Continuous epileptiform discharges are associated with worse neurodevelopmental findings in a congenital Zika syndrome prospective cohort.

PURPOSE: This study aimed to identify continuous epileptiform discharges (CEDs) on electroencephalograms (EEG) and to determine their clinical significance in children with congenital Zika syndrome (CZS). METHODS: This prospective cohort study included 75 children diagnosed with CZS born from March 2015 and followed up until September 2018 (age up to 36 months). EEG was performed to detect CEDs up to 24 months old. Data on obstetric, demographic, and clinical signs; cranial computed tomography (CT); ophthalmology examination; anti-seizure medication; growth; and motor development were collected. Fisher's exact test was used to verify the associations between categorical variables, and the T- test was used to compare the mean z-scores of anthropometric measurements between the groups with and without CED. RESULTS: CEDs were identified in 41 (54.67 %) children. The mean age of CEDs identification was 12.24 ± 6.86 months. Bilateral CEDs were shown in 62.89 % of EEGs. CEDs were associated with severe congenital microcephaly, defined by z-score >3 standard deviation of head circumference (HC) below the mean for sex and age (p = 0.025), and worse outcomes, including first seizure before 6 months (p = 0.004), drug-resistant epilepsy (p < 0.001), chorioretinal scarring or mottling (p = 0.002), and severe CT findings (p = 0.002). The CED group had lower mean z-scores of HC up to 24 months of age. CONCLUSION: This is the first description of the prevalence and significance of CEDs that also remains during wakefulness in patients with CZS. New investigations may suggest that it is more appropriate to classify the EEG not as a CED, but as a periodic pattern. Anyway, CEDs may be a marker of neurological severity in children with CSZ.

Molecular functions of ANKLE2 and its implications in human disease.

Ankyrin repeat and LEM domain-containing 2 (ANKLE2) is a scaffolding protein with established roles in cell division and development, the dysfunction of which is increasingly implicated in human disease. ANKLE2 regulates nuclear envelope disassembly at the onset of mitosis and its reassembly after chromosome segregation. ANKLE2 dysfunction is associated with abnormal nuclear morphology and cell division. It regulates the nuclear envelope by mediating protein-protein interactions with barrier to autointegration factor (BANF1; also known as BAF) and with the kinase and phosphatase that modulate the phosphorylation state of BAF. In brain development, ANKLE2 is crucial for proper asymmetric division of neural progenitor cells. In humans, pathogenic loss-of-function mutations in ANKLE2 are associated with primary congenital microcephaly, a condition in which the brain is not properly developed at birth. ANKLE2 is also linked to other disease pathologies, including congenital Zika syndrome, cancer and tauopathy. Here, we review the molecular roles of ANKLE2 and the recent literature on human diseases caused by its dysfunction.

High-throughput crystallographic fragment screening of Zika virus NS3 Helicase.

The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3 Hel ) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3 Hel . Here we applied high-throughput crystallographic fragment screening on ZIKV NS3 Hel , which yielded structures that reveal 3D binding poses of 46 fragments at multiple sites of the protein, including 11 unique fragments in the RNA-cleft site. These fragment structures provide templates for direct design of hit compounds and should thus assist the development of novel direct-acting antivirals against ZIKV and related flaviviruses, thus opening a promising avenue for combating future outbreaks.

Crystallographic fragment screening delivers diverse chemical scaffolds for Zika virus NS2B-NS3 protease inhibitor development.

Zika virus (ZIKV) infections cause microcephaly in new-borns and Guillain-Barre syndrome in adults raising a significant global public health concern, yet no vaccines or antiviral drugs have been developed to prevent or treat ZIKV infections. The viral protease NS3 and its co-factor NS2B are essential for the cleavage of the Zika polyprotein precursor into individual structural and non-structural proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 48 binders with diverse chemical scaffolds were identified in the active site of the protease, with another 6 fragment hits observed in a potential allosteric binding site. Our work provides potential starting points for the development of potent NS2B-NS3 protease inhibitors. Furthermore, we have structurally characterized a potential allosteric binding pocket, identifying opportunities for allosteric inhibitor development.

Antiviral drug discovery: Pyrimidine entry inhibitors for Zika and dengue viruses.

Vector-borne diseases, constituting over 17 % of infectious diseases, are caused by parasites, viruses, and bacteria, and their prevalence is shaped by environmental and social factors. Dengue virus (DENV) and Zika virus (ZIKV), some of the most prevalent infectious agents of this type of diseases, are transmitted by mosquitoes belonging to the genus Aedes. The highest prevalence is observed in tropical regions, inhabited by around 3 billion people. DENV infects millions of people annually and constitutes an additional sanitary challenge due to the circulation of four serotypes, which has complicated vaccine development. ZIKV causes large outbreaks globally and its infection is known to lead to severe neurological diseases, including microcephaly in newborns. Besides, not only mosquito control programs have proved to be not totally effective, but also, no antiviral drugs have been developed so far. The envelope protein (E) is a major component of DENV and ZIKV virion surface. This protein plays a key role during the virus cell entry, constituting an attractive target for the development of antiviral drugs. Our previous studies have identified two pyrimidine analogs (3e and 3h) as inhibitors; however, their activity was found to be hindered by their low water solubility. In this study, we performed a low-throughput antiviral screening, revealing compound 16a as a potent DENV-2 and ZIKV inhibitor (EC50 = 1.4 µM and 2.4 µM, respectively). This work was aimed at designing molecules with improved selectivity and pharmacokinetic properties, thus advancing the antiviral efficacy of compounds for potential therapeutic use.

Neuroinvasion of emerging and re-emerging arboviruses: A scoping review.

Background: Arboviruses are RNA viruses and some have the potential to cause neuroinvasive disease and are a growing threat to global health. Objectives: Our objective is to identify and map all aspects of arbovirus neuroinvasive disease, clarify key concepts, and identify gaps within our knowledge with appropriate future directions related to the improvement of global health. Methods: Sources of Evidence: A scoping review of the literature was conducted using PubMed, Scopus, ScienceDirect, and Hinari. Eligibility Criteria: Original data including epidemiology, risk factors, neurological manifestations, neuro-diagnostics, management, and preventive measures related to neuroinvasive arbovirus infections was obtained. Sources of evidence not reporting on original data, non-English, and not in peer-reviewed journals were removed. Charting Methods: An initial pilot sample of 30 abstracts were reviewed by all authors and a Cohen's kappa of κ = 0.81 (near-perfect agreement) was obtained. Records were manually reviewed by two authors using the Rayyan QCRI software. Results: A total of 171 records were included. A wide array of neurological manifestations can occur most frequently, including parkinsonism, encephalitis/encephalopathy, meningitis, flaccid myelitis, and Guillain-Barré syndrome. Magnetic resonance imaging of the brain often reveals subcortical lesions, sometimes with diffusion restriction consistent with acute ischemia. Vertical transmission of arbovirus is most often secondary to the Zika virus. Neurological manifestations of congenital Zika syndrome, include microcephaly, failure to thrive, intellectual disability, and seizures. Cerebrospinal fluid analysis often shows lymphocytic pleocytosis, elevated albumin, and protein consistent with blood-brain barrier dysfunction. Conclusions: Arbovirus infection with neurological manifestations leads to increased morbidity and mortality. Risk factors for disease include living and traveling in an arbovirus endemic zone, age, pregnancy, and immunosuppressed status. The management of neuroinvasive arbovirus disease is largely supportive and focuses on specific neurological complications. There is a need for therapeutics and currently, management is based on disease prevention and limiting zoonosis.

New records on toxic cyanobacteria from Brazil: Exploring their occurrence and geography.

Cyanobacterial Harmful Algal Blooms (CyanoHABs) pose a significant threat to communities globally, impacting ecosystems and public health. This study provides an in-depth review of the current state of cyanotoxins and the distribution of CyanoHABs species in Brazil, while also detailing the methods used for their detection. Four hundred and twenty-one incidents were analyzed from 1993 to 2021, compiling cyanotoxin records and toxic CyanoHABs occurrences. The investigation begins with the first detection of microcystins in 1994 and highlights pivotal moments, like the 1996 "Caruaru Syndrome" outbreak. This event encouraged research and updated cyanotoxin-monitoring guidelines. The Brazilian drought period of 2015-2016 exacerbated cyanobacterial growth and saxitoxin levels, coinciding with Zika-related microcephaly. This study delves into methods used for cyanotoxin analysis, including ELISA, bioassays, HPLC, and LC-MS. Additionally, we investigated the toxicity of 37 cyanobacterial strains isolated from various Brazilian environments. Extracts were tested against Artemia salina and analyzed by LC-MS. Results revealed toxicity in extracts from 49 % of cyanobacterial strains. LC-MS results were analyzed using GNPS MS/MS molecular networking for comparing experimental spectra with those of cyanotoxin standards against in-house databases and the existing literature. Our research underscores the variability in cyanotoxin production among species and over time, extending beyond microcystins. LC-MS results, interpreted through the GNPS platform, revealed six cyanotoxin groups in Brazilian strains. Yet, compounds present in 75 % of the toxic extracts remained unidentified. Further research is crucial for fully comprehending the impact of potentially harmful organisms on water quality and public health management strategies. The study highlights the urgent need for continuously monitoring cyanobacteria and the cyanotoxin inclusion of management in public health policies.

Characterization of Two Highly Specific Monoclonal Antibodies Targeting the Glycan Loop of the Zika Virus Envelope Protein.

Zika virus (ZIKV) is an emerging flavivirus associated with several neurological diseases such as Guillain-Barré syndrome in adults and microcephaly in newborn children. Its distribution and mode of transmission (via Aedes aegypti and Aedes albopictus mosquitoes) collectively cause ZIKV to be a serious concern for global health. High genetic homology of flaviviruses and shared ecology is a hurdle for accurate detection. Distinguishing infections caused by different viruses based on serological recognition can be misleading as many anti-flavivirus monoclonal antibodies (mAbs) discovered to date are highly cross-reactive, especially those against the envelope (E) protein. To provide more specific research tools, we produced ZIKV E directed hybridoma cell lines and characterized two highly ZIKV-specific mAb clones (mAbs A11 and A42) against several members of the Flavivirus genus. Epitope mapping of mAb A11 revealed glycan loop specificity in Domain I of the ZIKV E protein. The development of two highly specific mAbs targeting the surface fusion protein of ZIKV presents a significant advancement in research capabilities as these can be employed as essential tools to enhance our understanding of ZIKV identification on infected cells ex vivo or in culture.

Exploring Zika Virus Impact on Endothelial Permeability: Insights into Transcytosis Mechanisms and Vascular Leakage.

Treating brain disease is challenging, and the Zika virus (ZIKV) presents a unique obstacle due to its neuroinvasive nature. In this review, we discuss the immunopathogenesis of ZIKV and explore how the virus interacts with the body's immune responses and the role of the protein Mfsd2a in maintaining the integrity of the blood-brain barrier (BBB) during ZIKV neuroinvasion. ZIKV has emerged as a significant public health concern due to its association with severe neurological problems, including microcephaly and Gillain-Barré Syndrome (GBS). Understanding its journey through the brain-particularly its interaction with the placenta and BBB-is crucial. The placenta, which is designed to protect the fetus, becomes a pathway for ZIKV when infected. The BBB is composed of brain endothelial cells, acts as a second barrier, and protects the fetal brain. However, ZIKV finds ways to disrupt these barriers, leading to potential damage. This study explores the mechanisms by which ZIKV enters the CNS and highlights the role of transcytosis, which allows the virus to move through the cells without significantly disrupting the BBB. Although the exact mechanisms of transcytosis are unclear, research suggests that ZIKV may utilize this pathway.

Single-cell analysis reveals an antiviral network that controls Zika virus infection in human dendritic cells.

Zika virus (ZIKV) is a mosquito-borne flavivirus that caused an epidemic in the Americas in 2016 and is linked to severe neonatal birth defects, including microcephaly and spontaneous abortion. To better understand the host response to ZIKV infection, we adapted the 10× Genomics Chromium single-cell RNA sequencing (scRNA-seq) assay to simultaneously capture viral RNA and host mRNA. Using this assay, we profiled the antiviral landscape in a population of human monocyte-derived dendritic cells infected with ZIKV at the single-cell level. The bystander cells, which lacked detectable viral RNA, expressed an antiviral state that was enriched for genes coinciding predominantly with a type I interferon (IFN) response. Within the infected cells, viral RNA negatively correlated with type I IFN-dependent and -independent genes (the antiviral module). We modeled the ZIKV-specific antiviral state at the protein level, leveraging experimentally derived protein interaction data. We identified a highly interconnected network between the antiviral module and other host proteins. In this work, we propose a new paradigm for evaluating the antiviral response to a specific virus, combining an unbiased list of genes that highly correlate with viral RNA on a per-cell basis with experimental protein interaction data. IMPORTANCE: Zika virus (ZIKV) remains a public health threat given its potential for re-emergence and the detrimental fetal outcomes associated with infection during pregnancy. Understanding the dynamics between ZIKV and its host is critical to understanding ZIKV pathogenesis. Through ZIKV-inclusive single-cell RNA sequencing (scRNA-seq), we demonstrate on the single-cell level the dynamic interplay between ZIKV and the host: the transcriptional program that restricts viral infection and ZIKV-mediated inhibition of that response. Our ZIKV-inclusive scRNA-seq assay will serve as a useful tool for gaining greater insight into the host response to ZIKV and can be applied more broadly to the flavivirus field.

The ZIKV NS5 Protein Aberrantly Alters the Tubulin Cytoskeleton, Induces the Accumulation of Autophagic p62 and Affects IFN Production: HDAC6 Has Emerged as an Anti-NS5/ZIKV Factor.

Zika virus (ZIKV) infection and pathogenesis are linked to the disruption of neurogenesis, congenital Zika syndrome and microcephaly by affecting neural progenitor cells. Nonstructural protein 5 (NS5) is the largest product encoded by ZIKV-RNA and is important for replication and immune evasion. Here, we studied the potential effects of NS5 on microtubules (MTs) and autophagy flux, together with the interplay of NS5 with histone deacetylase 6 (HDAC6). Fluorescence microscopy, biochemical cell-fractionation combined with the use of HDAC6 mutants, chemical inhibitors and RNA interference indicated that NS5 accumulates in nuclear structures and strongly promotes the acetylation of MTs that aberrantly reorganize in nested structures. Similarly, NS5 accumulates the p62 protein, an autophagic-flux marker. Therefore, NS5 alters events that are under the control of the autophagic tubulin-deacetylase HDAC6. HDAC6 appears to degrade NS5 by autophagy in a deacetylase- and BUZ domain-dependent manner and to control the cytoplasmic expression of NS5. Moreover, NS5 inhibits RNA-mediated RIG-I interferon (IFN) production, resulting in greater activity when autophagy is inhibited (i.e., effect correlated with NS5 stability). Therefore, it is conceivable that NS5 contributes to cell toxicity and pathogenesis, evading the IFN-immune response by overcoming HDAC6 functions. HDAC6 has emerged as an anti-ZIKV factor by targeting NS5.

Design, development, and validation of multi-epitope proteins for serological diagnosis of Zika virus infections and discrimination from dengue virus seropositivity.

Zika virus (ZIKV), an arbovirus from the Flaviviridae family, is the causative agent of Zika fever, a mild and frequent oligosymptomatic disease in humans. Nonetheless, on rare occasions, ZIKV infection can be associated with Guillain-Barré Syndrome (GBS), and severe congenital complications, such as microcephaly. The oligosymptomatic disease, however, presents symptoms that are quite similar to those observed in infections caused by other frequent co-circulating arboviruses, including dengue virus (DENV). Moreover, the antigenic similarity between ZIKV and DENV, and even with other members of the Flaviviridae family, complicates serological testing due to the high cross-reactivity of antibodies. Here, we designed, produced in a prokaryotic expression system, and purified three multiepitope proteins (ZIKV-1, ZIKV-2, and ZIKV-3) for differential diagnosis of Zika. The proteins were evaluated as antigens in ELISA tests for the detection of anti-ZIKV IgG using ZIKV- and DENV-positive human sera. The recombinant proteins were able to bind and detect anti-ZIKV antibodies without cross-reactivity with DENV-positive sera and showed no reactivity with Chikungunya virus (CHIKV)- positive sera. ZIKV-1, ZIKV-2, and ZIKV-3 proteins presented 81.6%, 95%, and 66% sensitivity and 97%, 96%, and 84% specificity, respectively. Our results demonstrate the potential of the designed and expressed antigens in the development of specific diagnostic tests for the detection of IgG antibodies against ZIKV, especially in regions with the circulation of multiple arboviruses.

PfAgo-Based Zika Virus Detection.

As a mosquito-borne flavivirus, Zika virus (ZIKV) has been identified as a global health threat. The virus has been linked to severe congenital disabilities, including microcephaly and other congenital malformations, resulting in fatal intrauterine death. Therefore, developing sensitive and specific methods for the early detection and accurate diagnosis of the ZIKV is essential for controlling its spread and mitigating its impact on public health. Herein, we set up a novel nucleic acid detection system based on Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection, targeting the non-structural protein 5 (NS5) region of the ZIKV genome (abbreviated ZIKV-PAND). Without preamplification with the polymerase chain reaction (PCR), the minimum detection concentration (MDC) of ZIKV-PAND was about 10 nM. When introducing an amplification step, the MDC can be dramatically decreased to the aM level (8.3 aM), which is comparable to qRT-PCR assay (1.6 aM). In addition, the diagnostic findings from the analysis of simulated clinical samples or Zika virus samples using ZIKV-PAND show a complete agreement of 100% with qRT-PCR assays. This correlation can aid in the implementation of molecular testing for clinical diagnoses and the investigation of ZIKV infection on an epidemiological scale.

Feeding characteristics and growth among children with prenatal exposure to Zika virus with and without microcephaly in the microcephaly epidemic research group pediatric cohort.

OBJECTIVE: To describe the feeding characteristics and growth of children with prenatal exposure to Zika virus (ZIKV) from birth to 48 months. DESIGN: Using data from the prospective Microcephaly Epidemic Research Group Pediatric Cohort (MERG-PC), children without microcephaly born to mothers with evidence of ZIKV infection during pregnancy (ZIKV-exposed children without microcephaly) and children with Zika-related microcephaly were compared using repeated cross-sectional analyses within the following age strata: birth; 1 to 12; 13 to 24; 25 to 36; and 37 to 48 months. The groups were compared in relation to prematurity, birth weight, breastfeeding, alternative feeding routes, dysphagia and anthropometric profiles based on the World Health Organization Anthro z-scores (weight-length/height, weight-age, length/height-age and BMI-age). RESULTS: The first assessment included 248 children, 77 (31.05%) with microcephaly and 171 (68.95%) without microcephaly. The final assessment was performed on 86 children. Prematurity was 2.35 times higher and low birth weight was 3.49 times higher in children with microcephaly. The frequency of breastfeeding was high (> 80%) in both groups. On discharge from the maternity hospital, the frequency of children requiring alternative feeding route in both groups was less than 5%. After 12 months of age, children with microcephaly required alternative feeding route more often than children without microcephaly. In children with microcephaly, the z-score of all growth indicators was lower than in children without microcephaly. CONCLUSIONS: Children with Zika-related microcephaly were more frequently premature and low birth weight and remained with nutritional parameters, i.e., weight-for-age, weight-for-length/height and length/height-for-age below those of the children without microcephaly.

Ophthalmologic Findings in a Premature Infant leading to a Zika Diagnosis during the COVID-19 Pandemic: A Case Report.

We report on the first case of congenital Zika syndrome to be identified during the COVID-19 pandemic in Puerto Rico. The Zika virus (ZIKV) infection was first seen in Puerto Rico in December 2015. It is a flavivirus with vertical transmission, spreading from infected mothers to their fetuses and having a broad spectrum of clinical manifestations, of which microcephaly is the most worrisome. In Puerto Rico, routine ZIKV screening during pregnancy was implemented in October 2016. However, this practice has become less frequent over time. Nevertheless, the transmission of ZIKV continues, so it is important to ensure routine ZIKV screening in endemic regions, such as Puerto Rico.

The impact of ZIKV infection on gene expression in neural cells over time.

Zika virus (ZIKV) outbreak caused one of the most significant medical emergencies in the Americas due to associated microcephaly in newborns. To evaluate the impact of ZIKV infection on neuronal cells over time, we retrieved gene expression data from several ZIKV-infected samples obtained at different time point post-infection (pi). Differential gene expression analysis was applied at each time point, with more differentially expressed genes (DEG) identified at 72h pi. There were 5 DEGs (PLA2G2F, TMEM71, PKD1L2, UBD, and TNFAIP3 genes) across all timepoints, which clearly distinguished between infected and healthy samples. The highest expression levels of all five genes were identified at 72h pi. Taken together, our results indicate that ZIKV infection greatly impacts human neural cells at early times of infection, with peak perturbation observed at 72h pi. Our analysis revealed that all five DEGs, in samples of ZIKV-infected human neural stem cells, remained highly upregulated across the timepoints evaluated. Moreover, despite the pronounced inflammatory host response observed throughout infection, the impact of ZIKV is variable over time. Finally, the five DEGs identified herein play prominent roles in infection, and could serve to guide future investigations into virus-host interaction, as well as constitute targets for therapeutic drug development.

Viral infections in pregnancy and impact on offspring neurodevelopment: mechanisms and lessons learned.

Pregnant individuals with viral illness may experience significant morbidity and have higher rates of pregnancy and neonatal complications. With the growing number of viral infections and new viral pandemics, it is important to examine the effects of infection during pregnancy on both the gestational parent and the offspring. Febrile illness and inflammation during pregnancy are correlated with risk for autism, attention deficit/hyperactivity disorder, and developmental delay in the offspring in human and animal models. Historical viral epidemics had limited follow-up of the offspring of affected pregnancies. Infants exposed to seasonal influenza and the 2009 H1N1 influenza virus experienced increased risks of congenital malformations and neuropsychiatric conditions. Zika virus exposure in utero can lead to a spectrum of abnormalities, ranging from severe microcephaly to neurodevelopmental delays which may appear later in childhood and in the absence of Zika-related birth defects. Vertical infection with severe acute respiratory syndrome coronavirus-2 has occurred rarely, but there appears to be a risk for developmental delays in the infants with antenatal exposure. Determining how illness from infection during pregnancy and specific viral pathogens can affect pregnancy and neurodevelopmental outcomes of offspring can better prepare the community to care for these children as they grow. IMPACT: Viral infections have impacted pregnant people and their offspring throughout history. Antenatal exposure to maternal fever and inflammation may increase risk of developmental and neurobehavioral disorders in infants and children. The recent SARS-CoV-2 pandemic stresses the importance of longitudinal studies to follow pregnancies and offspring neurodevelopment.

New candidate genes potentially involved in Zika virus teratogenesis.

Despite efforts to elucidate Zika virus (ZIKV) teratogenesis, still several issues remain unresolved, particularly on the molecular mechanisms behind the pathogenesis of Congenital Zika Syndrome (CZS). To answer this question, we used bioinformatics tools, animal experiments and human gene expression analysis to investigate genes related to brain development potentially involved in CZS. Searches in databases for genes related to brain development and CZS were performed, and a protein interaction network was created. The expression of these genes was analyzed in a CZS animal model and secondary gene expression analysis (DGE) was performed in human cells exposed to ZIKV. A total of 2610 genes were identified in the databases, of which 1013 were connected. By applying centrality statistics of the global network, 36 candidate genes were identified, which, after selection resulted in nine genes. Gene expression analysis revealed distinctive expression patterns for PRKDC, PCNA, ATM, SMC3 as well as for FGF8 and SHH in the CZS model. Furthermore, DGE analysis altered expression of ATM, PRKDC, PCNA. In conclusion, systems biology are helpful tools to identify candidate genes to be validated in vitro and in vivo. PRKDC, PCNA, ATM, SMC3, FGF8 and SHH have altered expression in ZIKV-induced brain malformations.

Design, synthesis, and antiviral activity of 1-aryl-4-arylmethylpiperazine derivatives as Zika virus inhibitors with broad antiviral spectrum.

Zika virus (ZIKV) disease has been given attention due to the risk of congenital microcephaly and neurodevelopmental disorders after ZIKV infection in pregnancy, but no vaccine or antiviral drug is available. Based on a previously reported ZIKV inhibitor ZK22, a series of novel 1-aryl-4-arylmethylpiperazine derivatives was designed, synthesized, and investigated for antiviral activity by quantify cellular ZIKV RNA amount using RT-qPCR method in ZIKV-infected human venous endothelial cells (HUVECs) assay. Structure-activity relationship (SAR) analysis demonstrated that anti-ZIKV activity of 1-aryl-4-arylmethylpiperazine derivatives is not correlated with molecular hydrophobicity, multiple new derivatives with pyridine group to replace the benzonitrile moiety of ZK22 showed stronger antiviral activity, higher ligand lipophilicity efficiency as well as lower cytotoxicity. Two active compounds 13 and 33 were further identified as novel ZIKV entry inhibitors with the potential of oral available. Moreover, both ZK22 and newly active derivatives also possess of obvious inhibition on the viral replication of coronavirus and influenza A virus at low micromolar level. In summary, this work provided better candidates of ZIKV inhibitor for preclinical study and revealed the promise of 1-aryl-4-arylmethylpiperazine chemotype in the development of broad-spectrum antiviral agents.

Construction of exosome-loaded LL-37 and its protection against zika virus infection.

Zika virus (ZIKV) is an enveloped, single-stranded and positive-stranded RNA virus of the genus Flavivirus in the family Flaviviridae. ZIKV can cross the placental barrier and infect the fetus, causing microcephaly, congenital ZIKV syndrome, and even fetal death. ZIKV infection can also lead to testicular damage and male sterility. But no effective drugs and vaccines are available up to now. Previous studies have shown that the cathelicidin antimicrobial peptide LL-37 can protect against ZIKV infection. However, LL-37 is a secreted peptide, which can be easily degraded in vivo. We herein constructed exosome-loaded LL-37 (named LL-37-TM-exo and TM-LL-37-exo) using the transmembrane protein TM to load LL-37 onto the membrane of exosome. We found that exosome-loaded LL-37 could significantly inhibit ZIKV infection in vitro and in vivo, and LL-37-TM-exo had stronger antiviral activity than that of TM-LL-37-exo, which could significantly reduce ZIKV-induced testicular injury and sperm injury, and had broad-spectrum antiviral effect. Compared to free LL-37, exosome-loaded LL-37 showed a better serum stability, higher efficiency to cross the placental barrier, and stronger antiviral activity. The mechanism of exosome-loaded LL-37 against ZIKV infection was consistent with that of free LL-37, which could directly inactivate viral particles, reduce the susceptibility of host cells, and act on viral replication stage. Our study provides a novel strategy for the development of LL-37 against viral infection.