Impact of prior Dengue immunity on Zika vaccine protection in rhesus macaques and mice

Pre-existing immunity to flaviviruses can influence the outcome of subsequent flavivirus infections. Therefore, it is critical to determine whether baseline DENV immunity may influence subsequent ZIKV infection and the protective efficacy of ZIKV vaccines. In this study, we investigated the impact of pre-existing DENV immunity induced by vaccination on ZIKV infection and the protective efficacy of an inactivated ZIKV vaccine. Rhesus macaques and mice inoculated with a live attenuated DENV vaccine developed neutralizing antibodies (NAbs) to multiple DENV serotypes but no cross-reactive NAbs responses to ZIKV. Animals with baseline DENV NAbs did not exhibit enhanced ZIKV infection and showed no overall reduction in ZIKV vaccine protection. Moreover, passive transfer of purified DENV-specific IgG from convalescent human donors did not augment ZIKV infection in STAT2 ^-/- and BALB/c mice. In summary, these results suggest that baseline DENV immunity induced by vaccination does not significantly enhance ZIKV infection or impair the protective efficacy of candidate ZIKV vaccines in these models. These data can help inform immunization strategies in regions of the world with multiple circulating pathogenic flaviviruses.     

Carnosine exhibits significant antiviral activity against Dengue and Zika virus

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses transmitted to humans by their common vector, Aedes mosquitoes. DENV infection represents one of the most widely spread mosquito‐borne diseases whereas ZIKV infection occasionally re‐emerged in the past causing outbreaks. Although there have been considerable advances in understanding the pathophysiology of these viruses, no effective vaccines or antiviral drugs are currently available. In this study, we evaluated the antiviral activity of carnosine, an endogenous dipeptide (β‐alanyl‐l ‐histidine), against DENV serotype 2 (DENV2) and ZIKV infection in human liver cells (Huh7). Computational studies were performed to predict the potential interactions between carnosine and viral proteins. Biochemical and cell‐based assays were performed to validate the computational results. Mode‐of‐inhibition, plaque reduction, and immunostaining assays were performed to determine the antiviral activity of carnosine. Exogenous carnosine showed minimal cytotoxicity in Huh7 cells and rescued the viability of infected cells with EC₅₀ values of 52.3 and 59.5 μM for DENV2 and ZIKV infection, respectively. Based on the mode‐of‐inhibition assays, carnosine inhibited DENV2 mainly by inhibiting viral genome replication and interfering with virus entry. Carnosine antiviral activity was verified with immunostaining assay where carnosine treatment diminished viral fluorescence signal. In conclusion, carnosine exhibited significant inhibitory effects against DENV2 and ZIKV replication in human liver cells and could be utilized as a lead peptide for the development of effective and safe antiviral agents against DENV and ZIKV.     

An Integrated Systems Biology Approach Identifies the Proteasome as A Critical Host Machinery for ZIKV and DENV Replication

The Zika virus (ZIKV) and dengue virus (DENV) flaviviruses exhibit similar replicative processes but have distinct clinical outcomes. A systematic understanding of virus–host protein–pro-tein interaction networks can reveal cellular pathways critical to viral replication and disease patho-genesis. Here we employed three independent systems biology approaches toward this goal. First, protein array analysis of direct interactions between individual ZIKV/DENV viral proteins and 20,240 human proteins revealed multiple conserved cellular pathways and protein complexes, including proteasome complexes. Second, an RNAi screen of 10,415 druggable genes identified the host proteins required for ZIKV infection and uncovered that proteasome proteins were crucial in this process. Third, high-throughput screening of 6016 bioactive compounds for ZIKV inhibition yielded 134 effective compounds, including six proteasome inhibitors that suppress both ZIKV and DENV replication. Integrative analyses of these orthogonal datasets pinpoint proteasomes as crit-ical host machinery for ZIKV/DENV replication. Our study provides multi-omics datasets for fur-ther studies of flavivirus–host interactions, disease pathogenesis, and new drug targets.     

Transplacental Zika virus transmission in ex vivo perfused human placentas

A Zika virus (ZIKV) infection during pregnancy can result in severe birth defects such as microcephaly. To date, it is incompletely understood how ZIKV can cross the human placenta. Furthermore, results from studies in pregnant mice and non-human primates are conflicting regarding the role of cross-reactive dengue virus (DENV) antibodies on transplacental ZIKV transmission. Elucidating how ZIKV can cross the placenta and which risk factors contribute to this is important for risk assessment and for potential intervention strategies for transplacental ZIKV transmission. In this study we use an ex vivo human placental perfusion model to study transplacental ZIKV transmission and the effect that cross-reactive DENV antibodies have on this transmission. By using this model, we demonstrate that DENV antibodies significantly increase ZIKV uptake in perfused human placentas and that this increased uptake is neonatal Fc-receptor-dependent. Furthermore, we show that cross-reactive DENV antibodies enhance ZIKV infection in term human placental explants and in primary fetal macrophages but not in primary trophoblasts. Our data supports the hypothesis that presence of cross-reactive DENV antibodies could be an important risk factor for transplacental ZIKV transmission. Furthermore, we demonstrate that the ex vivo placental perfusion model is a relevant and animal friendly model to study transplacental pathogen transmission.     

Diagnostic differentiation of Zika and dengue virus exposure by analyzing T cell receptor sequences from peripheral blood of infected HLA-A2 transgenic mice

Zika virus (ZIKV) is a significant global health threat due to its potential for rapid emergence and association with severe congenital malformations during infection in pregnancy. Despite the urgent need, accurate diagnosis of ZIKV infection is still a major hurdle that must be overcome. Contributing to the inaccuracy of most serologically-based diagnostic assays for ZIKV, is the substantial geographic and antigenic overlap with other flaviviruses, including the four serotypes of dengue virus (DENV). Within this study, we have utilized a novel T cell receptor (TCR) sequencing platform to distinguish between ZIKV and DENV infections. Using high-throughput TCR sequencing of lymphocytes isolated from DENV and ZIKV infected mice, we were able to develop an algorithm which could identify virus-associated TCR sequences uniquely associated with either a prior ZIKV or DENV infection in mice. Using this algorithm, we were then able to separate mice that had been exposed to ZIKV or DENV infection with 97% accuracy. Overall this study serves as a proof-of-principle that T cell receptor sequencing can be used as a diagnostic tool capable of distinguishing between closely related viruses. Our results demonstrate the potential for this innovative platform to be used to accurately diagnose Zika virus infection and potentially the next emerging pathogen(s).     

Dengue and Zika virus multi-epitope antigen expression in insect cells

Dengue virus and Zika virus are arthropod-borne flaviviruses that cause millions of infections worldwide. The co-circulation of both viruses makes serological diagnosis difficult as they share high amino acid similarities in viral proteins. Antigens are one of the key reagents in the differential diagnosis of these viruses through the detection of IgG antibodies in serological assays during the convalescent-phase of infections. Here, we report the expression of Dengue virus (DENV) and Zika virus (ZIKV) antigens containing non-conserved and immunodominant amino acid sequences using the baculovirus expression vector system in insect cells. We designed DENV and ZIKV antigens based on the domain III of the E protein (EDIII) after analyzing previously reported epitopes and by multiple alignment of the most important flaviviruses. The ZIKV and DENV multi-epitope genes were designed as tandem repeats or impaired repeats separated by tetra- or hexa-glycine linkers. The biochemical analyses revealed adequate expression of the antigens. Then, the obtained multi-epitope antigens were semi-purified in a sucrose gradient and tested using patients’ sera collected during the convalescent-phase that were previously diagnosed positive for anti-DENV and -ZIKV IgG antibodies. The optimal serum dilution was 1:200, and the mean absorbance values in the preliminary tests show that multi-epitope antigens have been recognized by human sera. The production of both antigens using the multi-epitope strategy in the eukaryotic system and based on the EDIII regions provide a proof of concept for the use of antigens in the differentiation between DENV and ZIKV.

     

Identifying crucial E-protein residues responsible for unusual stability of Zika virus envelope

An outbreak of Zika virus (ZIKV) infections in 2015–16 that caused microcephaly and other congenital abnormalities in newborns prompted intense research across the globe. These studies have suggested that ZIKV can survive high temperatures and harsh physiological conditions, unlike the other flaviviruses such as dengue virus (DENV). In contrast, recent cryo-electron microscopy studies have shown very similar architecture of the ZIKV and DENV envelopes that constitute the primary level of viral protection. Encouraged by these findings, here we attempt to identify the crucial protein residues that make the ZIKV envelope so robust by employing coarse-grained and all-atomic molecular dynamics simulations and computational mutagenesis studies. In accordance with more recent cryo-electron microscopy findings, our simulation results exhibited stable ZIKV envelope protein shell both at 29^oC and 40°C, whereas the DENV2 shell loosened up significantly at 40°C. Subsequently, we simulated a series of ZIKV variants to identify the specific domain and residues involved in maintaining the structural integrity of the viral protein shell at high temperatures. Our results suggest that the DIII domain—more specifically, the CD- and FG-loop residues of the ZIKV protein shell—play a crucial role in making the virus envelope thermostable by inducing strong raft-raft interactions. These findings can accelerate the rational design of ZIKV therapeutics.     

Protein Kinase PKR Mediates the Apoptosis Induction and Growth Restriction Phenotypes of C Protein-Deficient Measles Virus

The measles virus (MV) accessory proteins V and C play important roles in MV replication and pathogenesis. Infection with recombinant MV lacking either V or C causes more cell death than infection with the parental vaccine-equivalent virus (MVvac), and C-deficient virus grows poorly relative to the parental virus. Here, we show that a major effector of the C phenotype is the RNA-dependent protein kinase PKR. Using human HeLa cells stably deficient in PKR as a result of RNA interference-mediated knockdown (PKR^kd cells), we demonstrated that a reduction in PKR partially rescued the growth defect of C knockout (C^ko) virus but had no effect on the growth of either wild-type (WT) or V knockout (V^ko) virus. Increased growth of the C^ko virus in PKR^kd cells correlated with increased viral protein expression, while defective growth and decreased protein expression in PKR-sufficient cells correlated with increased phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2. Furthermore, infection with WT, V^ko, or especially C^ko virus caused significantly less apoptosis in PKR^kd cells than in PKR-sufficient cells. Although apoptosis induced by C^ko virus infection in PKR-sufficient cells was blocked by a caspase antagonist, the growth of C^ko virus was not restored to the WT level by treatment with this pharmacologic inhibitor. Taken together, these results indicate that PKR plays an important antiviral role during MV infection but that the virus growth restriction by PKR is not dependent upon the induction of apoptosis. Furthermore, the results establish that a principal function of the MV C protein is to antagonize the proapoptotic and antiviral activities of PKR.     

Diagnostic performance of anti-Zika virus IgM,IgAM and IgG ELISAs during co-circulation of Zika,dengue, and chikungunya viruses in Brazil and Venezuela

BackgroundSerological diagnosis of Zika virus (ZIKV) infection is challenging because of the antibody cross-reactivity among flaviviruses. At the same time, the role of Nucleic Acid Testing (NAT) is limited by the low proportion of symptomatic infections and the low average viral load. Here, we compared the diagnostic performance of commercially available IgM, IgAM, and IgG ELISAs in sequential samples during the ZIKV and chikungunya (CHIKV) epidemics and co-circulation of dengue virus (DENV) in Brazil and Venezuela.Methodology/Principal findingsAcute (day of illness 1–5) and follow-up (day of illness ≥ 6) blood samples were collected from nine hundred and seven symptomatic patients enrolled in a prospective multicenter study between June 2012 and August 2016. Acute samples were tested by RT-PCR for ZIKV, DENV, and CHIKV. Acute and follow-up samples were tested for IgM, IgAM, and IgG antibodies to ZIKV using commercially available ELISAs. Among follow-up samples with a RT-PCR confirmed ZIKV infection, anti-ZIKV IgAM sensitivity was 93.5% (43/46), while IgM and IgG exhibited sensitivities of 30.3% (10/33) and 72% (18/25), respectively. An additional 24% (26/109) of ZIKV infections were detected via IgAM seroconversion in ZIKV/DENV/CHIKV RT-PCR negative patients. The specificity of anti-ZIKV IgM was estimated at 93% and that of IgAM at 85%.Conclusions/SignificanceOur findings exemplify the challenges of the assessment of test performance for ZIKV serological tests in the real-world setting, during co-circulation of DENV, ZIKV, and CHIKV. However, we can also demonstrate that the IgAM immunoassay exhibits superior sensitivity to detect ZIKV RT-PCR confirmed infections compared to IgG and IgM immunoassays. The IgAM assay also proves to be promising for detection of anti-ZIKV seroconversions in sequential samples, both in ZIKV PCR-positive as well as PCR-negative patients, making this a candidate assay for serological monitoring of pregnant women in future ZIKV outbreaks.     

Congenital abnormalities associated with Zika virus infection–Dengue as potential co-factor? A systematic review

Zika virus (ZIKV) emerged in Brazil during 2013–2014 causing an epidemic of previously unknown congenital abnormalities. The frequency of severe congenital abnormalities after maternal ZIKV infection revealed an unexplained geographic variability, especially between the Northeast and the rest of Brazil. Several reasons for this variability have been discussed. Prior immunity against Dengue virus (DENV) affecting ZIKV seems to be the most likely explanation. Here we summarise the current evidence regarding this prominent co-factor to potentially explain the geographic variability.This systematic review followed the PRISMA guidelines. The search was conducted up to May 15th, 2020, focussing on immunological interactions from Zika virus with previous Dengue virus infections as potential teratogenic effect for the foetus.Eight out of 339 screened studies reported on the association between ZIKV, prior DENV infection and microcephaly, mostly focusing on antibody-dependent enhancement (ADE) as potential pathomechanism. Prior DENV infection was associated with enhancement for ZIKV infection and increased neurovirulence in one included in vitro study only. Interestingly, the seven in vivo studies exhibited a heterogeneous picture with three studies showing a protective effect of prior DENV infections and others no effect at all. According to several studies, socio-economic factors are associated with increased risk for microcephaly.Very few studies addressed the question of unexplained variability of infection-related microcephaly. Many studies focussed on ADE as mechanism without measuring microcephaly as endpoint. Interestingly, three of the included studies reported a protective effect of prior DENV infection against microcephaly. This systematic review strengthens the hypothesis that immune priming after recent DENV infection is the crucial factor for determining protection or enhancement activity. It is of high importance that the currently ongoing prospective studies include a harmonised assessment of the potential candidate co-factors.     

Previous exposure to dengue virus is associated with increased Zika virus burden at the maternal-fetal interface in rhesus macaques

Concerns have arisen that pre-existing immunity to dengue virus (DENV) could enhance Zika virus (ZIKV) disease, due to the homology between ZIKV and DENV and the observation of antibody-dependent enhancement (ADE) among DENV serotypes. To date, no study has examined the impact of pre-existing DENV immunity on ZIKV pathogenesis during pregnancy in a translational non-human primate model. Here we show that macaques with a prior DENV-2 exposure had a higher burden of ZIKV vRNA in maternal-fetal interface tissues as compared to DENV-naive macaques. However, pre-existing DENV immunity had no detectable impact on ZIKV replication kinetics in maternal plasma, and all pregnancies progressed to term without adverse outcomes or gross fetal abnormalities detectable at delivery. Understanding the risks of ADE to pregnant women worldwide is critical as vaccines against DENV and ZIKV are developed and licensed and as DENV and ZIKV continue to circulate.     

Preventing Zika Virus Infection during Pregnancy Using a Seasonal Window of Opportunity for Conception

It has come to light that Zika virus (ZIKV) infection during pregnancy can result in trans-placental transmission to the fetus along with fetal death, congenital microcephaly, and/or Central Nervous System (CNS) malformations. There are projected to be >9,200,000 births annually in countries with ongoing ZIKV transmission. In response to the ZIKV threat, the World Health Organization (WHO) is strategically targeting prevention of infection in pregnant women and funding contraception in epidemic regions. I propose that the damaging effects of ZIKV can be reduced using a seasonal window of opportunity for conception that may minimize maternal exposure. Like other acute viral infections—including the related flavivirus, dengue virus (DENV)—the transmission of ZIKV is anticipated to be seasonal. By seasonally planning pregnancy, this aspect of pathogen ecology can be leveraged to align sensitive periods of gestation with the low-transmission season.     

Coordination of different ligands to copper(II) and cobalt(III) metal centers enhances Zika virus and dengue virus loads in both arthropod cells and human keratinocytes

Trace elements such as copper and cobalt have been associated with virus-host interactions. However, studies to show the effect of conjugation of copper(II) or cobalt(III) metal centers to thiosemicarbazone ligand(s) derived from either food additives or mosquito repellent such as 2-acetylethiazole or citral, respectively, on Zika virus (ZIKV) or dengue virus (serotype 2; DENV2) infections have not been explored. In this study, we show that four compounds comprising of thiosemicarbazone ligand derived from 2-acetylethiazole viz., (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide (acetylethTSC) (compound 1), a copper(II) complex with acetylethTSC as a ligand (compound 2), a thiosemicarbazone ligand-derived from citral (compound 3) and a cobalt(III) complex with a citral-thiosemicarbazone ligand (compound 4) increased DENV2 and ZIKV replication in both mosquito C6/36 cells and human keratinocytes (HaCaT cells). Treatment of both cell lines with compounds 2 or 4 showed increased dengue viral titers at all three tested doses. Enhanced dengue viral plaque formation was also noted at the tested dose of 100 μM, suggesting higher production of infectious viral particles. Treatment with the compounds 2 or 4 enhanced ZIKV and DENV2 RNA levels in HeLa cell line and primary cultures of mouse bone marrow derived dendritic cells. Also, pre- or post treatments with conjugated compounds 2 or 4 showed higher loads of ZIKV or DENV2 envelope (E) protein in HaCaT cells. No changes in loads of E-protein were found in ZIKV-infected C6/36 cells, when compounds were treated after infection. In addition, we tested bis(1,10-phenanthroline)copper(II) chloride ([Cu(phen)₂]Cl₂, (compound 5) and tris(1,10-phenanthroline)cobalt(III) chloride ([Co(phen)₃]Cl₃, (compound 6) that also showed enhanced DENV2 loads. Also, we found that copper(II) chloride dehydrate (CuCl₂·2H₂O) or cobalt(II) chloride hexahydrate (CoCl₂·6H₂O) alone had no effects as “free” cations. Taken together, these findings suggest that use of Cu(II) or Co(III) conjugation to organic compounds, in insect repellents and/or food additives could enhance DENV2/ZIKV loads in human cells and perhaps induce pathogenesis in infected individuals or individuals pre-exposed to such conjugated complexes.

Dengue and Zika viruses: lessons learned from the similarities between these <Emphasis Type="Italic">Aedes</Emphasis> mosquito-vectored arboviruses

The currently spreading arbovirus epidemic is having a severe impact on human health worldwide. The two most common flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), are transmitted through the same viral vector, Aedes spp. mosquitoes. Since the discovery of DENV in 1943, this virus has been reported to cause around 390 million human infections per year, approximately 500,000 of which require hospitalization and over 20,000 of which are lethal. The present DENV epidemic is primarily concentrated in Southeast Asia. ZIKV, which was discovered in 1952, is another important arthropod-borne flavivirus. The neurotropic role of ZIKV has been reported in infected newborns with microcephaly and in adults with Guillain-Barre syndrome. Despite DENV and ZIKV sharing the same viral vector, their complex pathogenic natures are poorly understood, and the infections they cause do not have specific treatments or effective vaccines. Therefore, this review will describe what is currently known about the clinical characteristics, pathogenesis mechanisms, and transmission of these two viruses. Better understanding of the interrelationships between DENV and ZIKV will provide a useful perspective for developing an effective strategy for controlling both viruses in the future.     

Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.     

Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1‐caspase‐1 axis

Viral infection triggers host innate immune responses, which primarily include the activation of type I interferon (IFN) signaling and inflammasomes. Here, we report that Zika virus (ZIKV) infection triggers NLRP3 inflammasome activation, which is further enhanced by viral non‐structural protein NS1 to benefit its replication. NS1 recruits the host deubiquitinase USP8 to cleave K11‐linked poly‐ubiquitin chains from caspase‐1 at Lys134, thus inhibiting the proteasomal degradation of caspase‐1. The enhanced stabilization of caspase‐1 by NS1 promotes the cleavage of cGAS, which recognizes mitochondrial DNA release and initiates type I IFN signaling during ZIKV infection. NLRP3 deficiency increases type I IFN production and strengthens host resistance to ZIKVin vitro and in vivo. Taken together, our work unravels a novel antagonistic mechanism employed by ZIKV to suppress host immune response by manipulating the interplay between inflammasome and type I IFN signaling, which might guide the rational design of therapeutics in the future.