The Relationship between HERV, Interleukin, and Transcription Factor Expression in ZIKV Infected versus Uninfected Trophoblastic Cells.

Zika virus (ZIKV) is an arbovirus with maternal, sexual, and TORCH-related transmission capabilities. After 2015, Brazil had the highest number of ZIVK-infected pregnant women who lost their babies or delivered them with Congenital ZIKV Syndrome (CZS). ZIKV triggers an immune defense in the placenta. This immune response counts with the participation of interleukins and transcription factors. Additionally, it has the potential involvement of human endogenous retroviruses (HERVS). Interleukins are immune response regulators that aid immune tolerance and support syncytial structure development in the placenta, where syncytin receptors facilitate vital cell-to-cell fusion events. HERVs are remnants of ancient viral infections that integrate into the genome and produce syncytin proteins crucial for placental development. Since ZIKV can infect trophoblast cells, we analyzed the relationship between ZIKV infection, HERV, interleukin, and transcription factor modulations in the placenta. To investigate the impact of ZIKV on trophoblast cells, we examined two cell types (BeWo and HTR8) infected with ZIKV-MR766 (African) and ZIKV-IEC-Paraíba (Asian-Brazilian) using Taqman and RT2 Profiler PCR Array assays. Our results indicate that early ZIKV infection (24-72 h) does not induce differential interleukins, transcription factors, and HERV expression. However, we show that the expression of a few of these host defense genes appears to be linked independently of ZIKV infection. Future studies involving additional trophoblastic cell lineages and extended infection timelines will illuminate the dynamic interplay between ZIKV, HERVs, interleukins, and transcription factors in the placenta.

Envelope Protein-Targeting Zika Virus Entry Inhibitors.

Zika virus (ZIKV; family, Flaviviridae), which causes congenital Zika syndrome, Guillain-Barré Syndrome, and other severe diseases, is transmitted mainly by mosquitoes; however, the virus can be transmitted through other routes. Among the three structural and seven nonstructural proteins, the surface envelope (E) protein of ZIKV plays a critical role in viral entry and pathogenesis, making it a key target for the development of effective entry inhibitors. This review article describes the life cycle, genome, and encoded proteins of ZIKV, illustrates the structure and function of the ZIKV E protein, summarizes E protein-targeting entry inhibitors (with a focus on those based on natural products and small molecules), and highlights challenges that may potentially hinder the development of effective inhibitors of ZIKV infection. Overall, the article will provide useful guidance for further development of safe and potent ZIKV entry inhibitors targeting the viral E protein.

Sustained Microglial Activation Promotes Synaptic Loss and Neuronal Dysfunction after Recovery from ZIKV Infection.

Zika virus (ZIKV), transmitted by Aedes mosquitoes, has been a global health concern since 2007. It primarily causes fetal microcephaly and neuronal defects through maternal transmission and induces neurological complications in adults. Recent studies report elevated proinflammatory cytokines and persistent neurological alterations post recovery, but the in vivo mechanisms remain unclear. In our study, viral RNA loads in the brains of mice infected with ZIKV peaked at 7 days post infection and returned to baseline by day 21, indicating recovery. RNA sequencing of the cerebral cortex at 7 and 21 days revealed upregulated genes related to neuroinflammation and microglial activation. Histological analyses indicated neuronal cell death and altered neurite morphology owing to severe neuroinflammation. Additionally, sustained microglial activation was associated with increased phospho-Tau levels, constituting a marker of neurodegeneration. These findings highlight how persistent microglial activation leads to neuronal dysfunction post ZIKV recovery, providing insights into the molecular pathogenesis of ZIKV-induced brain abnormalities.

Orthoflaviviral Inhibitors in Clinical Trials, Preclinical In Vivo Efficacy Targeting NS2B-NS3 and Cellular Antiviral Activity via Competitive Protease Inhibition.

Orthoflaviviruses, including zika (ZIKV), West Nile (WNV), and dengue (DENV) virus, induce severely debilitating infections and contribute significantly to the global disease burden, yet no clinically approved antiviral treatments exist. This review offers a comprehensive analysis of small-molecule drug development targeting orthoflaviviral infections, with a focus on NS2B-NS3 inhibition. We systematically examined clinical trials, preclinical efficacy studies, and modes of action for various viral replication inhibitors, emphasizing allosteric and orthosteric drugs inhibiting NS2B-NS3 protease with in vivo efficacy and in vitro-tested competitive NS2B-NS3 inhibitors with cellular efficacy. Our findings revealed that several compounds with in vivo preclinical efficacy failed to show clinical antiviral efficacy. NS3-NS4B inhibitors, such as JNJ-64281802 and EYU688, show promise, recently entering clinical trials, underscoring the importance of developing novel viral replication inhibitors targeting viral machinery. To date, the only NS2B-NS3 inhibitor that has undergone clinical trials is doxycycline, however, its mechanism of action and clinical efficacy as viral growth inhibitor require additional investigation. SYC-1307, an allosteric inhibitor, exhibits high in vivo efficacy, while temoporfin and methylene blue represent promising orthosteric non-competitive inhibitors. Compound 71, a competitive NS2B-NS3 inhibitor, emerges as a leading preclinical candidate due to its high cellular antiviral efficacy, minimal cytotoxicity, and favorable in vitro pharmacokinetic parameters. Challenges remain in developing competitive NS2B-NS3 inhibitors, including appropriate biochemical inhibition assays as well as the selectivity and conformational flexibility of the protease, complicating effective antiviral treatment design.

High-throughput peptide array analysis and computational techniques for serological profiling of flavivirus infections: Implications for diagnostics and vaccine development.

Arthropod-borne viruses, such as dengue virus (DENV), pose significant global health threats, with DENV alone infecting around 400 million people annually and causing outbreaks beyond endemic regions. This study aimed to enhance serological diagnosis and discover new drugs by identifying immunogenic protein regions of DENV. Utilizing a comprehensive approach, the study focused on peptides capable of distinguishing DENV from other flavivirus infections through serological analyses. Over 200 patients with confirmed arbovirus infection were profiled using high-density pan flavivirus peptide arrays comprising 6253 peptides and the computational method matrix of local coupling energy (MLCE). Twenty-four peptides from nonstructural and structural viral proteins were identified as specifically recognized by individuals with DENV infection. Six peptides were confirmed to distinguish DENV from Zika virus (ZIKV), West Nile virus (WNV), Yellow Fever virus (YFV), Usutu virus (USUV), and Chikungunya virus (CHIKV) infections, as well as healthy controls. Moreover, the combination of two immunogenic peptides emerged as a potential serum biomarker for DENV infection. These peptides, mapping to highly accessible regions on protein structures, show promise for diagnostic and prophylactic strategies against flavivirus infections. The described methodology holds broader applicability in the serodiagnosis of infectious diseases.

Inhibition of sterol O-acyltransferase 1 blocks Zika virus infection in cell lines and cerebral organoids.

Viruses depend on host metabolic pathways and flaviviruses are specifically linked to lipid metabolism. During dengue virus infection lipid droplets are degraded to fuel replication and Zika virus (ZIKV) infection depends on triglyceride biosynthesis. Here, we systematically investigated the neutral lipid-synthesizing enzymes diacylglycerol O-acyltransferases (DGAT) and the sterol O-acyltransferase (SOAT) 1 in orthoflavivirus infection. Downregulation of DGAT1 and SOAT1 compromises ZIKV infection in hepatoma cells but only SOAT1 and not DGAT inhibitor treatment reduces ZIKV infection. DGAT1 interacts with the ZIKV capsid protein, indicating that protein interaction might be required for ZIKV replication. Importantly, inhibition of SOAT1 severely impairs ZIKV infection in neural cell culture models and cerebral organoids. SOAT1 inhibitor treatment decreases extracellular viral RNA and E protein level and lowers the specific infectivity of virions, indicating that ZIKV morphogenesis is compromised, likely due to accumulation of free cholesterol. Our findings provide insights into the importance of cholesterol and cholesterol ester balance for efficient ZIKV replication and implicate SOAT1 as an antiviral target.

The TIRS trial: Enrollment procedures and baseline characterization of a pediatric cohort to quantify the epidemiologic impact of targeted indoor residual spraying on Aedes-borne viruses in Merida, Mexico.

Aedes mosquito-borne viruses (ABVs) place a substantial strain on public health resources in the Americas. Vector control of Aedes mosquitoes is an important public health strategy to decrease or prevent spread of ABVs. The ongoing Targeted Indoor Residual Spraying (TIRS) trial is an NIH-sponsored clinical trial to study the efficacy of a novel, proactive vector control technique to prevent dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) infections in the endemic city of Merida, Yucatan, Mexico. The primary outcome of the trial is laboratory-confirmed ABV infections in neighborhood clusters. Despite the difficulties caused by the COVID-19 pandemic, by early 2021 the TIRS trial completed enrollment of 4,792 children aged 2-15 years in 50 neighborhood clusters which were allocated to control or intervention arms via a covariate-constrained randomization algorithm. Here, we describe the makeup and ABV seroprevalence of participants and mosquito population characteristics in both arms before TIRS administration. Baseline surveys showed similar distribution of age, sex, and socio-economic factors between the arms. Serum samples from 1,399 children were tested by commercially available ELISAs for presence of anti-ABV antibodies. We found that 45.1% of children were seropositive for one or more flaviviruses and 24.0% were seropositive for CHIKV. Of the flavivirus-positive participants, most were positive for ZIKV-neutralizing antibodies by focus reduction neutralization testing which indicated a higher proportion of participants with previous ZIKV than DENV infections within the cohort. Both study arms had statistically similar seroprevalence for all viruses tested, similar socio-demographic compositions, similar levels of Ae. aegypti infestation, and similar observed mosquito susceptibility to insecticides. These findings describe a population with a high rate of previous exposure to ZIKV and lower titers of neutralizing antibodies against DENV serotypes, suggesting susceptibility to future outbreaks of flaviviruses is possible, but proactive vector control may mitigate these risks.

Fetal Zika virus inoculation in macaques revealed control of the fetal viral load during pregnancy.

BACKGROUND: Early pregnancy Zika virus (ZIKV) infection is associated with major brain damage in fetuses, leading to microcephaly in 0.6-5.0% of cases, but the underlying mechanisms remain largely unknown. METHODS: To understand the kinetics of ZIKV infection during fetal development in a nonhuman primate model, four cynomolgus macaque fetuses were exposed in utero through echo-guided intramuscular inoculation with 103 PFU of ZIKV at 70-80 days of gestation, 2 controls were mock inoculated. Clinical, immuno-virological and ultrasound imaging follow-ups of the mother/fetus pairs were performed until autopsy after cesarean section 1 or 2 months after exposure (n = 3 per group). RESULTS: ZIKV was transmitted from the fetus to the mother and then replicate in the peripheral blood of the mother from week 1 to 4 postexposure. Infected fetal brains tended to be smaller than those of controls, but not the femur lengths. High level of viral RNA ws found after the first month in brain tissues and placenta. Thereafter, there was partial control of the virus in the fetus, resulting in a decreased number of infected tissue sections and a decreased viral load. Immune cellular and humoral responses were effectively induced. CONCLUSIONS: ZIKV infection during the second trimester of gestation induces short-term brain injury, and although viral genomes persist in tissues, most of the virus is cleared before delivery.

Zika virus NS5 protein inhibits type I interferon signaling via CRL3 E3 ubiquitin ligase-mediated degradation of STAT2.

The ZIKA virus (ZIKV) evades the host immune response by degrading STAT2 through its NS5 protein, thereby inhibiting type I interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism underlying this process has remained elusive. In this study, we performed a genome-wide CRISPR/Cas9 screen, revealing that ZSWIM8 as the substrate receptor of Cullin3-RING E3 ligase is required for NS5-mediated STAT2 degradation. Genetic depletion of ZSWIM8 and CUL3 substantially impeded NS5-mediated STAT2 degradation. Biochemical analysis illuminated that NS5 enhances the interaction between STAT2 and the ZSWIM8-CUL3 E3 ligase complex, thereby facilitating STAT2 ubiquitination. Moreover, ZSWIM8 knockout endowed A549 and Huh7 cells with partial resistance to ZIKV infection and protected cells from the cytopathic effects induced by ZIKV, which was attributed to the restoration of STAT2 levels and the activation of IFN signaling. Subsequent studies in a physiologically relevant model, utilizing human neural progenitor cells, demonstrated that ZSWIM8 depletion reduced ZIKV infection, resulting from enhanced IFN signaling attributed to the sustained levels of STAT2. Our findings shed light on the role of ZIKV NS5, serving as the scaffold protein, reprograms the ZSWIM8-CUL3 E3 ligase complex to orchestrate STAT2 proteasome-dependent degradation, thereby facilitating evasion of IFN antiviral signaling. Our study provides unique insights into ZIKV-host interactions and holds promise for the development of antivirals and prophylactic vaccines.

Zika virus capsid protein closed structure modulates binding to host lipid systems.

Zika virus (ZIKV), a mosquito-borne Flavivirus of international concern, causes congenital microcephaly in newborns and Guillain-Barré syndrome in adults. ZIKV capsid (C) protein, one of three key structural proteins, is essential for viral assembly and encapsidation. In dengue virus, a closely related flavivirus, the homologous C protein interacts with host lipid systems, namely intracellular lipid droplets, for successful viral replication. Here, we investigate ZIKV C interaction with host lipid systems, showing that it binds host lipid droplets but, contrary to expected, in an unspecific manner. Contrasting with other flaviviruses, ZIKV C also does not bind very-low density-lipoproteins. Comparing with other Flavivirus, capsid proteins show that ZIKV C structure is particularly thermostable and seems to be locked into an auto-inhibitory conformation due to a disordered N-terminal, hence blocking specific interactions and supporting the experimental differences observed. Such distinct structural features must be considered when targeting capsid proteins in drug development.

The Role of TIM-1 and CD300a in Zika Virus Infection Investigated with Cell-Based Electrical Impedance.

Orthoflaviviruses cause a major threat to global public health, and no antiviral treatment is available yet. Zika virus (ZIKV) entry, together with many other viruses, is known to be enhanced by phosphatidylserine (PS) receptors such as T-cell immunoglobulin mucin domain protein 1 (TIM-1). In this study, we demonstrate for the first time, using cell-based electrical impedance (CEI) biosensing, that ZIKV entry is also enhanced by expression of CD300a, another PS receptor. Furthermore, inhibiting CD300a in immature monocyte-derived dendritic cells partially but significantly inhibits ZIKV replication. As we have previously demonstrated that CEI is a useful tool to study Orthoflavivirus infection in real time, we now use this technology to determine how these PS receptors influence the kinetics of in vitro ZIKV infection. Results show that ZIKV entry is highly sensitive to minor changes in TIM-1 expression, both after overexpression of TIM-1 in infection-resistant HEK293T cells, as well as after partial knockout of TIM-1 in susceptible A549 cells. These results are confirmed by quantification of viral copy number and viral infectivity, demonstrating that CEI is highly suited to study and compare virus-host interactions. Overall, the results presented here demonstrate the potential of targeting this universal viral entry pathway.

Low humidity enhances Zika virus infection and dissemination in Aedes aegypti mosquitoes.

As climate change alters Earth's biomes, it is expected the transmission dynamics of mosquito-borne viruses will change. While the effects of temperature changes on mosquito-virus interactions and the spread of the pathogens have been elucidated over the last decade, the impact of relative humidity changes is still relatively unknown. To overcome this knowledge gap, we exposed Aedes aegypti females to various humidity conditions. We measured different components of vectorial capacity such as survival, blood-feeding rates, and changes in infection and dissemination of Zika virus. Survival decreased as the humidity level decreased, while infection rates increased as the humidity level decreased. Alternatively, blood feeding rates and disseminated infection rates peaked at the intermediate 50% relative humidity treatment but were the same in the 30% and 80% relative humidity treatments. These results provide empirical evidence that Ae. aegypti exposure to low humidity can enhance Zika virus infection in the mosquito, which has important implications in predicting how climate change will impact mosquito-borne viruses.IMPORTANCEViruses transmitted by mosquitoes to humans are a major public health burden and are expected to increase under climate change. While we know that temperature is an important driver of variation in arbovirus replication in the mosquito, very little is known about how other relevant climate variables such as humidity will influence the interaction between mosquitoes and the viruses they transmit. Given the variability in humidity across environments, and the predicted changes in humidity under climate change, it is imperative that we also study the impact that it has on mosquito infection and transmission of arboviruses.

Multi-omics analysis of antiviral interactions of Elizabethkingia anophelis and Zika virus.

The microbial communities residing in the mosquito midgut play a key role in determining the outcome of mosquito pathogen infection. Elizabethkingia anophelis, originally isolated from the midgut of Anopheles gambiae possess a broad-spectrum antiviral phenotype, yet a gap in knowledge regarding the mechanistic basis of its interaction with viruses exists. The current study aims to identify pathways and genetic factors linked to E. anophelis antiviral activity. The understanding of E. anophelis antiviral mechanism could lead to novel transmission barrier tools to prevent arboviral outbreaks. We utilized a non-targeted multi-omics approach, analyzing extracellular lipids, proteins, metabolites of culture supernatants coinfected with ZIKV and E. anophelis. We observed a significant decrease in arginine and phenylalanine levels, metabolites that are essential for viral replication and progression of viral infection. This study provides insights into the molecular basis of E. anophelis antiviral phenotype. The findings lay a foundation for in-depth mechanistic studies.

Endogenous ZAP affects Zika virus RNA interactome.

One of the most recent advances in the analysis of viral RNA-cellular protein interactions is the Comprehensive Identification of RNA-binding Proteins by Mass Spectrometry (ChIRP-MS). Here, we used ChIRP-MS in mock-infected and Zika-infected wild-type cells and cells knockout for the zinc finger CCCH-type antiviral protein 1 (ZAP). We characterized 'ZAP-independent' and 'ZAP-dependent' cellular protein interactomes associated with flavivirus RNA and found that ZAP affects cellular proteins associated with Zika virus RNA. The ZAP-dependent interactome identified with ChIRP-MS provides potential ZAP co-factors for antiviral activity against Zika virus and possibly other viruses. Identifying the full spectrum of ZAP co-factors and mechanisms of how they act will be critical to understanding the ZAP antiviral system and may contribute to the development of antivirals.

The inoculum dose of Zika virus can affect the viral replication dynamics, cytokine responses and survival rate in immunocompromised AG129 mice.

Zika virus, a mosquito-borne arbovirus, has repeatedly caused large pandemics with symptoms worsening from mild and self-limiting diseases to Guillain-Barré syndrome in adults and fetal microcephaly in newborns. In recent years, Zika virus diseases have posed a serious threat to human health. The shortage of susceptible small animal models makes it difficult to study pathogenic mechanisms and evaluate potential therapies for Zika virus infection. Therefore, we chose immunocompromised mice (AG129 mice) deficient in IFN-α/ß and IFN-γ receptors, which can abolish the innate immune system that prevents Zika virus infection early. AG129 mice were infected with the Zika virus, and this mouse model exhibited replication dynamics, tissue tropism, pathological lesion and immune activation of the Zika virus. Our results suggest that the inoculum dose of Zika virus can affect the viral replication dynamics, cytokine responses and survival rate in AG129 mice. By testing the potential antiviral drug favipiravir, several critical indicators, including replication dynamics and survival rates, were identified in AG129 mice after Zika virus infection. It is suggested that the model is reliable for drug evaluation. In brief, this model provides a potential platform for studies of the infectivity, virulence, and pathogenesis of the Zika virus. Moreover, the development of an accessible mouse model of Zika virus infection will expedite the research and deployment of therapeutics and vaccines.

Computational investigation of stochastic Zika virus optimal control model using Legendre spectral method.

This study presents a computational investigation of a stochastic Zika virus along with optimal control model using the Legendre spectral collocation method (LSCM). By accumulation of stochasticity into the model through the proposed stochastic differential equations, we appropriating the random fluctuations essential in the progression and disease transmission. The stability, convergence and accuracy properties of the LSCM are conscientiously analyzed and also demonstrating its strength for solving the complex epidemiological models. Moreover, the study evaluates the various control strategies, such as treatment, prevention and treatment pesticide control, and identifies optimal combinations that the intervention costs and also minimize the proposed infection rates. The basic properties of the given model, such as the reproduction number, were determined with and without the presence of the control strategies. For R 0 < 0 , the model satisfies the disease-free equilibrium, in this case the disease die out after some time, while for R 0 > 1 , then endemic equilibrium is satisfied, in this case the disease spread in the population at higher scale. The fundamental findings acknowledge the significant impact of stochastic phonemes on the robustness and effectiveness of control strategies that accelerating the need for cost-effective and multi-faceted approaches. In last the results provide the valuable insights for public health department to enabling more impressive mitigation of Zika virus outbreaks and management in real-world scenarios.

Elucidating the inhibitory mechanism of Zika virus NS2B-NS3 protease with dipeptide inhibitors: Insights from molecular docking and molecular dynamics simulations.

Microcephaly, Guillain-Barré syndrome, and potential sexual transmission stand as prominent complications associated with Zika virus (ZIKV) infection. The absence of FDA-approved drugs or vaccines presents a substantial obstacle in combatting the virus. Furthermore, the inclusion of pregnancy in the pharmacological screening process complicates and extends the endeavor to ensure molecular safety and minimal toxicity. Given its pivotal role in viral assembly and maturation, the NS2B-NS3 viral protease emerges as a promising therapeutic target against ZIKV. In this context, a dipeptide inhibitor was specifically chosen as a control against 200 compounds for docking analysis. Subsequent molecular dynamics simulations extending over 200 ns were conducted to ascertain the stability of the docked complex and confirm the binding of the inhibitor at the protein's active site. The simulation outcomes exhibited conformity to acceptable thresholds, encompassing parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), ligand-protein interaction analysis, ligand characterization, and surface area analysis. Notably, analysis of ligand angles bolstered the identification of prospective ligands capable of inhibiting viral protein activity and impeding virus dissemination. In this study, the integration of molecular docking and dynamics simulations has pinpointed the dipeptide inhibitor as a potential candidate ligand against ZIKV protease, thereby offering promise for therapeutic intervention against the virus.

The human CD47 checkpoint is targeted by an immunosuppressive Aedes aegypti salivary factor to enhance arboviral skin infectivity.

The Aedes aegypti mosquito is a vector of many infectious agents, including flaviviruses such as Zika virus. Components of mosquito saliva have pleomorphic effects on the vertebrate host to enhance blood feeding, and these changes also create a favorable niche for pathogen replication and dissemination. Here, we demonstrate that human CD47, which is known to be involved in various immune processes, interacts with a 34-kilodalton mosquito salivary protein named Nest1. Nest1 is up-regulated in blood-fed female A. aegypti and facilitates Zika virus dissemination in human skin explants. Nest1 has a stronger affinity for CD47 than its natural ligand, signal regulatory protein α, competing for binding at the same interface. The interaction between Nest1 with CD47 suppresses phagocytosis by human macrophages and inhibits proinflammatory responses by white blood cells, thereby suppressing antiviral responses in the skin. This interaction elucidates how an arthropod protein alters the human response to promote arbovirus infectivity.

Zika virus vertical transmission induces neuroinflammation and synapse impairment in brain cells derived from children born with Congenital Zika Syndrome.

Zika virus (ZIKV) infection was first reported in 2015 in Brazil as causing microcephaly and other developmental abnormalities in newborns, leading to the identification of Congenital Zika Syndrome (CZS). Viral infections have been considered an environmental risk factor for neurodevelopmental disorders outcome, such as Autism Spectrum Disorder (ASD). Moreover, not only the infection per se, but maternal immune system activation during pregnancy, has been linked to fetal neurodevelopmental disorders. To understand the impact of ZIKV vertical infection on brain development, we derived induced pluripotent stem cells (iPSC) from Brazilian children born with CZS, some of the patients also being diagnosed with ASD. Comparing iPSC-derived neurons from CZS with a control group, we found lower levels of pre- and postsynaptic proteins and reduced functional synapses by puncta co-localization. Furthermore, neurons and astrocytes derived from the CZS group showed decreased glutamate levels. Additionally, the CZS group exhibited elevated levels of cytokine production, one of which being IL-6, already associated with the ASD phenotype. These preliminary findings suggest that ZIKV vertical infection may cause long-lasting disruptions in brain development during fetal stages, even in the absence of the virus after birth. These disruptions could contribute to neurodevelopmental disorders manifestations such as ASD. Our study contributes with novel knowledge of the CZS outcomes and paves the way for clinical validation and the development of potential interventions to mitigate the impact of ZIKV vertical infection on neurodevelopment.

Application of diceCT to Study the Development of the Zika Virus-Infected Mouse Brain.

Zika virus (ZIKV) impacts the developing brain. Here, a technique was applied to define, in 3D, developmental changes in the brains of ZIKV-infected mice. Postnatal day 1 mice were uninfected or ZIKV-infected, then analysed by iodine staining and micro-CT scanning (diffusible iodine contrast-enhanced micro-CT; diceCT) at 3-, 6-, and 10-days post-infection (dpi). Multiple brain regions were visualised using diceCT: the olfactory bulb, cerebrum, hippocampus, midbrain, interbrain, and cerebellum, along with the lens and retina of the eye. Brain regions were computationally segmented and quantitated, with increased brain volumes and developmental time in uninfected mice. Conversely, in ZIKV-infected mice, no quantitative differences were seen at 3 or 6 dpi when there were no clinical signs, but qualitatively, diverse visual defects were identified at 6-10 dpi. By 10 dpi, ZIKV-infected mice had significantly lower body weight and reduced volume of brain regions compared to 10 dpi-uninfected or 6 dpi ZIKV-infected mice. Nissl and immunofluorescent Iba1 staining on post-diceCT tissue were successful, but RNA extraction was not. Thus, diceCT shows utility for detecting both 3D qualitative and quantitative changes in the developing brain of ZIKV-infected mice, with the benefit, post-diceCT, of retaining the ability to apply traditional histology and immunofluorescent analysis to tissue.