Identification of Novel Natural Products as Effective and Broad-Spectrum Anti-Zika Virus Inhibitors

Zika virus (ZIKV) infection during pregnancy leads to severe congenital Zika syndrome, which includes microcephaly and other neurological malformations. No therapeutic agents have, so far, been approved for the treatment of ZIKV infection in humans; as such, there is a need for a continuous effort to develop effective and safe antiviral drugs to treat ZIKV-caused diseases. After screening a natural product library, we have herein identified four natural products with anti-ZIKV activity in Vero E6 cells, including gossypol, curcumin, digitonin, and conessine. Except for curcumin, the other three natural products have not been reported before to have anti-ZIKV activity. Among them, gossypol exhibited the strongest inhibitory activity against almost all 10 ZIKV strains tested, including six recent epidemic human strains. The mechanistic study indicated that gossypol could neutralize ZIKV infection by targeting the envelope protein domain III (EDIII) of ZIKV. In contrast, the other natural products inhibited ZIKV infection by targeting the host cell or cell-associated entry and replication stages of ZIKV. A combination of gossypol with any of the three natural products identified in this study, as well as with bortezomib, a previously reported anti-ZIKV compound, exhibited significant combinatorial inhibitory effects against three ZIKV human strains tested. Importantly, gossypol also demonstrated marked potency against all four serotypes of dengue virus (DENV) human strains in vitro. Taken together, this study indicates the potential for further development of these natural products, particularly gossypol, as the lead compound or broad-spectrum inhibitors against ZIKV and other flaviviruses, such as DENV.

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Immunogenicity and Efficacy of Zika Virus Envelope Domain III in DNA, Protein, and ChAdOx1 Adenoviral-Vectored Vaccines

Vaccines ◽

10.3390/vaccines8020307 ◽

2020 ◽

Vol 8 (2) ◽

pp. 307 ◽

Cited By ~ 3

Author(s):

César López-Camacho ◽

Giuditta De Lorenzo ◽

Jose Luis Slon-Campos ◽

Stuart Dowall ◽

Peter Abbink ◽

...

Keyword(s):

Immune Responses ◽

Dengue Virus ◽

Zika Virus ◽

Neutralizing Antibodies ◽

Vaccine Candidate ◽

Protein Domain ◽

Virus Envelope ◽

Zikv Infection ◽

Domain Iii ◽

Open Question

The flavivirus envelope protein domain III (EDIII) was an effective immunogen against dengue virus (DENV) and other related flaviviruses. Whether this can be applied to the Zika virus (ZIKV) vaccinology remains an open question. Here, we tested the efficacy of ZIKV-EDIII against ZIKV infection, using several vaccine platforms that present the antigen in various ways. We provide data demonstrating that mice vaccinated with a ZIKV-EDIII as DNA or protein-based vaccines failed to raise fully neutralizing antibodies and did not control viremia, following a ZIKV challenge, despite eliciting robust antibody responses. Furthermore, we showed that ZIKV-EDIII encoded in replication-deficient Chimpanzee adenovirus (ChAdOx1-EDIII) elicited anti-ZIKV envelope antibodies in vaccinated mice but also provided limited protection against ZIKV in two physiologically different mouse challenge models. Taken together, our data indicate that contrary to what was shown for other flaviviruses like the dengue virus, which has close similarities with ZIKV-EDIII, this antigen might not be a suitable vaccine candidate for the correct induction of protective immune responses against ZIKV.

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A combination of two human monoclonal antibodies limits fetal damage by Zika virus in macaques

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2000414117 ◽

2020 ◽

Vol 117 (14) ◽

pp. 7981-7989 ◽

Cited By ~ 4

Author(s):

Koen K. A. Van Rompay ◽

Lark L. Coffey ◽

Tania Kapoor ◽

Anna Gazumyan ◽

Rebekah I. Keesler ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Vertical Transmission ◽

Zika Virus ◽

Rhesus Macaques ◽

Passive Immunization ◽

Human Infection ◽

Zikv Infection ◽

Domain Iii ◽

Harmful Effects

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement (ADE) in vitro and extend their half-lives. Here we report on prophylactic coadministration of the Fc-modified antibodies to pregnant rhesus macaques challenged three times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission, protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV.

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Zika Virus Infection Leads to Demyelination and Axonal Injury in Mature CNS Cultures

Viruses ◽

10.3390/v13010091 ◽

2021 ◽

Vol 13 (1) ◽

pp. 91

Author(s):

Verena Schultz ◽

Stephanie L. Cumberworth ◽

Quan Gu ◽

Natasha Johnson ◽

Claire L. Donald ◽

...

Keyword(s):

Nervous System ◽

Neural Development ◽

Zika Virus ◽

Developmental Stages ◽

Cell Types ◽

Neural Cells ◽

Zikv Infection ◽

Axonal Pathology ◽

Time Frames

Understanding how Zika virus (Flaviviridae; ZIKV) affects neural cells is paramount in comprehending pathologies associated with infection. Whilst the effects of ZIKV in neural development are well documented, impact on the adult nervous system remains obscure. Here, we investigated the effects of ZIKV infection in established mature myelinated central nervous system (CNS) cultures. Infection incurred damage to myelinated fibers, with ZIKV-positive cells appearing when myelin damage was first detected as well as axonal pathology, suggesting the latter was a consequence of oligodendroglia infection. Transcriptome analysis revealed host factors that were upregulated during ZIKV infection. One such factor, CCL5, was validated in vitro as inhibiting myelination. Transferred UV-inactivated media from infected cultures did not damage myelin and axons, suggesting that viral replication is necessary to induce the observed effects. These data show that ZIKV infection affects CNS cells even after myelination—which is critical for saltatory conduction and neuronal function—has taken place. Understanding the targets of this virus across developmental stages including the mature CNS, and the subsequent effects of infection of cell types, is necessary to understand effective time frames for therapeutic intervention.

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The epidermal growth factor receptor is a relevant host factor in the early stages of Zika virus life cycle in vitro

Journal of Virology ◽

10.1128/jvi.01195-21 ◽

2021 ◽

Author(s):

Catarina Sabino ◽

Daniela Bender ◽

Marie-Luise Herrlein ◽

Eberhard Hildt

Keyword(s):

Life Cycle ◽

Zika Virus ◽

A549 Cells ◽

Growth Factor Receptor ◽

Viral Life Cycle ◽

Infection Process ◽

Zikv Infection ◽

Early Stages ◽

Egfr Expression

Zika virus (ZIKV) is a flavivirus well-known for the epidemic in the Americas in 2015-2016, where microcephaly in newborns and other neurological complications were connected to ZIKV infection. Many aspects of the viral life cycle, including binding and entry into the host cell, are still enigmatic. Based on the observation that CHO cells lack the expression of EGFR and are not permissive for various ZIKV strains, the relevance of EGFR for the viral life cycle was analyzed. Infection of A549 cells by ZIKV leads to a rapid internalization of EGFR that colocalizes with the endosomal marker EEA1. Moreover, the infection by different ZIKV strains is associated with an activation of EGFR and subsequent activation of the MAPK/ERK signaling cascade. However, treatment of the cells with MβCD, which on the one hand leads to an activation of EGFR but on the other hand prevents EGFR internalization, impairs ZIKV infection. Specific inhibition of EGFR or of the RAS-RAF-MEK-ERK signal transduction cascade hinders ZIKV infection by inhibition of ZIKV entry. In accordance to this, knockout of EGFR expression impedes ZIKV entry. In case of an already established infection, inhibition of EGFR or of downstream signaling does not affect viral replication. Taken together, these data demonstrate the relevance of EGFR in the early stages of ZIKV infection and identify EGFR as a target for antiviral strategies. Importance These data deepen the knowledge about the ZIKV infection process and demonstrate the relevance of EGFR for ZIKV entry. In light of the fact that a variety of specific and efficient inhibitors of EGFR and of EGFR-dependent signaling were developed and licensed, repurposing of these substances could be a helpful tool to prevent the spreading of ZIKV infection in an epidemic outbreak.

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Autophagy Contributes to Host Immunity and Protection against Zika Virus Infection via Type I IFN Signaling

Mediators of Inflammation ◽

10.1155/2020/9527147 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15 ◽

Cited By ~ 1

Author(s):

Yuyi Huang ◽

Yujie Wang ◽

Shuhui Meng ◽

Zhuohang Chen ◽

Haifan Kong ◽

...

Keyword(s):

Virus Infection ◽

Cell Line ◽

Zika Virus ◽

Fetal Brain ◽

Host Immunity ◽

Type I ◽

Type I Ifn ◽

Zikv Infection

Recent studies have indicated that the Zika virus (ZIKV) has a significant impact on the fetal brain, and autophagy is contributing to host immune response and defense against virus infection. Here, we demonstrate that ZIKV infection triggered increased LC3 punctuation in mouse monocyte-macrophage cell line (RAW264.7), mouse microglial cell line (BV2), and hindbrain tissues, proving the occurrence of autophagy both in vitro and in vivo. Interestingly, manual intervention of autophagy, like deficiency inhibited by 3-MA, can reduce viral clearance in RAW264.7 cells upon ZIKV infection. Besides, specific siRNA strategy confirmed that autophagy can be activated through Atg7-Atg5 and type I IFN signaling pathway upon ZIKV infection, while knocking down of Atg7 and Atg5 effectively decreased the ZIKV clearance in phagocytes. Furthermore, we analyzed that type I IFN signaling could contribute to autophagic clearance of invaded ZIKV in phagocytes. Taken together, our findings demonstrate that ZIKV-induced autophagy is favorable to activate host immunity, particularly through type I IFN signaling, which participates in host protection and defense against ZIKV infection.

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Zika Virus-Derived E-DIII Protein Displayed on Immunologically Optimized VLPs Induces Neutralizing Antibodies without Causing Enhancement of Dengue Virus Infection

Vaccines ◽

10.3390/vaccines7030072 ◽

2019 ◽

Vol 7 (3) ◽

pp. 72 ◽

Cited By ~ 11

Author(s):

Gustavo Cabral-Miranda ◽

Stephanie M. Lim ◽

Mona O. Mohsen ◽

Ilya V. Pobelov ◽

Elisa S. Roesti ◽

...

Keyword(s):

Dengue Virus ◽

Zika Virus ◽

Neutralizing Antibodies ◽

Clinical Manifestations ◽

First Trimester ◽

Neurological Complications ◽

Zikv Infection ◽

First Trimester Of Pregnancy ◽

Specific Igg

Zika virus (ZIKV) is a flavivirus similar to Dengue virus (DENV) in terms of transmission and clinical manifestations, and usually both viruses are found to co-circulate. ZIKV is usually transmitted by mosquitoes bites, but may also be transmitted by blood transfusion, via the maternal–foetal route, and sexually. After 2015, when the most extensive outbreak of ZIKV had occurred in Brazil and subsequently spread throughout the rest of South America, it became evident that ZIKV infection during the first trimester of pregnancy was associated with microcephaly and other neurological complications in newborns. As a result, the development of a vaccine against ZIKV became an urgent goal. A major issue with DENV vaccines, and therefore likely also with ZIKV vaccines, is the induction of antibodies that fail to neutralize the virus properly and cause antibody-dependent enhancement (ADE) of the infection instead. It has previously been shown that antibodies against the third domain of the envelope protein (EDIII) induces optimally neutralizing antibodies with no evidence for ADE for other viral strains. Therefore, we generated a ZIKV vaccine based on the EDIII domain displayed on the immunologically optimized Cucumber mosaic virus (CuMVtt) derived virus-like particles (VLPs) formulated in dioleoyl phosphatidylserine (DOPS) as adjuvant. The vaccine induced high levels of specific IgG after a single injection. The antibodies were able to neutralise ZIKV without enhancing infection by DENV in vitro. Thus, the here described vaccine based on EDIII displayed on VLPs was able to stimulate production of antibodies specifically neutralizing ZIKV without potentially enhancing disease caused by DENV.

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Tryptophan Trimers and Tetramers Inhibit Dengue and Zika Virus Replication by Interfering with Viral Attachment Processes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02130-19 ◽

2020 ◽

Vol 64 (3) ◽

Author(s):

Antonios Fikatas ◽

Peter Vervaeke ◽

Belén Martínez-Gualda ◽

Olaia Martí-Marí ◽

Sam Noppen ◽

...

Keyword(s):

Antiviral Activity ◽

Zika Virus ◽

Antiviral Agent ◽

Viral Envelope ◽

Pharmacokinetic Studies ◽

Host Cell Membrane ◽

Viral Attachment ◽

Virus Attachment ◽

Domain Iii

ABSTRACT Here, we report a class of tryptophan trimers and tetramers that inhibit (at low micromolar range) dengue and Zika virus infection in vitro. These compounds (AL family) have three or four peripheral tryptophan moieties directly linked to a central scaffold through their amino groups; thus, their carboxylic acid groups are free and exposed to the periphery. Structure-activity relationship (SAR) studies demonstrated that the presence of extra phenyl rings with substituents other than COOH at the N1 or C2 position of the indole side chain is a requisite for the antiviral activity against both viruses. The molecules showed potent antiviral activity, with low cytotoxicity, when evaluated on different cell lines. Moreover, they were active against laboratory and clinical strains of all four serotypes of dengue virus as well as a selected group of Zika virus strains. Additional mechanistic studies performed with the two most potent compounds (AL439 and AL440) demonstrated an interaction with the viral envelope glycoprotein (domain III) of dengue 2 virus, preventing virus attachment to the host cell membrane. Since no antiviral agent is approved at the moment against these two flaviviruses, further pharmacokinetic studies with these molecules are needed for their development as future therapeutic/prophylactic drugs.

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Embryotoxic impact of Zika virus in a rhesus macaque in vitro implantation model†

Biology of Reproduction ◽

10.1093/biolre/ioz236 ◽

2020 ◽

Vol 102 (4) ◽

pp. 806-816 ◽

Cited By ~ 4

Author(s):

Lindsey N Block ◽

Matthew T Aliota ◽

Thomas C Friedrich ◽

Michele L Schotzko ◽

Katherine D Mean ◽

...

Keyword(s):

Rhesus Macaque ◽

Pregnancy Outcomes ◽

Zika Virus ◽

Negative Impact ◽

Culture Media ◽

Blastocyst Formation ◽

Placental Development ◽

Zikv Infection ◽

The Impact

Abstract Zika virus (ZIKV) infection is associated with adverse pregnancy outcomes in humans, and infection in the first trimester can lead to miscarriage and stillbirth. Vertical and sexual transmissions of ZIKV have been demonstrated, yet the impact of infection during the initial stages of pregnancy remains unexplored. Here we defined the impact of ZIKV on early embryonic and placental development with a rhesus macaque model. During in vitro fertilization (IVF), macaque gametes were inoculated with a physiologically relevant dose of 5.48log10 plaque-forming units (PFU) of Zika virus/H.sapiens-tc/PUR/2015/PRVABC59_v3c2. Exposure at fertilization did not alter blastocyst formation rates compared to controls. To determine the impact of ZIKV exposure at implantation, hatched blastocysts were incubated with 3.26log10, 4.26log10, or 5.26log10 PFU, or not exposed to ZIKV, followed by extended embryo culture for 10 days. ZIKV exposure negatively impacted attachment, growth, and survival in comparison to controls, with exposure to 5.26log10 PFU ZIKV resulting in embryonic degeneration by day 2. Embryonic secretion of pregnancy hormones was lower in ZIKV-exposed embryos. Increasing levels of infectious virus were detected in the culture media post-exposure, suggesting that the trophectoderm is susceptible to productive ZIKV infection. These results demonstrate that ZIKV exposure severely impacts the zona-free blastocyst, whereas exposure at the time of fertilization does not hinder blastocyst formation. Overall, early stages of pregnancy may be profoundly sensitive to infection and pregnancy loss, and the negative impact of ZIKV infection on pregnancy outcomes may be underestimated.

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Dengue virus envelope protein domain III-elicited antibodies mediate cross-protection against Zika virus in a mouse model

Virus Research ◽

10.1016/j.virusres.2020.197882 ◽

2020 ◽

Vol 278 ◽

pp. 197882

Author(s):

Yongchao Zhou ◽

Dong Chen ◽

Lan Yang ◽

Weiwei Zou ◽

Zhiliang Duan ◽

...

Keyword(s):

Mouse Model ◽

Dengue Virus ◽

Zika Virus ◽

Envelope Protein ◽

Cross Protection ◽

Protein Domain ◽

Virus Envelope ◽

Virus Envelope Protein ◽

Domain Iii

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N-Methyl-d-Aspartate (NMDA) Receptor Blockade Prevents Neuronal Death Induced by Zika Virus Infection

mBio ◽

10.1128/mbio.00350-17 ◽

2017 ◽

Vol 8 (2) ◽

Cited By ~ 34

Author(s):

Vivian V. Costa ◽

Juliana L. Del Sarto ◽

Rebeca F. Rocha ◽

Flavia R. Silva ◽

Juliana G. Doria ◽

...

Keyword(s):

Global Health ◽

Drug Treatment ◽

Zika Virus ◽

Neuronal Death ◽

Neuronal Damage ◽

Neurological Complications ◽

Zikv Infection ◽

Experimental Animals

ABSTRACT Zika virus (ZIKV) infection is a global health emergency that causes significant neurodegeneration. Neurodegenerative processes may be exacerbated by N-methyl-d-aspartate receptor (NMDAR)-dependent neuronal excitoxicity. Here, we have exploited the hypothesis that ZIKV-induced neurodegeneration can be rescued by blocking NMDA overstimulation with memantine. Our results show that ZIKV actively replicates in primary neurons and that virus replication is directly associated with massive neuronal cell death. Interestingly, treatment with memantine or other NMDAR blockers, including dizocilpine (MK-801), agmatine sulfate, or ifenprodil, prevents neuronal death without interfering with the ability of ZIKV to replicate in these cells. Moreover, in vivo experiments demonstrate that therapeutic memantine treatment prevents the increase of intraocular pressure (IOP) induced by infection and massively reduces neurodegeneration and microgliosis in the brain of infected mice. Our results indicate that the blockade of NMDARs by memantine provides potent neuroprotective effects against ZIKV-induced neuronal damage, suggesting it could be a viable treatment for patients at risk for ZIKV infection-induced neurodegeneration. IMPORTANCE Zika virus (ZIKV) infection is a global health emergency associated with serious neurological complications, including microcephaly and Guillain-Barré syndrome. Infection of experimental animals with ZIKV causes significant neuronal damage and microgliosis. Treatment with drugs that block NMDARs prevented neuronal damage both in vitro and in vivo. These results suggest that overactivation of NMDARs contributes significantly to the neuronal damage induced by ZIKV infection, and this is amenable to inhibition by drug treatment. IMPORTANCE Zika virus (ZIKV) infection is a global health emergency associated with serious neurological complications, including microcephaly and Guillain-Barré syndrome. Infection of experimental animals with ZIKV causes significant neuronal damage and microgliosis. Treatment with drugs that block NMDARs prevented neuronal damage both in vitro and in vivo. These results suggest that overactivation of NMDARs contributes significantly to the neuronal damage induced by ZIKV infection, and this is amenable to inhibition by drug treatment.

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