity of the DAS-ELISA: well 1 represents the recombinant ZIKV-NS1 protein; well 2 represents the natural NS1 protein in the cell culture supernatant that infected with ZIKV; well 3−6 represent the natural NS1 protein in the cell culture supernatant infected with DEN-1, DEN-2, DEN-3, and JEV, respectively; and well 7 represents the blank control. (B) Optical density for the detection of different viruses. (C) Sensitivity of the DAS-ELISA: well 1−13 represent the different concentrations of recombinant ZIKV-NS1 proteins (500 µg mL−1–0.122 µg mL−1), well 14 represents the blank control. (D) Optical density for the detection of different concentrations of recombinant ZIKV-NS1 protein (500 µg mL−1–0.122 µg mL−1). (E) The plotted linear curve based on the different concentrations of recombinant ZIKV-NS1 protein. Full article ">Figure 6
Detection of the ZIKV-NS1 protein in cell culture supernatants and lyates, as well as sera of animal infection model by the DAS-ELISA. Supernatants (A,B) and cell lysates (C,D) of Vero and BHK cells infected with ZIKV from 12, 24, 36, 48, 60, and 72 h were detected by DAS-ELISA. (E,F) Sera of tree shrews infected with ZIKV from 1 to 12 days were detected by DAS-ELISA. A Real-time quantitative reverse transcription polymerase chain reaction analysis of the supernatant of Vero, BHK (G) and the sera of tree shrews (H) after infected with ZIKV. (I) Western blot assay of supernatants and cell lysates of BHK and Vero cells infected with ZIKV. Full article ">

16 pages, 3397 KiB

Open AccessArticle

Persistence and Intra-Host Genetic Evolution of Zika Virus Infection in Symptomatic Adults: A Special View in the Male Reproductive System

by Danielle B. L. Oliveira, Giuliana S. Durigon, Érica A. Mendes, Jason T. Ladner, Robert Andreata-Santos, Danielle B. Araujo, Viviane F. Botosso, Nicholas D. Paola, Daniel F. L. Neto, Marielton P. Cunha, Carla T. Braconi, Rúbens P. S. Alves, Monica R. Jesus, Lennon R. Pereira, Stella R. Melo, Flávio S. Mesquita, Vanessa B. Silveira, Luciano M. Thomazelli, Silvana R. Favoretto, Franciane B. Almonfrey, Regina C. R. M. Abdulkader, Joel M. Gabrili, Denise V. Tambourgi, Sérgio F. Oliveira, Karla Prieto, Michael R. Wiley, Luís C. S. Ferreira, Marcos V. Silva, Gustavo F. Palacios, Paolo M. A. Zanotto and Edison L. Durigon Show full author list Hide full author list

Viruses 2018, 10(11), 615; https://doi.org/10.3390/v10110615 - 7 Nov 2018

Cited by 32 | Viewed by 5967

Abstract

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, [...] Read more.

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Figure 1
Timelines of ZIKV exposure, symptoms and sample collection in the four subjects enrolled in the present study. Periods of exposure, symptom onset, serology and molecular detection results of ZIKV in the followed subjects are described as: (A) case 1—female (ZIKV01), (B) case 2—male (ZIKV17), (C) case 3—female (ZIKV18) and case 4—male (ZIKV19). Day 0 denotes the onset of symptoms. Full article ">Figure 2
ZIKV RNA load in patient’s body fluids and clinical isolates on culture. The graph shows the viral load (genome copies/mL) versus excretion time (days after symptoms onset) of the weekly collection of urine (orange), saliva (green) and serum (red) samples from patients ZIKV01 (A), ZIKV17 (B), ZIKV18 (C) and ZIKV19 (D), in addition to the semen (blue) of the two men involved in the study (C,D). To confirm the viability of the excreted virus, the urine samples collected from days 18 and 25 after symptoms, saliva from day 25 and the semen from days 18, 25, 32, 53 and 117 for patient ZIKV17 and semen samples from patient ZIKV19 from days 19, 26 and 40 after symptoms onset were tested and the ones with positive results in cell culture are exhibited. All clinical samples and isolated samples were analyzed by qRT-PCR. Full article ">Figure 3
Detection of ZIKV in semen and C636 cells. (A) Detection by Indirect Immunofluorescence assay using anti-ZIKV specific antibody. Spermatozoa from semen sample collected from patient ZIKV17 at day 39 stained with FITC conjugate (in green) for virus location and with DAPI for nucleus staining (in blue). The viruses were located in the cytoplasm and flagella. C6/36 cell culture infected with virus from ZIKV17 semen sample. The cell infected presents with a green color (lower right panel). (B) Electron Microscopy of ultrathin sections of semen sample. (B1) A lower-power view of ZIKV particles inside an infected cell, with the characteristic of an immature sperm cell. (B2) Viral particles in a magnified view of the same cell in (B1). (B3) C6/36 cell infected with a semen sample from patient ZIKV17 with a cluster of dense virions located in the cytoplasm (red arrow). Full article ">Figure 3 Cont.
Detection of ZIKV in semen and C636 cells. (A) Detection by Indirect Immunofluorescence assay using anti-ZIKV specific antibody. Spermatozoa from semen sample collected from patient ZIKV17 at day 39 stained with FITC conjugate (in green) for virus location and with DAPI for nucleus staining (in blue). The viruses were located in the cytoplasm and flagella. C6/36 cell culture infected with virus from ZIKV17 semen sample. The cell infected presents with a green color (lower right panel). (B) Electron Microscopy of ultrathin sections of semen sample. (B1) A lower-power view of ZIKV particles inside an infected cell, with the characteristic of an immature sperm cell. (B2) Viral particles in a magnified view of the same cell in (B1). (B3) C6/36 cell infected with a semen sample from patient ZIKV17 with a cluster of dense virions located in the cytoplasm (red arrow). Full article ">Figure 4
Concentration of cytokines, chemokines and RNA viral load determined on semen of patients ZIKV17 (A) and ZIKV19 (B). The levels of the following cytokines and chemokines were measured in blood and seminal plasma—IL-2, IL-4, IL-6, CXCL8 (IL-8), IL-10, IL-17, IFN-γ, TNF–α, CCL2 (MCP-1), CCL5 (RANTES), CXCL9 (MIG), CXCL10 (IP-10). The results are representative of two distinct experiments performed in duplicate. Values of p less them 0.05 were considered statistically significant (* p < 0.05; ** p < 0.001; *** p < 0.0001—concentration of cytokines/chemokines in ZIKV patients versus control (semen from Zika—uninfected individual), ### p < 0.0001 correlation of concentration of cytokine/chemokines in different days after symptoms onset). IL-2, IL-4, IL17A and TNF–α were not detected in the seminal plasma of both patients. No cytokines or chemokines were detected in serum of both patients—serum results were below the limit of detection of the kit (20 pg/mL for cytokines and 10 pg/mL for chemokines). Full article ">Figure 4 Cont.
Concentration of cytokines, chemokines and RNA viral load determined on semen of patients ZIKV17 (A) and ZIKV19 (B). The levels of the following cytokines and chemokines were measured in blood and seminal plasma—IL-2, IL-4, IL-6, CXCL8 (IL-8), IL-10, IL-17, IFN-γ, TNF–α, CCL2 (MCP-1), CCL5 (RANTES), CXCL9 (MIG), CXCL10 (IP-10). The results are representative of two distinct experiments performed in duplicate. Values of p less them 0.05 were considered statistically significant (* p < 0.05; ** p < 0.001; *** p < 0.0001—concentration of cytokines/chemokines in ZIKV patients versus control (semen from Zika—uninfected individual), ### p < 0.0001 correlation of concentration of cytokine/chemokines in different days after symptoms onset). IL-2, IL-4, IL17A and TNF–α were not detected in the seminal plasma of both patients. No cytokines or chemokines were detected in serum of both patients—serum results were below the limit of detection of the kit (20 pg/mL for cytokines and 10 pg/mL for chemokines). Full article ">Figure 5
Evolution of ZIKV populations throughout the infection course. Median-joining haplotype networks constructed from full genome alignments of the consensus sequences from patients ZIKV19 (A) and ZIKV17 (B). Colors indicate sample type and collection date relative to symptoms onset. Each dash represents a single nucleotide substitution differentiating consensus sequences from different samples. (C) Intra host single nucleotide variant (iSNV) frequencies over time in semen samples collected from patient ZIKV17. The legend indicates the nucleotide position of each iSNV relative to KX197192.1 (GenBank) and for non-synonymous changes, the affected protein and amino acid change. Only positions with a minimum frequency ≥25% in at least one sample are shown. See <a href="#app1-viruses-10-00615" class="html-app">Table S3</a> for details about these mutations and others present at lower frequencies. (D) Proportion of nonsynonymous and synonymous ZIKV iSNVs observed in semen samples from patients ZIKV17 and ZIKV19. The relative counts of nonsynonymous and synonymous variants observed above (consensus-level) and below 50% frequency (minor population) in at least one sample were significantly different (Fisher’s exact test p-value = 0.01). Full article ">Figure 6
Dendogram showing the most parsimonious unique amino acid changes with high consistency index (CI=1) (black framed red boxes). Reconstructions were made using a set of ZIKV polyproteins from African and Asian lineage viruses. Branch lengths are shown proportional to the number of most parsimonious reconstructions (MPR) of amino acid changes. Amino acid changes that define patient clades are shown as well as the viral proteins affected. Each patient clade (which had 100% support in ML tree shown in <a href="#app1-viruses-10-00615" class="html-app">Figure S3</a>) was supported by four synapomorphic changes. For both patients ZIKV19 and ZIKV17 changes were observed in the NS5 protein. Although we only show the results for selection detection methods for the three patients, elevated rates of non-synonymous changes were detected for all of the codons containing unique amino acid changes shown. The multiple EM for motif elicitation (MEME) algorithm detected significant positive selection (p-value = 0.03) acting on the codons containing the two NS5 changes observed during infection of patient ZIKV17. All MPRs were detected with FUBAR with a Bayes factor >3 and had elevated dN. Sites detected by 2-rates FEL had nonsynonymous changes in the absence of detectable synonymous changes. Significant negative, purifying selection was detected by all methods used on several sites of the polyprotein. Full article ">

15 pages, 2343 KiB

Open AccessArticle

Strain-Dependent Consequences of Zika Virus Infection and Differential Impact on Neural Development

by Forrest T. Goodfellow, Katherine A. Willard, Xian Wu, Shelley Scoville, Steven L. Stice and Melinda A. Brindley

Viruses 2018, 10(10), 550; https://doi.org/10.3390/v10100550 - 9 Oct 2018

Cited by 35 | Viewed by 5337

Abstract

Maternal infection with Zika virus (ZIKV) during pregnancy can result in neonatal abnormalities, including neurological dysfunction and microcephaly. Experimental models of congenital Zika syndrome identified neural progenitor cells as a target of viral infection. Neural progenitor cells are responsible for populating the developing [...] Read more.

Maternal infection with Zika virus (ZIKV) during pregnancy can result in neonatal abnormalities, including neurological dysfunction and microcephaly. Experimental models of congenital Zika syndrome identified neural progenitor cells as a target of viral infection. Neural progenitor cells are responsible for populating the developing central nervous system with neurons and glia. Neural progenitor dysfunction can lead to severe birth defects, namely, lissencephaly, microcephaly, and cognitive deficits. For this study, the consequences of ZIKV infection in human pluripotent stem cell-derived neural progenitor (hNP) cells and neurons were evaluated. ZIKV isolates from Asian and African lineages displayed lineage-specific replication kinetics, cytopathic effects, and impacts on hNP function and neuronal differentiation. The currently circulating ZIKV isolates exhibit a unique profile of virulence, cytopathic effect, and impaired cellular functions that likely contribute to the pathological mechanism of congenital Zika syndrome. The authors found that infection with Asian-lineage ZIKV isolates impaired the proliferation and migration of hNP cells, and neuron maturation. In contrast, the African-lineage infections resulted in abrupt and extensive cell death. This work furthers the understanding of ZIKV-induced brain pathology. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Zika virus (ZIKV) isolate-specific growth and cytotoxicity in human pluripotent stem cell-derived neural progenitor (hNP) cells at six days post-infection. (A) hNP cells are maintained as a proliferating population in media with fibroblast growth factor (FGF) and leukemia inhibitory factor (LIF). Withdrawal of FGF and LIF from the media leads to differentiation and a population of immature neurons after 14 DIV, then post-mitotic neurons exclusively emerge after 28 DIV. (B–E) African-lineage ZIKV isolate (IbH) grew robustly and induced cell death in undifferentiated hNP cells and differentiating hNP cells, while Asian isolate (SPH) replicated more slowly with less extensive cell death. * demonstrates p < 0.05. Full article ">Figure 2
ZIKV isolate-specific growth and cytotoxicity in human neurons. (A,B) Viral replication and viability of hNP-derived nascent neurons (14 DIV) and (C,D) mature neurons (28 DIV) six days post-infection. * demonstrates p < 0.05. Full article ">Figure 3
Isolate-dependent ability of ZIKV to infect hNP cells and mature neurons. (A) ZIKV isolates IbH and SPH readily infect Vero cells within 48 h. (B) hNP cells are more susceptible to IbH infection than SPH infection, however neither isolate infected more than 13% of the population. (C) Mature neurons (28 DIV) were similarly more susceptible to ZIKV IbH infection than SPH infection. (D) Non-infected (0 MOI) did not demonstrate ZIKV E protein’s presence, but infected (SPH 10 MOI) cells after 48 h did contain ZIKV E protein in all three cell types. Full article ">Figure 4
ZIKV infection decreased hNP cell proliferation and migration. (A) Only infection with MOI 10 of African-lineage ZIKV demonstrated a significant impact on cell viability after 48 h, whereas infection with SPH did not decrease viability. (B) The proliferation of hNP cells after ZIKV infection was significantly decreased following infection with SPH as determined by quantification of Ki67 expression. (C) The migration of hNP cells was disrupted by both IbH and SPH infections. IbH infection severely inhibited migration at MOI 10 to such an extent that no acceptable data were collected (shown as ND). * demonstrates p < 0.05 significance between IbH and SPH and # demonstrates p < 0.05 significance difference from non-infected control. Full article ">Figure 5
ZIKV infection perturbs neurite outgrowth in human neural progenitor cell-derived neurons. (A) At 48 h post-infection, both African (IbH) and Asian (SPH) isolates had minimal impact on neuron viability. (B) Representative images of neurite outgrowth quantification. Hoechst stain labels nuclei of neurons and MAP2 identifies neurites. Both images contribute to the enumeration and quantification of neurite outgrowth characteristics. Scale bar = 50 micrometers (C–F). Infection with Asian-lineage ZIKV (SPH) did not reduce the number of valid neurons observed, yet infection with SPH (MOI 10) proved detrimental to neurite outgrowth by decreasing the quantity, length, and number of branch points per neuron. * demonstrates p < 0.05 significance between IbH and SPH and # demonstrates p < 0.05 significance difference from non-infected control. Full article ">

21 pages, 10263 KiB

Open AccessArticle

An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

by Silvia Márquez-Jurado, Aitor Nogales, Ginés Ávila-Pérez, Francisco J. Iborra, Luis Martínez-Sobrido and Fernando Almazán

Viruses 2018, 10(10), 547; https://doi.org/10.3390/v10100547 - 7 Oct 2018

Cited by 30 | Viewed by 5826

Abstract

The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish [...] Read more.

The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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10 pages, 1836 KiB

Open AccessCommunication

Fetal Brain Infection Is Not a Unique Characteristic of Brazilian Zika Viruses

by Yin Xiang Setoh, Nias Y. Peng, Eri Nakayama, Alberto A. Amarilla, Natalie A. Prow, Andreas Suhrbier and Alexander A. Khromykh

Viruses 2018, 10(10), 541; https://doi.org/10.3390/v10100541 - 3 Oct 2018

Cited by 14 | Viewed by 5266

Abstract

The recent emergence of Zika virus (ZIKV) in Brazil was associated with an increased number of fetal brain infections that resulted in a spectrum of congenital neurological complications known as congenital Zika syndrome (CZS). Herein, we generated de novo from sequence data an [...] Read more.

The recent emergence of Zika virus (ZIKV) in Brazil was associated with an increased number of fetal brain infections that resulted in a spectrum of congenital neurological complications known as congenital Zika syndrome (CZS). Herein, we generated de novo from sequence data an early Asian lineage ZIKV isolate (ZIKV-MY; Malaysia, 1966) not associated with microcephaly and compared the in vitro replication kinetics and fetal brain infection in interferon α/β receptor 1 knockout (IFNAR1^−/−) dams of this isolate and of a Brazilian isolate (ZIKV-Natal; Natal, 2015) unequivocally associated with microcephaly. The replication efficiencies of ZIKV-MY and ZIKV-Natal in A549 and Vero cells were similar, while ZIKV-MY replicated more efficiently in wild-type (WT) and IFNAR^−/− mouse embryonic fibroblasts. Viremias in IFNAR1^−/− dams were similar after infection with ZIKV-MY or ZIKV-Natal, and importantly, infection of fetal brains was also not significantly different. Thus, fetal brain infection does not appear to be a unique feature of Brazilian ZIKV isolates. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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De novo generation and characterization of ZIKV-MY. (a) Schematic of Circular Polymerase Extension Reaction (CPER) fragments used for recovering ZIKV-MY. All fragments, except for the UTR-linker, are drawn to scale; (b) Plaque morphology on a Vero cell monolayer of ZIKV-MY recovered from the culture supernatant of CPER-transfected Vero cells, compared to ZIKV-Natal; Growth kinetics of ZIKV-MY versus ZIKV-Natal was performed on (c) Vero, (d) A549, (e) WT MEF, and (f) IFNAR−/− MEF cells at their indicated multiplicity of infection (MOI), and culture supernatants were harvested at the indicated time points post-infection and titered by plaque assay. The dashed lines represent the limit of detection of the assay. Means and ± SE are shown. Statistical analyses were performed using t-tests (n = three biological replicates); statistically significant are differences shown in panel (f) **—p = 0.008, ***—p < 0.001. Full article ">Figure 2
ZIKV-MY and ZIKV-Natal replicate to similar levels in vivo. (a) Interferon α/β receptor 1 knockout (IFNAR1−/−) C57BL/6 female mice were infected with ZIKV-MY or ZIKV-Natal. Pregnant mice were infected at E12.5 of gestation. Viremia was determined daily for five days post-infection. Pregnant mice were euthanized at E17.5, and their (b) spleen, (c) spinal cord, (d) muscle, (e) liver, (f) kidney, (g) eye, and (h) brain samples were processed to determine the viral titers by the CCID50 assay. Full article ">Figure 3
Fetal and placental virus titers after infection with ZIKV-MY or ZIKV-Natal. IFNAR1−/− C57BL/6 pregnant mice were infected with ZIKV-MY or ZIKV-Natal at E12.5 and were euthanized at E17.5 of gestation. (a) Fetal heads, (b) placenta, and (c) deformed fetal–placental masses were collected, and tissue viral titers determined by the CCID50 assay. *—p = 0.002; **—p = 0.045. Full article ">

18 pages, 1119 KiB

Open AccessArticle

The Effect of Permethrin Resistance on Aedes aegypti Transcriptome Following Ingestion of Zika Virus Infected Blood

by Liming Zhao, Barry W. Alto, Dongyoung Shin and Fahong Yu

Viruses 2018, 10(9), 470; https://doi.org/10.3390/v10090470 - 1 Sep 2018

Cited by 16 | Viewed by 5268

Abstract

Aedes aegypti (L.) is the primary vector of many emerging arboviruses. Insecticide resistance among mosquito populations is a consequence of the application of insecticides for mosquito control. We used RNA-sequencing to compare transcriptomes between permethrin resistant and susceptible strains of Florida Ae. aegypti [...] Read more.

Aedes aegypti (L.) is the primary vector of many emerging arboviruses. Insecticide resistance among mosquito populations is a consequence of the application of insecticides for mosquito control. We used RNA-sequencing to compare transcriptomes between permethrin resistant and susceptible strains of Florida Ae. aegypti in response to Zika virus infection. A total of 2459 transcripts were expressed at significantly different levels between resistant and susceptible Ae. aegypti. Gene ontology analysis placed these genes into seven categories of biological processes. The 863 transcripts were expressed at significantly different levels between the two mosquito strains (up/down regulated) more than 2-fold. Quantitative real-time PCR analysis was used to validate the Zika-infection response. Our results suggested a highly overexpressed P450, with AAEL014617 and AAEL006798 as potential candidates for the molecular mechanism of permethrin resistance in Ae. aegypti. Our findings indicated that most detoxification enzymes and immune system enzymes altered their gene expression between the two strains of Ae. aegypti in response to Zika virus infection. Understanding the interactions of arboviruses with resistant mosquito vectors at the molecular level allows for the possible development of new approaches in mitigating arbovirus transmission. This information sheds light on Zika-induced changes in insecticide resistant Ae. aegypti with implications for mosquito control strategies. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Zika virus titers in infectious blood meals and blood fed mosquitoes for permethrin resistant (KW) and susceptible (OR) strains of Aedes aegypti, including initial dose in bloodmeal, freshly fed, 7 days post infection (7 dpi), and 10 days post infection (10 dpi). Zika virus (strain PRVABC59, GenBank accession # KU501215.1) isolated from a human infected in Puerto Rico in 2015. Full article ">Figure 2
Validation of the expression of transcripts between the permethrin resistant (KW) and susceptible (OR) strains of Aedes aegypti by qRT-PCR. * p < 0.05. ** p < 0.01. Full article ">Figure 3
Overview of the functional categories of differentially expressed (DE) transcripts in response to ZIKV infection and between the permethrin resistant (KW) and susceptible (OR) strains of Aedes aegypti blood-feeding control. DE transcripts were determined based on statistical analysis by DESeq package. The total number of DE transcripts for each comparison is shown in parentheses in each figure. Gene ontology analysis of DE genes was performed based on the database of AmiGO 2 (<a href="http://amigo.geneontology.org/amigo" target="_blank">http://amigo.geneontology.org/amigo</a>), and pie charts were generated using Excel. Up, upregulated DE genes; Down, downregulated DE genes. Please also notice the details in the <a href="#app1-viruses-10-00470" class="html-app">Supplementary Figure S1</a>. GO analyses for RNA-seq data. (A) 12 h post injection KW-Control compared with OR-Control; (B) 12 h post infection, KW-ZIKV compared with OR-ZIKV; (C) 7 dpi, KW-ZIKV compared with KW-Control; (D) 7 dpi, OR-ZIKV compared with OR-Control; (E) 7 dpi, KW-Control compared with OR-Control; (F) 7 dpi, KW-ZIKV compared with OR-ZIKV. Full article ">

28 pages, 7627 KiB

Open AccessArticle

Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection

by Sang-Im Yun, Byung-Hak Song, Jordan C. Frank, Justin G. Julander, Aaron L. Olsen, Irina A. Polejaeva, Christopher J. Davies, Kenneth L. White and Young-Min Lee

Viruses 2018, 10(8), 422; https://doi.org/10.3390/v10080422 - 11 Aug 2018

Cited by 15 | Viewed by 5587

Abstract

Zika virus (ZIKV) causes no-to-mild symptoms or severe neurological disorders. To investigate the importance of viral and host genetic variations in determining ZIKV infection outcomes, we created three full-length infectious cDNA clones as bacterial artificial chromosomes for each of three spatiotemporally distinct and [...] Read more.

Zika virus (ZIKV) causes no-to-mild symptoms or severe neurological disorders. To investigate the importance of viral and host genetic variations in determining ZIKV infection outcomes, we created three full-length infectious cDNA clones as bacterial artificial chromosomes for each of three spatiotemporally distinct and genetically divergent ZIKVs: MR-766 (Uganda, 1947), P6-740 (Malaysia, 1966), and PRVABC-59 (Puerto Rico, 2015). Using the three molecularly cloned ZIKVs, together with 13 ZIKV region-specific polyclonal antibodies covering nearly the entire viral protein-coding region, we made three conceptual advances: (i) We created a comprehensive genome-wide portrait of ZIKV gene products and their related species, with several previously undescribed gene products identified in the case of all three molecularly cloned ZIKVs. (ii) We found that ZIKV has a broad cell tropism in vitro, being capable of establishing productive infection in 16 of 17 animal cell lines from 12 different species, although its growth kinetics varied depending on both the specific virus strain and host cell line. More importantly, we identified one ZIKV-non-susceptible bovine cell line that has a block in viral entry but fully supports the subsequent post-entry steps. (iii) We showed that in mice, the three molecularly cloned ZIKVs differ in their neuropathogenicity, depending on the particular combination of viral and host genetic backgrounds, as well as in the presence or absence of type I/II interferon signaling. Overall, our findings demonstrate the impact of viral and host genetic variations on the replication kinetics and neuropathogenicity of ZIKV and provide multiple avenues for developing and testing medical countermeasures against ZIKV. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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9 pages, 228 KiB

Open AccessArticle

Comparative Evaluation of Indirect Immunofluorescence and NS-1-Based ELISA to Determine Zika Virus-Specific IgM

by Fernando De Ory, María Paz Sánchez-Seco, Ana Vázquez, María Dolores Montero, Elena Sulleiro, Miguel J. Martínez, Lurdes Matas, Francisco J. Merino and Working Group for the Study of Zika Virus Infections

Viruses 2018, 10(7), 379; https://doi.org/10.3390/v10070379 - 19 Jul 2018

Cited by 12 | Viewed by 4669

Abstract

Differential diagnosis of the Zika virus (ZIKV) is hampered by cross-reactivity with other flaviviruses, mainly dengue viruses. The aim of this study was to compare two commercial methods for detecting ZIKV immunoglobulin M (IgM), an indirect immunofluorescence (IIF) and an enzyme immunoassay (ELISA), [...] Read more.

Differential diagnosis of the Zika virus (ZIKV) is hampered by cross-reactivity with other flaviviruses, mainly dengue viruses. The aim of this study was to compare two commercial methods for detecting ZIKV immunoglobulin M (IgM), an indirect immunofluorescence (IIF) and an enzyme immunoassay (ELISA), using the non-structural (NS) 1 protein as an antigen, both from EuroImmun, Germany. In total, 255 serum samples were analyzed, 203 of which showed laboratory markers of ZIKV infections (PCR-positive in serum and/or in urine and/or positive or indeterminate specific IgM). When tested with IIF, 163 samples were IgM-positive, while 13 samples were indeterminate and 78 were negative. When IIF-positive samples were tested using ELISA, we found 61 positive results, 14 indeterminate results, and 88 negative results. Among the indeterminate cases tested with IIF, ELISA analysis found two positive, two indeterminate, and nine negative results. Finally, 74 of the 78 IIF-negative samples proved also to be negative using ELISA. For the calculations, all indeterminate results were considered to be positive. The agreement, sensitivity, and specificity between ELISA and IIF were 60.2%, 44.9%, and 94.9%, respectively. Overall, 101 samples showed discrepant results; these samples were finally classified on the basis of other ZIKV diagnostic approaches (PCR-positive in serum and/or in urine, IgG determinations using IIF or ELISA, and ZIKV Plaque Reduction Neutralization test—positive), when available. A final classification of 228 samples was possible; 126 of them were positive and 102 were negative. The corresponding values of agreement, sensitivity, and specificity of IIF were 86.0%, 96.8%, and 72.5%, respectively. The corresponding figures for ELISA were 81.1%, 65.9%, and 100%, respectively. The ELISA and IIF methods are both adequate approaches for detecting ZIKV-specific IgM. However, considering their respective weaknesses (low sensitivity in ELISA and low specificity in IIF), serological results must be considered jointly with other laboratory results. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

17 pages, 2651 KiB

Open AccessArticle

A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

by Maximilian Münster, Anna Płaszczyca, Mirko Cortese, Christopher John Neufeldt, Sarah Goellner, Gang Long and Ralf Bartenschlager

Viruses 2018, 10(7), 368; https://doi.org/10.3390/v10070368 - 12 Jul 2018

Cited by 36 | Viewed by 7838

Abstract

The Zika virus (ZIKV) has recently attracted major research interest as infection was unexpectedly associated with neurological manifestations in developing foetuses and with Guillain-Barré syndrome in infected adults. Understanding the underlying molecular mechanisms requires reverse genetic systems, which allow manipulation of infectious cDNA [...] Read more.

The Zika virus (ZIKV) has recently attracted major research interest as infection was unexpectedly associated with neurological manifestations in developing foetuses and with Guillain-Barré syndrome in infected adults. Understanding the underlying molecular mechanisms requires reverse genetic systems, which allow manipulation of infectious cDNA clones at will. In the case of flaviviruses, to which ZIKV belongs, several reports have indicated that the construction of full-length cDNA clones is difficult due to toxicity during plasmid amplification in Escherichia coli. Toxicity of flaviviral cDNAs has been linked to the activity of cryptic prokaryotic promoters within the region encoding the structural proteins leading to spurious transcription and expression of toxic viral proteins. Here, we employ an approach based on in silico prediction and mutational silencing of putative promoters to generate full-length cDNA clones of the historical MR766 strain and the contemporary French Polynesian strain H/PF/2013 of ZIKV. While for both strains construction of full-length cDNA clones has failed in the past, we show that our approach generates cDNA clones that are stable on single bacterial plasmids and give rise to infectious viruses with properties similar to those generated by other more complex assembly strategies. Further, we generate luciferase and fluorescent reporter viruses as well as sub-genomic replicons that are fully functional and suitable for various research and drug screening applications. Taken together, this study confirms that in silico prediction and silencing of cryptic prokaryotic promoters is an efficient strategy to generate full-length cDNA clones of flaviviruses and reports novel tools that will facilitate research on ZIKV biology and development of antiviral strategies. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Figure 1
Construction and stability of synthetic full length Zika virus (synZIKV) cDNA clones. (A) Schematic representation of the synZIKV MR766 construct and the four fragments used to assemble the genome. The 5′ and 3′UTRs are indicated with bold black lines, the promoter for the T7 RNA polymerase with a black arrow. Restriction sites used for the assembly of the fragments are indicated. An enlargement of fragment #1 is shown below with putative CEPs (score > 0.85) indicated by red arrow heads. CEP 1 was not mutated (indicated with the pink arrow head). (B) Same as in panel (A) but for synZIKV-H/PF/2013. (C) Restriction patterns of pFK-synZIKV constructs obtained after digest with EcoRI (MR766) or XmnI (H/PF/2013) and agarose gel electrophoresis. Plasmids were analysed directly after assembly (original prep) and after five passages (P5) in E. coli (five DNA clones of P5 are shown). Full article ">Figure 2
Replication kinetics of viruses obtained with the full-length synZIKV clones. (A) Replication kinetics of the two synZIKV clones as determined by plaque assay. VeroE6 cells were transfected with in vitro transcribed synZIKV RNAs and virus contained in culture supernatant at different time points after transfection was measured. Mean ± SEM of two independent experiments is shown. (B,C) Comparison of replication kinetics of synZIKV and parental viruses. Huh7 cells were infected with either ZIKV using a multiplicity of infection (MOI) of 1. Supernatants from infected cells were harvested at indicated times post-infection and titres were determined by plaque assay. Mean ± SEM of three independent experiments is shown. (D) Comparison of plaque morphology of synZIKV and the parental viruses. (E,F) Replication kinetics of passaged synZIKVs. Virus stocks were prepared as described in Materials and methods (P0). Huh7 cells were infected with MOI = 0.1 of P0 virus, cell culture supernatants were collected 72 h post-infection (P1) and passaged two more times by infection of Huh7 cells (P2–P3) in 72 h intervals. Huh7 cells were then infected using a MOI of 0.01 of P0 and P3 virus, respectively and virus titres were measured at indicated time points by plaque assay. Full article ">Figure 3
Construction and characterization of synZIKV-R2A reporter virus genomes. (A) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. (B) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. (C) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of three independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the RNA-dependent-RNA polymerase in NS5 (GAA) served as negative controls. (D) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. (E) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. (F) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. (G) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown. Full article ">Figure 4
Construction and characterization of synZIKV-FP635 reporter viruses suitable for live cell imaging. (A) Schematic representation of the synZIKV-FP635 reporter genomes. The FP635 gene fused at the 3′ end to the coding sequence of the SV40 NLS (not indicated) was inserted into the synZIKV constructs via NotI/NruI restriction sites. (B) Detection of E-antigen by immunofluorescence analysis of VeroE6 cells 96 h post-transfection with synZIKV-FP635 RNAs. The FP635 signal (red) was detected by its fluorescence. Note the accumulation of FP635 in distinct nuclear sites, most likely corresponding to nucleoli. Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. (C) Quantification of E- and FP635-positive VeroE6 cells 96 h post-transfection of synZIKV-FP635 RNAs. Results show the mean from two independent experiments ± SEM. At least 150 cells per condition were counted. Full article ">Figure 5
Properties of synZIKV sub-genomic reporter replicons. (A) Schematic representation of the synZIKV-sgR2A subgenomic reporter replicons. The reporter cassette (grey) was inserted into the synZIKV genomes via the MLuI and AgeI restriction sites and replaces the region encoding the structural proteins. (B) RLuc activity in Huh7 cells transfected with wild-type or replication-deficient (mutant GAA) synZIKV-sgR2A replicon RNAs measured at given times post-transfection. Shown RLuc values were normalized to the 4 h value to correct for transfection efficiency. Mean ± SEM of three independent experiments is presented. (C) Western blot showing the abundance of ZIKV NS3 and NS4B proteins in Huh7 cells transfected with synZIKV-sgR2A replicon RNAs. Cells were lysed at indicated times post-transfection and ZIKV-specific antibodies were used to detect viral proteins. β actin served as loading control. Numbers on the left refer to the positions of marker proteins that are given in kilodalton (kDa). (D) Immunofluorescence analysis of Huh7 cells 48 h post-transfection of synZIKV-sgR2A RNAs. Cells were stained with a dsRNA- (green) and a NS3-specific antibody (red). Nuclear DNA was stained with DAPI (grey). Scale bars = 15 μm. Boxed areas indicate regions that are shown in the left panels as enlargements. Full article ">

15 pages, 854 KiB

Open AccessArticle

Detection of Specific ZIKV IgM in Travelers Using a Multiplexed Flavivirus Microsphere Immunoassay

by Carmel T. Taylor, Ian M. Mackay, Jamie L. McMahon, Sarah L. Wheatley, Peter R. Moore, Mitchell J. Finger, Glen R. Hewitson and Frederick A. Moore

Viruses 2018, 10(5), 253; https://doi.org/10.3390/v10050253 - 12 May 2018

Cited by 13 | Viewed by 5997

Abstract

Zika virus (ZIKV) has spread widely in the Pacific and recently throughout the Americas. Unless detected by RT-PCR, confirming an acute ZIKV infection can be challenging. We developed and validated a multiplexed flavivirus immunoglobulin M (IgM) microsphere immunoassay (flaviMIA) which can differentiate ZIKV-specific [...] Read more.

Zika virus (ZIKV) has spread widely in the Pacific and recently throughout the Americas. Unless detected by RT-PCR, confirming an acute ZIKV infection can be challenging. We developed and validated a multiplexed flavivirus immunoglobulin M (IgM) microsphere immunoassay (flaviMIA) which can differentiate ZIKV-specific IgM from that due to other flavivirus infections in humans. The flaviMIA bound 12 inactivated flavivirus antigens, including those from ZIKV and yellow fever virus (YFV), to distinct anti-flavivirus antibody coupled beads. These beads were used to interrogate sera from patients with suspected ZIKV infection following travel to relevant countries. FlaviMIA results were validated by comparison to the ZIKV plaque reduction neutralization test (PRNT). The results highlight the complexity of serological ZIKV diagnosis, particularly in patients previously exposed to or vaccinated against other flaviviruses. We confirmed 99 patients with ZIKV infection by a combination of RT-PCR and serology. Importantly, ZIKV antibodies could be discriminated from those ascribed to other flavivirus infections. Serological results were sometimes confounded by the presence of pre-existing antibodies attributed to previous flavivirus infection or vaccination. Where RT-PCR results were negative, testing of appropriately timed paired sera was necessary to demonstrate seroconversion or differentiation of recent from past infection with or exposure to ZIKV. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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16 pages, 2251 KiB

Open AccessArticle

Comparative Pathogenesis of Asian and African-Lineage Zika Virus in Indian Rhesus Macaque’s and Development of a Non-Human Primate Model Suitable for the Evaluation of New Drugs and Vaccines

by Jonathan O. Rayner, Raj Kalkeri, Scott Goebel, Zhaohui Cai, Brian Green, Shuling Lin, Beth Snyder, Kimberly Hagelin, Kevin B. Walters and Fusataka Koide

Viruses 2018, 10(5), 229; https://doi.org/10.3390/v10050229 - 1 May 2018

Cited by 25 | Viewed by 5848

Abstract

The establishment of a well characterized non-human primate model of Zika virus (ZIKV) infection is critical for the development of medical interventions. In this study, challenging Indian rhesus macaques (IRMs) with ZIKV strains of the Asian lineage resulted in dose-dependent peak viral loads [...] Read more.

The establishment of a well characterized non-human primate model of Zika virus (ZIKV) infection is critical for the development of medical interventions. In this study, challenging Indian rhesus macaques (IRMs) with ZIKV strains of the Asian lineage resulted in dose-dependent peak viral loads between days 2 and 5 post infection and a robust immune response which protected the animals from homologous and heterologous re-challenge. In contrast, viremia in IRMs challenged with an African lineage strain was below the assay’s lower limit of quantitation, and the immune response was insufficient to protect from re-challenge. These results corroborate previous observations but are contrary to reports using other African strains, obviating the need for additional studies to elucidate the variables contributing to the disparities. Nonetheless, the utility of an Asian lineage ZIKV IRM model for countermeasure development was verified by vaccinating animals with a formalin inactivated reference vaccine and demonstrating sterilizing immunity against a subsequent subcutaneous challenge. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Figure 1
Key Study Challenge and Sample Collection Time Points. Challenge Phase I animals were subdivided into cohorts and inoculated on day 0 with a primary challenge ZIKV isolate. After the primary challenge and a resting period, animals were regrouped and challenged on day 45 with either the same isolate or cross-challenged with an isolate of different geographic origin. Blood, saliva and urine samples were collected as indicated for viral load analysis by qRT-PCR or plaque assay. Full article ">Figure 2
Serum viral RNA copies in IRMs infected with different ZIKV isolates: Serum viral RNA copies in the IRM’s (N = 4 per dose group) infected with different doses of ZIKV isolates as indicated in the figures. (A) PRVABC59 (B) PLCal_ZV (C) IbH_30656. Viral RNA is reported as copies/mL from serum purified from blood collected at multiple time points post challenge between days 0–30. Lower Limit of Quantitation (LLOQ) is shown by the dotted line. Full article ">Figure 3
Viral RNA shedding in PRVABC59 infected IRM: Urine and saliva were collected at multiple time points post challenge between days 0–30 from IRM infected with ZIKV PRVABC59 and subjected to qRT-PCR assay. ZIKV RNA concentrations (copies/mL) in the urine (A) and saliva (B). Circles represent data from single animals in the 1 × 104 dose group, squares represent the 1 × 105 dose group, and triangles represent the 1 × 106 dose group. Urine could not be collected from one IRM on day 5 (dose 1 × 104) and day 25 (dose 1 × 105). Lower Limit of Quantitation (LLOQ) is shown by the dotted line. Full article ">Figure 4
Anti-ZIKV IgG production after initial challenge with ZIKV. Animals were challenged with 1 × 104 PFU, 1 × 105 PFU, or 1 × 106 PFU with either ZIKV (A) PRVABC59, (B) PLCal_ZV or (C) IbH_30656 and anti-ZIKV IgG was detected in serum by ELISA. Bars with no fill 1 × 104 PFU, hatched lines 1 × 105 PFU, or horizontal lines 1 × 106 PFU. Full article ">Figure 5
Serum viral RNA copies post rechallenge in IRMs: Serum viral RNA copies were determined by qRT-PCR in IRMs (N = 6 per group) previously infected and re-challenged with either PRVABC59, PLCal_ZV, or IbH_30656 at a dose of 1 × 106 PFU/IRM. Viral RNA is reported as copies/mL of serum purified from blood collected at multiple time points post re-challenge between days 45–50. Note, for the IbH_30656:IbH_30656 group day (50) viral RNA copies were determined from N = 3. Lower Limit of Quantitation (LLOQ) is shown by the dotted line. Full article ">Figure 6
Anti-ZIKV IgG production after secondary challenge with ZIKV. Antibody titers on Study days 50 and 75 during Phase II. Animals from each group in Phase I were reassigned based on previous challenge strain and titer, then re-challenged with 1 × 106 PFU of PRVABC59, PLCal_ZV, or IbH_30656. (A) Primary challenge with PRVABC59 followed by secondary challenge with PRVABC59 (No fill) or PLCal_ZV (Hatched lines); (B) Primary challenge with PLCal_ZV followed by secondary challenge with PLCal_ZV (No fill) or PRVABC59 (Hatched lines); and (C) Primary challenge with IbH_30656 followed by secondary challenge with IbH_30656 (No fill) or PRVABC59 (Hatched lines). Full article ">Figure 7
Immunogenicity (A) and Efficacy (B) of Formalin-inactivated ZIKV vaccine in the IRM model: IRMs were vaccinated with formalin-inactivated ZIKV vaccine or the sham control on day 0, followed by challenge with 1 × 105 PFU of PRVABC59 per IRM on day 54. Serum viral RNA copies after the challenge and FRNT50 titers after vaccination were measured by using qRT-PCR assay and ZIKV neutralization assay as described in the method section. Triangles—Vaccinated animals, Circles—Vehicle control. Full article ">

14 pages, 10575 KiB

Open AccessArticle

Inhibition of Zika Virus Replication by Silvestrol

by Fabian Elgner, Catarina Sabino, Michael Basic, Daniela Ploen, Arnold Grünweller and Eberhard Hildt

Viruses 2018, 10(4), 149; https://doi.org/10.3390/v10040149 - 27 Mar 2018

Cited by 55 | Viewed by 8160

Abstract

The Zika virus (ZIKV) outbreak in 2016 in South America with specific pathogenic outcomes highlighted the need for new antiviral substances with broad-spectrum activities to react quickly to unexpected outbreaks of emerging viral pathogens. Very recently, the natural compound silvestrol isolated from the [...] Read more.

The Zika virus (ZIKV) outbreak in 2016 in South America with specific pathogenic outcomes highlighted the need for new antiviral substances with broad-spectrum activities to react quickly to unexpected outbreaks of emerging viral pathogens. Very recently, the natural compound silvestrol isolated from the plant Aglaia foveolata was found to have very potent antiviral effects against the (−)-strand RNA-virus Ebola virus as well as against Corona- and Picornaviruses with a (+)-strand RNA-genome. This antiviral activity is based on the impaired translation of viral RNA by the inhibition of the DEAD-box RNA helicase eukaryotic initiation factor-4A (eIF4A) which is required to unwind structured 5´-untranslated regions (5′-UTRs) of several proto-oncogenes and thereby facilitate their translation. Zika virus is a flavivirus with a positive-stranded RNA-genome harboring a 5′-capped UTR with distinct secondary structure elements. Therefore, we investigated the effects of silvestrol on ZIKV replication in A549 cells and primary human hepatocytes. Two different ZIKV strains were used. In both infected A549 cells and primary human hepatocytes, silvestrol has the potential to exert a significant inhibition of ZIKV replication for both analyzed strains, even though the ancestor strain from Uganda is less sensitive to silvestrol. Our data might contribute to identify host factors involved in the control of ZIKV infection and help to develop antiviral concepts that can be used to treat a variety of viral infections without the risk of resistances because a host protein is targeted. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Cytostatic effect of silvestrol in A549 cells. (A) Time- and concentration-dependent decrease of cellular metabolic activity after silvestrol treatment in A549 cells determined by the PrestoBlue assay. Cycloheximid (CHX) served as positive control in a concentration of 35 µM to inhibit cell proliferation; (B) Lactate dehydrogenase (LDH) release assay with the supernatants of silvestrol treated A549 cells did not show an increased cell death. Treatment with 1% Triton X-100 served as positive control for complete cell death. LDH activity in the samples was normalized to the LDH activity in the supernatant of Triton X-100 treated cells. ns = not significant = p > 0.05; * = p ≤ 0.05; **** = p ≤ 0.0001. Full article ">Figure 2
Silvestrol treatment reduces the number of Zika virus (ZIKV) positive cells. (A) A549 cells were infected with ZIKV French Polynesia isolate (FP) and treated with the indicated amount of silvestrol. Cells were fixed on the indicated time points and nuclei were stained with DAPI (blue) and NS1 was stained with a specific antibody in green, scale bar = 200 µm; (B) Ratio of NS1-positive cells in at least four fields of view of the respective samples exemplary shown in (A); (C) A549 cells were infected with ZIKV Uganda isolate (U) and treated with the indicated amount of silvestrol. Cells were fixed on the indicated time points and nuclei were stained with DAPI (blue) and NS1 was stained with a specific antibody in green, scale bar = 200 µm; (D) Ratio of NS1-positive cells in at least four fields of view of the respective samples exemplary shown in (C). * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Full article ">Figure 3
Significant reduction of intra- and extracellular ZIKV RNA levels and released virions. (A) Quantification of intracellular ZIKV RNA of A549 cells infected with ZIKV FP and treated with the indicated concentrations of silvestrol. ZIKV RNA was quantified by RT-qPCR and normalized to the amount of RPL27 transcripts; (B) Quantification of intracellular ZIKV RNA of A549 cells infected with ZIKV U and treated with the indicated concentrations of silvestrol. ZIKV RNA was quantified by RT-qPCR and normalized to the amount of RPL27 transcripts; (C) RT-qPCR quantification of extracellular ZIKV RNA of A549 cells infected with ZIKV FP and treated with the indicated concentrations of silvestrol; (D) RT-qPCR quantification of extracellular ZIKV RNA of A549 cells infected with ZIKV U and treated with the indicated concentrations of silvestrol; (E) Relative extracellular titers of A549 cells infected with ZIKV FP and treated with the indicated concentrations of silvestrol. Titers were quantified by plaque assay in Vero cells; (F) Relative extracellular titers of A549 cells infected with ZIKV U and treated with the indicated concentrations of silvestrol. Titers were quantified by plaque assay in Vero cells. $: only two experiments were performed. ns = not significant = p > 0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Full article ">Figure 4
Silvestrol treatment reduces the amount of intracellular NS1 protein. (A) A549 cells were infected with ZIKV U and treated with the indicated amount of silvestrol. Cell lysates of the indicated time points were analyzed by Western blot with specific antibodies against NS1 and β-actin; (B) Quantification of densitometry scans examples are shown in (A); (C) A549 cells were infected with ZIKV FP and treated with the indicated amount of silvestrol. Cell lysates of the indicated time points were analyzed by Western blot with specific antibodies against NS1 and β-actin; (D) Quantification of densitometry scans examples are shown in (C). ns = not significant = p > 0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Full article ">Figure 5
Silvestrol exerts an anti-ZIKV effect also in primary cells. Primary human hepatocytes were infected with ZIKV FP and treated with the indicated concentrations of silvestrol. The extracellular ZIKV RNA was quantified by RT-qPCR after 24 (A), 48 (B), and 72 h (C); (D) Extracellular titers of infected and treated primary human hepatocytes (PHHs) after 48 and 72 h. Shown are titers from just one PHH donor quantified by plaque assay in Vero cells; (E) ALT activity in PHH supernatants. ns = not significant = p > 0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Full article ">

Review

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16 pages, 263 KiB

Open AccessReview

Suppression of Type I Interferon Signaling by Flavivirus NS5

by Stephanie Thurmond, Boxiao Wang, Jikui Song and Rong Hai

Viruses 2018, 10(12), 712; https://doi.org/10.3390/v10120712 - 14 Dec 2018

Cited by 39 | Viewed by 5123

Abstract

Type I interferon (IFN-I) is the first line of mammalian host defense against viral infection. To counteract this, the flaviviruses, like other viruses, have encoded a variety of antagonists, and use a multi-layered molecular defense strategy to establish their infections. Among the most [...] Read more.

Type I interferon (IFN-I) is the first line of mammalian host defense against viral infection. To counteract this, the flaviviruses, like other viruses, have encoded a variety of antagonists, and use a multi-layered molecular defense strategy to establish their infections. Among the most potent antagonists is non-structural protein 5 (NS5), which has been shown for all disease-causing flaviviruses to target different steps and players of the type I IFN signaling pathway. Here, we summarize the type I IFN antagonist mechanisms used by flaviviruses with a focus on the role of NS5 in regulating one key regulator of type I IFN, signal transducer and activator of transcription 2 (STAT2). Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

13 pages, 406 KiB

Open AccessReview

Recent Advances in Zika Virus Vaccines

by Himanshu Garg, Tugba Mehmetoglu-Gurbuz and Anjali Joshi

Viruses 2018, 10(11), 631; https://doi.org/10.3390/v10110631 - 14 Nov 2018

Cited by 39 | Viewed by 5072

Abstract

The recent outbreaks of Zika virus (ZIKV) infections and associated microcephaly in newborns has resulted in an unprecedented effort by researchers to target this virus. Significant advances have been made in developing vaccine candidates, treatment strategies and diagnostic assays in a relatively short [...] Read more.

The recent outbreaks of Zika virus (ZIKV) infections and associated microcephaly in newborns has resulted in an unprecedented effort by researchers to target this virus. Significant advances have been made in developing vaccine candidates, treatment strategies and diagnostic assays in a relatively short period of time. Being a preventable disease, the first line of defense against ZIKV would be to vaccinate the highly susceptible target population, especially pregnant women. Along those lines, several vaccine candidates including purified inactivated virus (PIV), live attenuated virus (LAV), virus like particles (VLP), DNA, modified RNA, viral vectors and subunit vaccines have been in the pipeline with several advancing to clinical trials. As the primary objective of Zika vaccination is the prevention of vertical transmission of the virus to the unborn fetus, the safety and efficacy requirements for this vaccine remain unique when compared to other diseases. This review will discuss these recent advances in the field of Zika vaccine development. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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22 pages, 1614 KiB

Open AccessReview

Reverse Genetic Approaches for the Generation of Recombinant Zika Virus

by Ginés Ávila-Pérez, Aitor Nogales, Verónica Martín, Fernando Almazán and Luis Martínez-Sobrido

Viruses 2018, 10(11), 597; https://doi.org/10.3390/v10110597 - 31 Oct 2018

Cited by 29 | Viewed by 11705

Abstract

Zika virus (ZIKV) is an emergent mosquito-borne member of the Flaviviridae family that was responsible for a recent epidemic in the Americas. ZIKV has been associated with severe clinical complications, including neurological disorder such as Guillain-Barré syndrome in adults and severe fetal abnormalities [...] Read more.

Zika virus (ZIKV) is an emergent mosquito-borne member of the Flaviviridae family that was responsible for a recent epidemic in the Americas. ZIKV has been associated with severe clinical complications, including neurological disorder such as Guillain-Barré syndrome in adults and severe fetal abnormalities and microcephaly in newborn infants. Given the significance of these clinical manifestations, the development of tools and reagents to study the pathogenesis of ZIKV and to develop new therapeutic options are urgently needed. In this respect, the implementation of reverse genetic techniques has allowed the direct manipulation of the viral genome to generate recombinant (r)ZIKVs, which have provided investigators with powerful systems to answer important questions about the biology of ZIKV, including virus-host interactions, the mechanism of transmission and pathogenesis or the function of viral proteins. In this review, we will summarize the different reverse genetic strategies that have been implemented, to date, for the generation of rZIKVs and the applications of these platforms for the development of replicon systems or reporter-expressing viruses. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Figure 1
Zika virus (ZIKV) virion structure and genome organization. (A) Schematic representation of ZIKV virion structure: ZIKV virion surface is decorated with the E and M proteins, anchored in a lipid bilayer with an icosahedral-like symmetry. Under the viral lipid bilayer is the nucleocapsid composed of the vRNA genome associated with the C protein. (B) Genome organization and polyprotein processing: ZIKV genome (approximately 10.8 kb) is translated as a single polyprotein that is cleaved co- and post-translationally by viral (arrows) and host (diamonds) proteases to yield the three structural proteins C, M and E; and seven NS proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The 5′ and 3′ untranslated regions (UTR) are indicated as black lines at the end of the viral genome. Full article ">Figure 2
Schematic representation of ZIKV reverse genetic approaches. (A) Infectious RNA transcripts from full-length cDNA clones: A full-length genomic cDNA clone containing the ZIKV genome flanked by a prokaryotic promoter (e.g., T7 or SP6) and the hepatitis delta virus ribozyme (HDVr) is usually assembled in a low-copy plasmid. In this approach, in vitro transcription is required to produce viral RNA that is transfected into susceptible cells to initiate viral replication and transcription in the cytoplasm of transfected cells. (B) Full-length infectious genomic cDNA clones: A full-length infectious genomic cDNA clone containing the viral genome flanked by a polymerase II-driven promoter from cytomegalovirus (CMV) and the HDVr followed by a polymerase II terminator and polyadenylation signal (pA) is assembled in a low-copy plasmid. In this case, the full-length cDNA clone is transcribed in the nucleus of transfected cells by the cellular RNA polymerase II. Primary transcripts are translocated to the cytoplasm where further amplifications steps are conducted by the viral polymerase. (C) Infectious Subgenomic Amplicons (ISA): The ISA approach can be used for the production of infectious viruses from genomic DNA material, including pre-existing infectious cDNA clones, viral RNA or de novo synthesized DNA genomic sequences. The entire viral genome is amplified by overlapping PCR reactions with each PCR product containing 30–40 base pairs overlapping regions [<a href="#B45-viruses-10-00597" class="html-bibr">45</a>]. The first and last PCR products are flanked by the CMV promoter and the HDVr followed by a polymerase II terminator and pA signal, respectively. Co-transfected cDNAs result in self-assembly in the cytoplasm of susceptible cells and virus production. Full article ">Figure 3
Schematic representation for the generation of rZIKV: Mammalian Vero cells are transiently transfected with infectious RNA transcribed in vitro from a full-length cDNA clone (top), full-length infectious cDNA clones (left) or infectious subgenomic amplicons (right) (see also <a href="#viruses-10-00597-f002" class="html-fig">Figure 2</a>). After 3–4 days post-transfection, when cytopathic effect is observed, rZIKVs present in tissue-culture supernatants can be recover for titration or viral amplification in mammalian (Vero, left) or insect (C6/36, right) cells. Full article ">Figure 4
ZIKV replicons. (A,B) Schematic diagram of Rluc-expressing ZIKV replicons: ZIKV replicons expressing Rluc constructed by Xie et al. [<a href="#B101-viruses-10-00597" class="html-bibr">101</a>] are shown. In these replicons, ZIKV structural proteins were replaced by the Rluc reporter gene followed by the FMDV 2A protease sequence (2A, yellow). This cassette was flanking by the N-terminal 38 amino acids of C protein (C38, dark gray) and the last 30 amino acids of E protein (E30, orange) fused in-frame with the viral downstream NS proteins (A). A stable reporter ZIKV Rluc-expressing replicon was constructed inserting the encephalomyocarditis virus internal ribosomal site (IRES, blue) followed by a neomycin resistance gene (Neo, pink) into the first 28 nucleotides of 3′ UTR (B). (C) Schematic representation of a PAC- and Rluc-expressing ZIKV replicon: The ZIKV replicon expressing a PAC and a Rluc genes separated by the FMDV 2A protease sequence is shown [<a href="#B105-viruses-10-00597" class="html-bibr">105</a>]. Both foreign viral genes, PAC and Rluc, followed by the FMDV 2A protease sequence were introduced between of the first N-terminal 38 amino acids of C protein (C38, dark gray) and the last 30 amino acids of E protein (E30, orange) in-frame with the viral NS1 protein. (D) Schematic diagram of a Gluc-expressing ZIKV replicon: The ZIKV replicon expressing the Gluc reporter gene constructed by Mutso et al. is indicated [<a href="#B48-viruses-10-00597" class="html-bibr">48</a>]. The Gluc reporter gene followed by the FMDV 2A protease sequence was introduced downstream of the C protein (dark gray) and the last N-terminal 30 amino acids of E protein (E30, orange) fused in-frame with the NS1 ZIKV protein. T7 and SP6: prokaryotic T7 or SP6 promoters. HDVr: Hepatitis delta virus ribozyme sequence. Full article ">Figure 5
Reporter gene-expressing rZIKVs. (A) Schematic diagram of a Rluc-expressing ZIKV cDNA clone: A full-length cDNA clone expressing the Rluc reporter gene (white box) was constructed by Shan et al. [<a href="#B46-viruses-10-00597" class="html-bibr">46</a>]. Rluc was introduced downstream of the first 25 amino acids of the C protein (C25, dark gray) followed by the FMDV 2A protease sequence (yellow box) fused in-frame with the viral ORF. Silent mutations changing the flavivirus-cyclization sequence were introduced in the full-length C protein (mut CS). (B) Schematic representation of a full-length ZIKV cDNA expressing Rluc or FP635: Full-length cDNA clones expressing Rluc (white box) or FP635 (red box) were generated by Münters et al. [<a href="#B50-viruses-10-00597" class="html-bibr">50</a>]. Reporter genes were introduced downstream the first 34 amino acid of the C protein (C34, dark gray) followed by the FMDV 2A protease sequence in-frame with the viral downstream proteins. (C) Schematic diagram of a GFP-expressing ZIKV generated by ISA: A ZIKV cDNA clone expressing GFP was constructed by Gadea et al. [<a href="#B45-viruses-10-00597" class="html-bibr">45</a>] using the ISA approach. The GFP was introduced downstream of the first 33 amino acid of the C protein (C33, dark gray) followed by the FMDV 2A protease sequence in-frame with the viral ORF. (D) Representation of full-length ZIKV cDNAs expressing luciferase and fluorescent proteins: Full-length ZIKV cDNA clones expressing NanoLuc, FfLuc, RSLuc (white boxes), GFP (green box) or mCherry (red box) were constructed by Mutso et al. [<a href="#B48-viruses-10-00597" class="html-bibr">48</a>]. Reporter genes were individually introduced downstream of the complete C protein followed by the FMDV 2A protease sequence fused in-frame with the downstream viral ORF. The codon sequence of the downstream copy of the viral C protein was altered to reduce potential recombination (dotted gray). T7 and SP6: prokaryotic T7 or SP6 promoters. CMV: cytomegalovirus promoter. HDVr: Hepatitis delta virus ribozyme sequence. pA: simian virus 40 late polyadenylation signal. Full article ">

30 pages, 2763 KiB

Open AccessReview

Research Models and Tools for the Identification of Antivirals and Therapeutics against Zika Virus Infection

by Marco P. Alves, Nathalie J. Vielle, Volker Thiel and Stephanie Pfaender

Viruses 2018, 10(11), 593; https://doi.org/10.3390/v10110593 - 30 Oct 2018

Cited by 15 | Viewed by 7565

Abstract

Zika virus recently re-emerged and caused global outbreaks mainly in Central Africa, Southeast Asia, the Pacific Islands and in Central and South America. Even though there is a declining trend, the virus continues to spread throughout different geographical regions of the world. Since [...] Read more.

Zika virus recently re-emerged and caused global outbreaks mainly in Central Africa, Southeast Asia, the Pacific Islands and in Central and South America. Even though there is a declining trend, the virus continues to spread throughout different geographical regions of the world. Since its re-emergence in 2015, massive advances have been made regarding our understanding of clinical manifestations, epidemiology, genetic diversity, genomic structure and potential therapeutic intervention strategies. Nevertheless, treatment remains a challenge as there is no licensed effective therapy available. This review focuses on the recent advances regarding research models, as well as available experimental tools that can be used for the identification and characterization of potential antiviral targets and therapeutic intervention strategies. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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20 pages, 1628 KiB

Open AccessReview

Ocular Manifestations of Emerging Flaviviruses and the Blood-Retinal Barrier

by Sneha Singh, Dustin Farr and Ashok Kumar

Viruses 2018, 10(10), 530; https://doi.org/10.3390/v10100530 - 28 Sep 2018

Cited by 55 | Viewed by 9208

Abstract

Despite flaviviruses remaining the leading cause of systemic human infections worldwide, ocular manifestations of these mosquito-transmitted viruses are considered relatively uncommon in part due to under-reporting. However, recent outbreaks of Zika virus (ZIKV) implicated in causing multiple ocular abnormalities, such as conjunctivitis, retinal [...] Read more.

Despite flaviviruses remaining the leading cause of systemic human infections worldwide, ocular manifestations of these mosquito-transmitted viruses are considered relatively uncommon in part due to under-reporting. However, recent outbreaks of Zika virus (ZIKV) implicated in causing multiple ocular abnormalities, such as conjunctivitis, retinal hemorrhages, chorioretinal atrophy, posterior uveitis, optic neuritis, and maculopathies, has rejuvenated a significant interest in understanding the pathogenesis of flaviviruses, including ZIKV, in the eye. In this review, first, we summarize the current knowledge of the major flaviviruses (Dengue, West Nile, Yellow Fever, and Japanese Encephalitis) reported to cause ocular manifestations in humans with emphasis on recent ZIKV outbreaks. Second, being an immune privilege organ, the eye is protected from systemic infections by the presence of blood-retinal barriers (BRB). Hence, we discuss how flaviviruses modulate retinal innate response and breach the protective BRB to cause ocular or retinal pathology. Finally, we describe recently identified infection signatures of ZIKV and discuss whether these system biology-predicted genes or signaling pathways (e.g., cellular metabolism) could contribute to the pathogenesis of ocular manifestations and assist in the development of ocular antiviral therapies against ZIKV and other flaviviruses. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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Figure 1
Eye anatomy and ocular complications caused by flaviviruses. Various components of the human eye are labelled in black. The flaviviruses responsible for causing ocular manifestations are shown in green whereas specific ocular tissue pathology is highlighted in red. Full article ">Figure 2
Flavivirus replication cycle and genome structure. (A) The flavivirus enters the host cell by attaching to specific receptors (1) which then leads to its endocytosis (2) followed by fusion to a lysosome into an acidic environment (3). The genome is released from the endolysosome (4) which is then translated on the Endoplasmic reticulum membrane (5) and post translational processing is done in the Golgi apparatus (6). The mature virus then buds off from the Golgi network (7) to the extracellular space via exocytosis (8, 9). (B) The genome consists of three structural proteins (Envelope (E), Capsid (C) and pre-membrane (prM)) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NSB, and NS5). Full article ">Figure 3
Probable mechanisms for the breach of blood-retinal barriers by ZIKV. Upon infection and peak viremia, there is an increased circulation of Zika virus, ZIKV NS1 protein, and immune cells in the blood (1). The virus in the retinal blood capillaries infect the endothelial lining (2) and the immune cells reach the site of infection/inflammation by diapedesis through the capillaries (3). It is followed by infection of RPE, the cell lining the outer BRB, resulting in chorioretinal atrophy. The viral infections might cause a BRB weakening by decreasing intercellular junction integrity. Being a neurotrophic virus, at later stages ZIKV can infect retinal Muller glia or neurons inside of the eye (4). The complications are worsened with the involvement of immune cells which get activated upon infection and release a “cytokine storm” as an antiviral response which damages the host cells by altering the barrier integrity (5). The virus along with the circulating immune cells can cross the inner BRB (retinal blood vessels) and infect neuronal cells such as ganglion cells (6, 7). The Image has been created with BioRender software. Full article ">

7 pages, 213 KiB

Open AccessReview

External Quality Assessment (EQA) for Molecular Diagnostics of Zika Virus: Experiences from an International EQA Programme, 2016–2018

by Oliver Donoso Mantke, Elaine McCulloch, Paul S. Wallace, Constanze Yue, Sally A. Baylis and Matthias Niedrig

Viruses 2018, 10(9), 491; https://doi.org/10.3390/v10090491 - 13 Sep 2018

Cited by 8 | Viewed by 4767

Abstract

Quality Control for Molecular Diagnostics (QCMD), an international provider for External Quality Assessment (EQA) programmes, has introduced a programme for molecular diagnostics of Zika virus (ZIKV) in 2016, which has been continuously offered to interested laboratories since that time. The EQA schemes provided [...] Read more.

Quality Control for Molecular Diagnostics (QCMD), an international provider for External Quality Assessment (EQA) programmes, has introduced a programme for molecular diagnostics of Zika virus (ZIKV) in 2016, which has been continuously offered to interested laboratories since that time. The EQA schemes provided from 2016 to 2018 revealed that 86.7% (92/106), 82.4% (89/108), and 88.2% (90/102) of the participating laboratories reported correct results for all samples, respectively in 2016, 2017, and 2018. The review of results indicated a need for improvement concerning analytical sensitivity and specificity of the test methods. Comparison with the outcomes of other EQA initiatives briefly summarized here show that continuous quality assurance is important to improve laboratory performance and to increase preparedness with reliable diagnostic assays for effective patient management, infection and outbreak control. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

18 pages, 1369 KiB

Open AccessReview

Host-Directed Antivirals: A Realistic Alternative to Fight Zika Virus

by Juan-Carlos Saiz, Nereida Jiménez de Oya, Ana-Belén Blázquez, Estela Escribano-Romero and Miguel A. Martín-Acebes

Viruses 2018, 10(9), 453; https://doi.org/10.3390/v10090453 - 24 Aug 2018

Cited by 39 | Viewed by 6443

Abstract

Zika virus (ZIKV), a mosquito-borne flavivirus, was an almost neglected pathogen until its introduction in the Americas in 2015, where it has been responsible for a threat to global health, causing a great social and sanitary alarm due to its increased virulence, rapid [...] Read more.

Zika virus (ZIKV), a mosquito-borne flavivirus, was an almost neglected pathogen until its introduction in the Americas in 2015, where it has been responsible for a threat to global health, causing a great social and sanitary alarm due to its increased virulence, rapid spread, and an association with severe neurological and ophthalmological complications. Currently, no specific antiviral therapy against ZIKV is available, and treatments are palliative and mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration. Nevertheless, lately, search for antivirals has been a major aim in ZIKV investigations. To do so, screening of libraries from different sources, testing of natural compounds, and repurposing of drugs with known antiviral activity have allowed the identification of several antiviral candidates directed to both viral (structural proteins and enzymes) and cellular elements. Here, we present an updated review of current knowledge about anti-ZIKV strategies, focusing on host-directed antivirals as a realistic alternative to combat ZIKV infection. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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26 pages, 1495 KiB

Open AccessReview

Probing Molecular Insights into Zika Virus–Host Interactions

by Ina Lee, Sandra Bos, Ge Li, Shusheng Wang, Gilles Gadea, Philippe Desprès and Richard Y. Zhao

Viruses 2018, 10(5), 233; https://doi.org/10.3390/v10050233 - 2 May 2018

Cited by 69 | Viewed by 9962

Abstract

The recent Zika virus (ZIKV) outbreak in the Americas surprised all of us because of its rapid spread and association with neurologic disorders including fetal microcephaly, brain and ocular anomalies, and Guillain–Barré syndrome. In response to this global health crisis, unprecedented and world-wide [...] Read more.

The recent Zika virus (ZIKV) outbreak in the Americas surprised all of us because of its rapid spread and association with neurologic disorders including fetal microcephaly, brain and ocular anomalies, and Guillain–Barré syndrome. In response to this global health crisis, unprecedented and world-wide efforts are taking place to study the ZIKV-related human diseases. Much has been learned about this virus in the areas of epidemiology, genetic diversity, protein structures, and clinical manifestations, such as consequences of ZIKV infection on fetal brain development. However, progress on understanding the molecular mechanism underlying ZIKV-associated neurologic disorders remains elusive. To date, we still lack a good understanding of; (1) what virologic factors are involved in the ZIKV-associated human diseases; (2) which ZIKV protein(s) contributes to the enhanced viral pathogenicity; and (3) how do the newly adapted and pandemic ZIKV strains alter their interactions with the host cells leading to neurologic defects? The goal of this review is to explore the molecular insights into the ZIKV–host interactions with an emphasis on host cell receptor usage for viral entry, cell innate immunity to ZIKV, and the ability of ZIKV to subvert antiviral responses and to cause cytopathic effects. We hope this literature review will inspire additional molecular studies focusing on ZIKV–host Interactions. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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15 pages, 2331 KiB

Open AccessReview

Zika Virus in the Male Reproductive Tract

by Liesel Stassen, Charles W. Armitage, David J. Van der Heide, Kenneth W. Beagley and Francesca D. Frentiu

Viruses 2018, 10(4), 198; https://doi.org/10.3390/v10040198 - 16 Apr 2018

Cited by 46 | Viewed by 9054

Abstract

Arthropod-borne viruses (arboviruses) are resurging across the globe. Zika virus (ZIKV) has caused significant concern in recent years because it can lead to congenital malformations in babies and Guillain-Barré syndrome in adults. Unlike other arboviruses, ZIKV can be sexually transmitted and may persist [...] Read more.

Arthropod-borne viruses (arboviruses) are resurging across the globe. Zika virus (ZIKV) has caused significant concern in recent years because it can lead to congenital malformations in babies and Guillain-Barré syndrome in adults. Unlike other arboviruses, ZIKV can be sexually transmitted and may persist in the male reproductive tract. There is limited information regarding the impact of ZIKV on male reproductive health and fertility. Understanding the mechanisms that underlie persistent ZIKV infections in men is critical to developing effective vaccines and therapies. Mouse and macaque models have begun to unravel the pathogenesis of ZIKV infection in the male reproductive tract, with the testes and prostate gland implicated as potential reservoirs for persistent ZIKV infection. Here, we summarize current knowledge regarding the pathogenesis of ZIKV in the male reproductive tract, the development of animal models to study ZIKV infection at this site, and prospects for vaccines and therapeutics against persistent ZIKV infection. Full article

(This article belongs to the Special Issue New Advances on Zika Virus Research)

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